Citation
Oxalate Degrading Enzymes of Oxalobacter formigenes and Escherichia coli

Material Information

Title:
Oxalate Degrading Enzymes of Oxalobacter formigenes and Escherichia coli
Creator:
Toyota, Cory
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (169 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Richards, Nigel G.
Committee Members:
Horenstein, Nicole A.
Dolbier, William R.
Lyons, Thomas J.
McKenna, Robert
Graduation Date:
8/9/2008

Subjects

Subjects / Keywords:
Active sites ( jstor )
Anhydrides ( jstor )
Biochemistry ( jstor )
Coenzymes ( jstor )
Crystal structure ( jstor )
Crystals ( jstor )
Enzymes ( jstor )
Escherichia coli ( jstor )
Kinetics ( jstor )
Oxalates ( jstor )
Chemistry -- Dissertations, Academic -- UF
Genre:
Electronic Thesis or Dissertation
born-digital ( sobekcm )
Chemistry thesis, Ph.D.

Notes

Abstract:
Oxalate is a toxic organic diacid found in a variety of foods; build up of oxalate in the gut is linked to the formation of calcium oxalate kidney stones. Humans have no innate mechanism for metabolizing oxalate, so enzymes that catalyze the degradation of oxalate may represent a form of therapy. Oxalobacter formigenes and Escherichia coli may influence oxalate homeostasis in man. The focus of this work is on understanding the kinetic mechanisms and metabolic roles of the proteins involved in oxalate metabolism in these organisms. Two enzymes are essential to O. formigenes metabolism: formyl-CoA transferase (FRC) and oxalyl-CoA decarboxylase (OXC). FRC catalyzes the transfer of CoA from formyl-CoA to oxalate to yield one molecule each of formate and oxalyl-CoA. The thioester product is subsequently decarboxylated by OXC in a proton-consuming reaction that is essential in energy generation for the organism. In addition to recombinant wild-type and His-tagged fusion proteins, a series of active site and truncation mutants were prepared for steady state kinetic analysis. Hydroxylamine and borohydride trapping experiments in conjunction with high resolution X-ray crystallographic freeze trapping experiments have outlined the complete kinetic mechanisms for both OXC and FRC. This work has demonstrated that despite differing in kinetic mechanism both Family I and Family III CoA transferase reactions proceed through an enzyme-CoA thioester intermediate. In addition, flexible loops in both enzymes, the C-terminal peptide loop of OXC and the tetraglycine loop (GGGGQ) of FRC, have been identified as critical to catalysis and substrate specificity. Structural genomics studies have shown that YfdW, encoded by the gene yfdW from an operon that appears to enhance the ability of Escherichia coli MG1655 to survive under acidic conditions, is structurally homologous to FRC. This work confirms that YfdW is a formyl-CoA transferase that appears to be more stringent than FRC in employing formyl-CoA and oxalate as substrates. Replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein shows that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Finally, in addition to demonstrating the value of utilizing structural homology in assigning protein function, this work suggests that the yfdW and yfdU genes in E. coli may be be involved in conferring oxalate-dependent acid resistance to the bacterium. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2008.
Local:
Adviser: Richards, Nigel G.
Statement of Responsibility:
by Cory Toyota.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Toyota, Cory. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Classification:
LD1780 2008 ( lcc )

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Full Text





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LIST OF FIGURES


Figure page

1-1 Cartoon representation of the key metabolic enzymes of 0. formigenes.........................21

1-2 C o en zy m e A ........................................................................... 2 4

1-3 Ping-pong kinetics of Family I glutaconate-CoA transferase (GCT) from
Acidaminococcus fermentans....... ................................................... ............... 26

1-4 Prosthetic cofactor of citrate lyase.......................... ............................... .. ............. 32

1-5 Double-reciprocal plots of initial velocity data consistent with an ordered mechanism
in Family II citramalate-CoA transferase from Klebsiella aerogenes (64). ....................33

1-6 Possible reaction m echanism s for FRC ........................................ ......................... 35

1-7 Cartoon representations of Family III CoA transferases ................................................41

1-8 Structure-based sequence alignment of Family III CoA transferase family members ......44

2-1 Summary of kinetic mechanisms for the three known CoA transferase Families............47

2-2 Double-reciprocal plot for the inhibition of FRC by free CoA against varied [formyl-
CoA ] at constant saturating [oxalate] ........................................ .......................... 50

2-3 Two formyl-CoA transferase monomers displayed separately and in the dimer..............51

2-4 Stereoview of the overlay of the two active site conformations of the 0-aspartyl-CoA
thioester complex ...................................... ................................. ..........52

2-5 Mass spectrum of formyl-CoA transferase incubated with formyl-CoA...........................54

2-6 Electron density maps of P-aspartyl-CoA thioester and chloride ions ............................ 54

2-7 Double-reciprocal plot of competitive C1- inhibition of FRC against varied [oxalate] .....55

2-8 Steroview overlay of FRC aspartyl-formyl and aspartyl-oxalyl anhydride active sites ....59

2-9 Stereoview of oxalate and format modeled into the FRC active site..............................60

2-10 Initial velocity plot of initial velocities of G261A variant against varied [oxalate] ..........61

2-11 Substrate inhibition of the G261A variant by oxalate against varied formyl-CoA ...........62

2-12 Ramachandran plot showing loop glycine residues (258GGGG261) ................................... 63

2-13 Stereoview of G 260A tetraglycine loop ................................................. .....................64









acetate could resume, and further, that the process could be facilitated by the addition of

oxidizable substrates such as ethyl, propyl, or butyl alcohol (158). This lead to the theory that

acetate must be "activated" in some fashion in order to be used as a substrate and, indeed, Lynen

was later able to identify acetyl-CoA from starved yeast (159). At the same time, several

independent research groups confirmed that some form of "active acetic acid" was required for

the enzymatic acetylation of both sulfanilamide (152) and choline (80, 176, 177) and it was

theorized that the same water soluble activator was involved in all cases. Fritz Lipmann (154)

was able to purify this activator-coenzyme for acetylation-and demonstrated that it comprised

pantothenic acid, or vitamin B5, whose origin and nature had been reported in the 1930's (263),

and, upon acid hydrolysis, P-alanine. The complete purification protocol, the presence of sulfur,

as well as the identification of a pyrophosphate bridge to an adenylate group was reported in

1950 (153, 155). Snell and coworkers were able to show that the sulfur-containing component of

CoA is cysteamine, or 2-amino-ethanethiol, linked to pantothenic acid by a peptide bond (228).

Baddiley used a combination of analysis and synthesis to show that the pyrophosphate group

binds to the 4-position of pantothenate (8). Kaplan demonstrated that the third phosphate is

bound to the 3'-position of the adenylyl-bound ribose (223). With the composition of CoA

understood and the availability of isolated acetyl-CoA, researchers would now have the tools

necessary to unravel the mysteries of the processes involved in P-oxidation.

Coenzyme A Pool in Bacteria

In vivo CoA pools have been determined for E. coli by three methods. Radioisotopic

methods were employed to assay the total CoA pool in P-alanine auxotrophs (panD2) grown on

glucose-minimal medium. Concentrations were about 380 iM with about 80% found as acetyl-

CoA(118). The three other major species were CoA, 52 riM; succinyl-CoA, 22 riM; and

malonyl-CoA, 2 iM. Chohnan and coworkers employed their malonate decarboxylase-









and subsequent gel filtration on a Superdex 200 column eluting with 5 mM HEPES buffer

containing 150 mM NaC1, pH 7.5.

Expression and Purification of His-Tagged FRC

The gene encoding Oxalobacterformigenes FRC (225) was cloned into the pET-28b

vector (Novagen, San Diego, CA) so as to introduce a His-tag with 10-amino acid linker at the

N-terminus of the recombinant protein. Primers used were as follows: 5'-Ndel 5'-AGG AGA

TAT ACA TAT GAC TAA ACC ATT AGA TGG AAT TAA TGT GC and 3'-BamH](stop) 5'-

AAG TCT GGA TCC TCA AAC TAC CTG T. BL21(DE3) competent cells were transformed

with the resulting construct and protein expression was induced by the addition of IPTG at an

OD600 of 0.6. After harvesting and pelleting by centrifugation at 5000 g for 15 min, the cells

were re-suspended in lysis buffer (50 mM potassium phosphate, pH 7.2, containing 300 mM

NaC1, 10 mM imidazole and 1 mM P-mercaptoethanol) and sonicated. Cell debris was removed

by centrifugation at 10,000 g for 15 min, and the supernatant was loaded on to an 0.5 mL Ni-

NTA column (Novagen) equilibrated with lysis buffer at 4 C. The column was washed with

lysis buffer containing 50 mM imidazole, and His-FRC was eluted (5 x 0.5 mL) with elution

buffer (50 mM potassium phosphate, pH 7.2, containing 300 mM NaCl and 250 mM imidazole).

Size-exclusion chromatography on a Sephadex G-25 column (30 mL) equilibrated with storage

buffer (25 mM sodium phosphate, pH 6.7, 300 mM NaC1, and 1 mM DTT) removed the

imidazole, and the purified protein was stored at -80 C in 10% glycerol.

Expression and Purification of FRC Variants

The expression and purification of wild-type FRC and the W48F and W48Q FRC variants

lacking the N-terminal histidine fusion tag were performed by following procedures described in

the literature (124, 197).









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mole of oxalate degraded, 1 mol of protons was also consumed with the concomitant production

of about equimolar CO2 and 0.9 mol of format. Carbon assimilation is mediated by conversion

of oxalyl-CoA into 3-phosphoglycerate by way of glycerate (51).

Formate appears to be an end product of oxalate catabolism in 0. formigenes rather than a

substrate for NAD-linked format dehydrogenase and source of cellular reducing equivalents (1).

Careful studies with native membrane vesicles in reconstituted proteoliposomes have

demonstrated that energy generation in 0. formigenes is the result of both electrostatic and

proton gradients created by the oxalate-2:formate-1 membrane-bound antiporter Oxlt (3).

Key Metabolic Proteins of Oxalobacterformigenes

0. formigenes depends on three identified proteins for the generation of metabolic energy

from oxalate: an oxalate-2:formate-1 antiporter (OxlT), formyl-CoA transferase (FRC), and

oxalyl-CoA decarboxylase (OXC). In the metabolic cycle, OxlT transports one molecule of

divalent extracellular oxalate into the cell with the concomitant transfer of one molecule of

monovalent format out of the cytosol (3). Oxalate is activated by transfer to coenzyme A from

formyl-CoA in a reaction catalyzed by FRC. The thioester product is subsequently

decarboxylated by OXC producing C02, regenerating formyl-CoA for subsequent cycles, and

consuming one equivalent of cytosolic protons (3, 9). Anion exchange promotes a polarization

of the membrane. The net extrusion of protons has the effect of increasing cytosolic pH and

drives an F1FoATP synthase with a proposed stoichiometry of 3H+/ATP. Decarboxylation of

oxalate and the assumption that CO2 diffuses irreversibly from the cell ensures a near

stoichiometric link between oxalate and the combination proton-motive and chemiosmotic

gradient. The importance of these three proteins is evident: together FRC and OXC make up

20% of the total cytosolic protein (9) and OxlT constitutes a major fraction (-10%) of the inner

membrane proteins in 0. formigenes (205).









Thefrc gene has since been cloned from 0. formigenes; recombinant wild-type FRC has

428 amino acids, pi of 5.2, a calculated monomeric mass of 47.3 kDa, and exists in solution as a

homodimer (225). Steady state kinetics and product inhibition studies by J6nsson et al.,

established that the enzyme exhibits an ordered sequential Bi-Bi kinetic mechanism where

formyl-CoA binds first followed by oxalate (124). Subsequent to the chemistry step, format is

released first followed by oxalyl-CoA. FRC was initially reported to be a monomer (9), but later

crystal structure data suggested that the catalytic subunit is dimeric (197). Size-exclusion

chromatography experiments have since confirmed that active FRC exists as the dimer (124).

High resolution X-ray crystal structures of YfdW, a putative formyl-CoA transferase in E.

coli, have been published independently by two groups (92, 97). YfdW, identified by structural

genomics as part of the effort to annotate the many unknown genes in E. coli (251), shares 63%

identity with FRC (see Figure 1-8) and is proposed to be a formyl-CoA transferase (97). YfdW

is the gene product of the yfdXWUVE operon, where the yfdX gene is under the control of the

EvGAS two-component regulatory system (see Figure 3-3), and has been implicated in acid

resistance in E. coli (169).

In addition, frc and oxc genes also appear to be involved in acid protection mechanisms in

Lactobacillus acidophilus. Variants withfrc and oxc deletions challenged by exposure to pH 3.5

showed a reduced ability to survive compared to the wild type (7). Interest in possible probiotic

treatments for oxalate stones has also prompted study of formyl-CoA transferase activity in L.

acidophilus. Of the 60 Lactobacillus strains evaluated, several showed high oxalate degrading

activity (247).

BaiF

Some prokaryotes are able to metabolize bile acids utilizing the bile acid inducible (bai)

operon (see the review by Ridlon et al. (199)). BaiF, the protein product of the baiF gene, is




























Figure. 4-2. OXC tetramer and active site. A, the OXC tetramer represented with one of the
catalytic dimers in a surface mode. ADP, dzThDP, and oxalyl-CoA are represented
as balls-and-sticks. The C-terminal region that undergoes organization upon
substrate binding is shown in red. B, the substrate binding site with side chains
interacting with oxalyl-CoA shown as sticks. The C-terminal residues after Arg-555
have been omitted for clarity. The main chain of two residues in the newly organized
C-terminus is shown coloured in red. Taken from Berthold 2007 (17).

The structure of OXC with bound dzThDP, refined to 2.2 A resolution (Table 4-1), is virtually

identical to the holoenzyme structure solved previously (rmsd, 0.24 A for 546 Ca atoms) (15).

Structure of the Oxalyl-CoA Complex and the CoA Complex

The substrate binding site in OXC was identified by crystallizing OXC inhibited with

dzThDP and then soaking oxalyl-CoA into the crystals. The structure was refined to 2.0 A

resolution. The substrate is bound with the CoA carrier in the cleft between the regulatory (R)-

and pyrophosphate (PP)-domain of one subunit, and, with a length of approximately 30 A, it

reaches all the way into the active site where the oxalyl group is well positioned for attack by the

cofactor (Figure 4-2). No significant reorganization of the active site takes place upon substrate

binding and superposition of the holostructure to the substrate complex results in an rmsd of

0.286 A for 546 Ca atoms. The only significant structural changes upon substrate binding are









In order to form the anhydride complex 2-3 ptL of a formyl-CoA solution was slowly

added to the crystals in the drop and crystals were then transferred to an ethylene glycol cryo

solution (1 M sodium citrate, 75 mM HEPES buffer, pH 7.2 and 25 % ethylene glycol) after

approximately 10 minutes. The formyl-CoA solution was prepared by mixing equal volumes of

20 mM formyl-CoA in 50 mM sodium acetate buffer, pH 5.0, and well solution.

Crystals of the G260A and Q17A mutants of FRC were obtained using the same conditions

as for the aspartyl-formyl anhydride complex. Crystals of the G260A mutant were directly frozen

in liquid nitrogen after transfer through silicon oil while crystals of Q17A were used for complex

formation. For the ternary complex the drops containing the Q17A mutant crystals were

supplemented with formyl-CoA as for the wild type aspartyl-formyl anhydride complex,

followed by the addition of 1 ptL 40 mM potassium oxalate mixed into the well solution. For this

complex, the ethylene glycol cryo-solution was supplemented with 40 mM potassium oxalate.

Crystals of the Q17A mutant belong to the space group 14 with unit cell dimensions a = b =

153.6 A and c = 98.1 A while the G260A mutant crystallized in space group P43212 with cell

dimensions of a = b = 97.3 A and c = 193.4 A.

Data collection, Structure Determination, and Refinement

Data were collected at beamlines ID14 ehl and ID23 eh2 at the European Synchrotron

Research Facility, Grenoble, France and at beamline 1911-2 at MAX-lab, Lund, Sweden. Data

collection and refinement statistics are summarized in Table 2-1. All images were integrated with

MOSFLM (146) and further processed using SCALA (11). Phases from the originally

determined apoenzyme (pdb accession code: lp5h) (197) were used to solve the structures by

molecular replacement using MOLREP (248). Refinement by the maximum likelihood method

was carried out in REFMAC5 (174) interspersed with manual model building in WinCoot (157)












QCOA

Asp ms 1

Mechanisms 1 and 1A


Mechanism 2


0Asp
Asp,$ vJ O


0
,CoA



0COP
o


sp H
Asp, &,oA


A
ASP,69 ,


Mechanism 3
0

Asp16 S _
-o


O
O H
0


H
H0 CoA
^ HS'
coo


3SH ,CoA


Aspl, %
ASie^^O


0 O l Note: lAformate
C 2 remains in active site

0 Note: Formate is
O .H released in Mech 1
0-


co




oa ^H
^-^ -
Asp 9 ..CoA Asp,$




O


AsP169 O CoA


co?


0
-H 0'H


oY
COS


Figure. 1-6. Possible reaction mechanisms for FRC. Mechanism 1 corresponds to the Family
III CoA transferase pathway that exhibits ping-pong kinetics. Mechanism 1A is the
ordered sequential variation of Mechanism 1 where format must remain in the active
site until oxalyl-CoA is formed. Mechanism 2 is analogous to the enzyme stabilized
direct-attack mechanism of Family II CoA transferases. Mechanism 3 is a third
proposed mechanism that avoids the enzyme-CoA thioester intermediate of
Mechanism 1 (124). Enzyme aspartyl-oxalyl mixed anhydride intermediates in the
reaction pathways are highlighted. Putative tetrahedral intermediates have been
omitted to save space.

Formyl-CoA transferase (FRC; EC 2.8.3.16)

Formyl-CoA transferase activity was first suggested by studies of Pseudomonas oxalaticus


(192) and limited kinetic characterization was carried out on native formyl-CoA transferase


(FRC) isolated from Oxalobacterformigenes (9). FRC has been detected in Enteroccusfaecalis


and identified by Western blotting using antibodies against frc from 0. formigenes (111).


CoA
c S
CO2


oe,CoA









339. Trp-48 is located in a flexible loop near the active site. The native fluorescence of Trp-48

may act as a reporter on gross conformational changes in the enzyme or AEDANS-labelled

cysteine mutants could be used to monitor changes in the environment of Trp-48. Trp-48 is

found on a loop connecting helices a2 and a3 and is located near both the tetraglycine loop and

catalytic Asp-169. Trp-48 is found packed against the glycine loop residues when in the closed

position (124), and if these environments differ enough to cause discernable changes in the

FRET donor characteristics of Trp-48, it may be useful as a reporter on conformational changes

linked to catalysis.









52. Cornick, N. A., and M. J. Allison. 1996. Assimilation of oxalate, acetate, and CO2 by
Oxalobacterformigenes. Can J Microbiol 42:1081-6.

53. Coros, A. M., L. Swenson, W. T. Wolodko, and M. E. Fraser. 2004. Structure of the
CoA transferase from pig heart to 1.7 A resolution. Acta Crystallogr D Biol Crystallogr
60:1717-25.

54. Corthesy-Theulaz, I. E., G. E. Bergonzelli, H. Henry, D. Bachmann, D. F.
Schorderet, A. L. Blum, and L. N. Ornston. 1997. Cloning and characterization of
Helicobacterpylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic
member of the CoA-transferase family. J Biol Chem 272:25659-67.

55. Davis, J. T., R. N. Moore, B. Imperiali, A. J. Pratt, K. Kobayashi, S. Masamune, A.
J. Sinskey, C. T. Walsh, T. Fukui, and K. Tomita. 1987. Biosynthetic Thiolase from
Zoogloea-Ramigera .1. Preliminary Characterization and Analysis of Proton-Transfer
Reaction. Journal of Biological Chemistry 262:82-89.

56. Dawson, K. A., M. J. Allison, and P. A. Hartman. 1980. Characteristics of anaerobic
oxalate-degrading enrichment cultures from the rumen. Appl Environ Microbiol 40:840-
6.

57. Dawson, K. A., M. J. Allison, and P. A. Hartman. 1980. Isolation and some
characteristics of anaerobic oxalate-degrading bacteria from the rumen. Appl Environ
Microbiol 40:833-9.

58. Dehning, I., and B. Schink. 1989. 2 New Species of Anaerobic Oxalate-Fermenting
Bacteria, Oxalobacter-vibrioformis Sp-Nov and Clostridium-oxalicum Sp-Nov, from
Sediment Samples. Archives of Microbiology 153:79-84.

59. DeLano, W. L. 2002. PYMOL. DeLano Scientific LLC, Palo Alto, CA.

60. Dickert, S., A. J. Pierik, D. Linder, and W. Buckel. 2000. The involvement of
coenzyme A esters in the dehydration of (R)-phenyllactate to (E)-cinnamate by
Clostridium sporogenes. Eur J Biochem 267:3874-84.

61. Dimroth, P. 1976. The prosthetic group of citrate-lyase acyl-carrier protein. Eur J
Biochem 64:269-81.

62. Dimroth, P., W. Buckel, R. Loyal, and H. Eggerer. 1977. Isolation and function of the
subunits of citramalate lyase and formation of hybrids with the subunits of citrate lyase.
Eur J Biochem 80:469-77.

63. Dimroth, P., and H. Eggerer. 1975. Isolation of subunits of citrate lyase and
characterization of their function in the enzyme complex. Proc Natl Acad Sci U S A
72:3458-62.

64. Dimroth, P., and H. Hilbi. 1997. Enzymic and genetic basis for bacterial growth on
malonate. Mol Microbiol 25:3-10.









3-16 Initial velocity plot of His-YfdU activity with varied oxalyl-CoA ............................. 101

3-17 Comparison of the active-site residues in YfdW and FRC................................103

3.18 Active-site structure in the W48Q FRC variant...........................................................105

4-1 Scheme of OXC mechanism for ThDP-dependent oxalyl-CoA decarboxylation ...........16

4-2 OX C tetram er and active site ........................................................................ 117

4-3 Three snapshots of OX C interim ediates .................................. ...................................... 118

4-4 Annealed composite omit maps calculated for the structures shown around the active
site ............... ...................................................................................................... . 1 1 9

4-5 Stereoview of postdecarboxylation intermediate complex.............................................121

4-6 Initial velocity plot of S553A OXC variant ....................................... ...............122

4-7 Stereoview of the aligned OXC structures.................................................................... 127

5-1 Pair-wise sequence alignment 10 A around the active sites of FRC and YfdW............134

5-2 Phylogram of FRC, putative formyl-CoA transferases, and known Family III CoA
transferases from pair-wise sequence alignment of polypeptide sequences....................135

5-4 IA E D A N S and A N S. ............................................................................ ......................139

C-l Phylogram of Family III CoA transferases from pair-wise sequence alignment of
nucleotide sequences......... .................................................................. ........ ........ 144

C-2 Phylogram of Family III CoA transferases from pair-wise sequence alignment of
am ino acid sequences......... .................................................................... ........ .. ...... .. 145

C-3 Phylogram of ThDP-dependent decarboxylases from pair-wise sequence alignment
of nucleotide sequences. ...................................................................................................... 146

C-4 Phylogram of ThDP-dependent decarboxylases from pair-wise sequence alignment
of am in o acid sequ en ces. ............................................ ............................................... 14 7









Size-Exclusion Chromatography Measurements.

A BIOSEP SEC-S2000 column (300 x 7.8 mm with 75 x 7.8-mm guard column) was

calibrated using lysozyme (14.4 kDa), carbonic anhydrase (29.0 kDa), peroxidase (44.0 kDa),

bovine serum albumin (66.0 kDa), alcohol dehydrogenase (150 kDa), amylase (200 kDa),

apoferritin (443 kDa), and thyroglobulin (669 kDa) in 100 mM potassium phosphate buffer, pH

7, with 100 mM KC1 at a flow rate of 1 mL/min. A 75 tL aliquot of 53.8 gg/tL YfdW in 100

mM potassium phosphate, pH 6.7 was injected to give a single peak with retention time

corresponding to a molecular mass of 85 kDa. KD is calculated as (Velution Vvoid)/(Vcolumn -

Vvoid).

Confirmation of Quench Conditions

Kinetic assays of CoA transferase activity in FRC were quenched in 10% acetic acid (124).

Conditions for stopping the transferase reaction with YfdW were examined. Reactions were run

as below: 90 tL of reaction mixture were quenched in 10 pL of either 10% or 20% HAc,

followed by incubation at either 0 oC or 32 oC. Aliquots were removed at times of up to 100

minutes and oxalyl-CoA concentration was ascertained by measurement of absorbance at A260

(vida infra).

Steady-State Kinetic Assays

All kinetic measurements were performed using an HPLC-based assay, as reported in

previous studies on FRC (124, 197). For measurements of YfdW-catalyzed CoA transfer, assay

mixtures consisted of YfdW (54 ng) and the carboxylic acid acceptor in 100 mM potassium

phosphate, pH 6.7 (total volume 100 tL). The concentration of free CoA in all samples was

normalized to that present as a contaminant in the assay mixtures containing the largest amount

of formyl-CoA. After incubating this solution at 30 oC, reaction was initiated by the addition of

formyl-CoA. An aliquot (90 pL) was taken after 60 seconds, and quenched by addition to 20%









Figure. 1-8. Structure-based sequence alignment of Family III CoA transferase family
members. This alignment, following page, was generated by the superimposition of
the crystal structures ofFRC (pdb lp5h), YfdW (pdb lpt5), MCR (pdb 1x74) (210),
and CaiB (pdb lxa3) (235). a-Helical and P-strand secondary structural elements are
colored orange and blue, respectively. Specific residues discussed in the text are
highlighted. Asterisks indicate residues identical in all four transferases. Taken from
Toyota 2008 (245).









that was similar to that determined for YfdW. Two variants were prepared in which Trp-48 was

replaced by phenylalanine (W48F) and glutamine (W48Q), and characterized under steady-state

conditions. Relatively little change in the specificity constants (kcat/KM) of the two FRC variant

enzymes for formyl-CoA and oxalate was evident when compared with the wild type enzyme

(Table 3-3). Perhaps more importantly, the W48Q FRC variant exhibited substrate inhibition

with oxalate as observed for YfdW having a Ki value of 74 mM (Figure 3-15). In contrast, the

W48F FRC variant was not inhibited by oxalate at concentrations up to 154 mM, suggesting that

hydrogen bonding to the Gln-48 side chain is an essential element for the interaction of this

substrate with the site, as suggested by the YfdW/acetyl-CoA/oxalate crystal structure (97). So as

to understand the structural effects of changing the tryptophan residue in more detail, the crystal

structures of the two FRC variants were obtained. Neither the W48Q nor the W48F FRC variant

displayed any major structural changes when compared to wild type FRC, with the rmsd of the

Ca atoms being 0.2-0.3 A2 and 0.6-0.7 A2 relative to subunit A and subunit B of apo-FRC,

respectively. In both variant enzymes, the tetraglycine loop (corresponding to residues

258GGGGQ262 in FRC) was seen to adopt a "closed" conformation (197). In wild type FRC, a 900

reorientation of the Trp-48 side chain seems to be important in controlling the tetraglycine loop

conformation. This "flipping" of the indole moiety, however, is accompanied by repositioning of

Met-44 when the loop adopts its "open" conformation. For YfdW, in which a glutamine residue

(Gln-48) replaces tryptophan however, oxalate can bind to the tetraglycine loop in the closed

conformation, even though Gln-48 adopts the rotamer conformation corresponding to that of

Trp-48 in FRC when the cognate loop is "open". Comparison of the YfdW and W48Q FRC

variant structures showed that Gln-48 in W48Q, in the absence of oxalate, does not take the side

chain rotamer conformation seen for the cognate residue in YfdW, presumably because of the









130. Kaplun, A., E. Binshtein, M. Vyazmensky, A. Steinmetz, Z. Barak, D. M. Chipman,
K. Tittmann, and B. Shaanan. 2008. Glyoxylate carboligase lacks the canonical active
site glutamate of thiamine-dependent enzymes. Nat Chem Biol 4:113-8.

131. Kaschabek, S. R., B. Kuhn, D. Muller, E. Schmidt, and W. Reineke. 2002.
Degradation of aromatics and chloroaromatics by Pseudomonas sp. strain B13:
purification and characterization of 3-oxoadipate:succinyl-coenzyme A (CoA) transferase
and 3-oxoadipyl-CoA thiolase. J Bacteriol 184:207-15.

132. Katagiri, M., and 0. Hayaishi. 1957. Enzymatic degradation ofbeta-ketoadipic acid. J
Biol Chem 226:439-48.

133. Kaur, J., and R. Sharma. 2006. Directed evolution: An approach to engineer enzymes.
Critical Reviews in Biotechnology 26:165-199.

134. Kern, D., G. Kern, H. Neef, K. Tittmann, M. Killenberg-Jabs, C. Wikner, G.
Schneider, and G. Hubner. 1997. How thiamine diphosphate is activated in enzymes.
Science 275:67-70.

135. Kern, D., G. Kern, G. Scherer, G. Fischer, and T. Drakenberg. 1995. Kinetic-
Analysis of Cyclophilin-Catalyzed Prolyl Cis/Trans Isomerization by Dynamic Nmr-
Spectroscopy. Biochemistry 34:13594-13602.

136. Kim, J., D. G. Beak, Y. T. Kim, J. D. Choi, and M. Y. Yoon. 2004. Effects of deletions
at the C-terminus of tobacco acetohydroxyacid synthase on the enzyme activity and
cofactor binding. Biochemical Journal 384:59-68.

137. Kim, J., D. Darley, T. Selmer, and W. Buckel. 2006. Characterization of (R)-2-
hydroxyisocaproate dehydrogenase and a family III coenzyme A transferase involved in
reduction of L-leucine to isocaproate by Clostridium difficile. Appl Environ Microbiol
72:6062-9.

138. Kleber, H. P. 1997. Bacterial carnitine metabolism. FEMS Microbiol Lett 147:1-9.

139. Knoop, F. 1904/05. Der Abbau aromatischer Fettsauren im Tierkorper. Beitr. Chem.
Physiol. Pathol. 6:150-62.

140. Kosaka, T., T. Uchiyama, S. Ishii, M. Enoki, H. Imachi, Y. Kamagata, A. Ohashi, H.
Harada, H. Ikenaga, and K. Watanabe. 2006. Reconstruction and regulation of the
central catabolic pathway in the thermophilic propionate-oxidizing syntroph
Pelotomaculum thermopropionicum. J Bacteriol 188:202-10.

141. Koshland, D. E., Jr. 1996. The structural basis of negative cooperativity: receptors and
enzymes. Curr Opin Struct Biol 6:757-61.

142. Kuhner, C. H., P. A. Hartman, and M. J. Allison. 1996. Generation of a proton motive
force by the anaerobic oxalate-degrading bacterium Oxalobacterformigenes. Appl
Environ Microbiol 62:2494-500.









the accessibility of the active site. Mutations in the loop seriously affect the activity, proving its

importance during catalysis. A modified mechanism in concordance with all information

obtained is proposed, where catalysis includes formation of both the aspartyl-formyl and -oxalyl

anhydrides and the P-aspartyl-CoA thioester and where the carboxylate product remains bound to

the enzyme until release of the acceptor thioester.

Results

Wild-Type FRC Activity

By first determining the inhibitory effects of free CoA (Table 2-5), a ubiquitous

contaminant resulting from the hydrolysis of formyl-CoA, the kinetic parameters for FRC and

mutant variants were improved (see Figure 2-2). The values obtained for FRC by this method

were similar to previously reported values (124). Fortuitiously, the inhibition constant for CoA

(KiCoA) was 16.7 + 0.7 iM and high enough that there was little effect on the original kinetic

analysis.

Enzyme-P-Aspartyl-CoA Thioester Complexes

Crystals, produced with a precipitant mixture of polyethylene glycol and magnesium

chloride, were soaked with formyl-CoA for 1, 5, and 10 min respectively and with oxalyl-CoA

for 2, 4, and 10 min, respectively. Inspection of the crystal structures from different soaking

times revealed that all formyl-CoA soaked crystals contained the same intermediate and all

oxalyl-CoA soaked crystals contained the same intermediate, with no difference over time. The

best data set of each, formyl-CoA soaked for 2 min and oxalyl-CoA soaked for 5 min, were used

for further analysis.

Close inspection of the freeze-trapped formyl-CoA and oxalyl-CoA intermediates, show

that the formyl- as well as the oxalyl moieties are cleaved off by the enzyme, and a covalent

bond is formed between the carboxyl group of Asp-169 and the thiol-group of the CoA carrier.









solution of 1.9 M malic acid, pH 7.0, gave crystals belonging to the monoclinic space group C2.

The crystallization drops containing the W48F mutant were covered in silicon oil, through which

the crystals were dragged before being flash-frozen. X-ray data were collected in a nitrogen

stream at the beamlines ID14 ehl and ID23 eh2 at the European Synchrotron Research Facility,

Grenoble. All crystallographic data were processed with MOSFLM (147) followed by SCALA

of the CCP4 program suit (11). The structure of the apoenzyme (pdb code: lp5h) (197) was used

to retrieve the phases by molecular replacement using the program MOLREP (248). Refinement

was carried out with REFMAC (174) and manual model building was performed in COOT (76)

where water molecules were assigned and the structures were validated. The stereochemistry of

the structures was checked with PROCHECK (144).

Formyl-CoA Hydrolysis in the Presence and Absence of FRC, D169S, and YfdW

Pseudo-first order rate constants for the hydrolysis of formyl-CoA at pH 6.7 and 30C

were determined by standard fitting procedures. Half-lives for hydrolysis in the presence of

FRC, D169A, and YfdW were 51, 58, and 198 minutes, respectively. Formyl-CoA hydrolysis

has been reported with a half-life of 150 minutes at pH 6.7 and 300C (124).

Cloning, Expression, and Purification of HisYfdU and HisYfdW

The E. coli genes yfdW and yfdUwere cloned from genomic DNA isolated from

BL21(DE3) by nested PCR. DNA was purified by phenol-chloroform extraction (208). The first

PCR primers were 5'- CGC CTG GCC GGT GTT GGC GTA ATG G and 3'-5'- CCC TGT

TTG CCC GAG TAA TAG ATA CAA ATA GAG CCG C. Nested primers were designed to

include upstream Ndel and downstream HindIIl restriction endonuclease sites: HisYfdW 5'- A

GGT ATT CAT ATG TCA ACT CCA CTT CAA GGA ATT AAA GTT CTC GAT TTC, His

YfdW 3'-5'- GGG AGC AAG CTT CCC CCG TTA ATA TCA GAT GGC G, HisYfdU 5'-

CGA GGT TAT TAC ATA TGT CAG ATC AAC TTC AAA TGA CAG ATG G, and HisYfdU











'open loop' B


COAS L


'edosed loop' F


Formats


CoAS t


Figure. 2-17. Models and crystal structures showing assumed important features in the active site between the catalytic steps in
Figure 2-15. For clarity, the amino acid residues are only labeled in A. Glycine loop is shown as Ca trace. A, model of
formyl-CoA in the active site. B, aspartyl-formyl anhydride formed after step 1; C, the enzyme-CoA thioester; D, the
activated conformation of the enzyme-CoA thioester observed in subunit B of the crystal structure; E, the second
anhydride; and, F, the apoenzyme with both products modeled in the active site. Taken from Berthold 2008 (18).









Table 2-1. Data collection and refinement statistics. Values in parentheses represent the highest resolution shell.
Data Collection P-aspartyl-CoA P-aspartyl- Aspartyl- Q17A-3- G260A FRC
thioester from CoA thioester formyl aspartyl-CoA
formyl-CoA from oxalyl- anhydride thioester and
CoA oxalate
Beamline ID14 ehl ID14 ehl 1911-2 1911-2 ID23 eh2
Space group 14 14 14 14 P43212
Unit cell a,b,c (A) 151.8, 151.8, 151.9, 151.9, 151.7, 151.7, 153.6, 153.6, 97.3, 97.3,
100.1 99.5 98.9 98.1 193.4
Resolution (A) 2.0 (2.11-2.0) 2.0 (2.11-2.0) 1.87 (1.97- 2.2 (2.32-2.2) 2.0 (2.11-2.0)
1.87)
Rsym 0.11 (0.39) 0.11(0.30) 0.067 (0.36) 0.12(0.57) 0.15 (0.50)
Mn(I/o(I)) 8.7 (1.8) 10.1 (2.4) 15.9 (4.2) 9.3 (1.9) 7.2 (2.5)
Completeness (%) 98.3 (94.3) 99.2 (97.2) 97.1 (81.1) 99.5 (100) 99.6 (99.9)
Wilson B-factor 28 25 23 34 14
Refinement
Resolution (A) 30-2.0 30.0-2.0 30-1.87 30-2.2 30-2.0
Reflections in working set 71459 74942 85088 54545 59799
Reflections in test set 3765 3910 4473 2894 3206
R-factor / R-free (%) 19.7/24.1 17.3/21.4 17.2/20.3 20.7/24.8 16.9/21.5
Atoms modeled 7565 7565 7509 7197 7566
No. of amino acids/ B-factor (A2) 854 / 35.4* 854 / 29.7* 854 / 25.1* 854/ 33.0 854 / 12.5
Number of ligands / B-factor (A2) 2 / 47.3** 2 / 42.4** 4 / 30.5** 3/41.3 0/-
Number of waters / B-factor (A2) 693 / 38.2 788 / 35.5 737 / 31.6 430 / 29.2 873 /21.8
RMS Deviations from ideals
Bonds (A) 0.007 0.008 0.008 0.008 0.009
Angles (0) 1.08 1.12 1.06 1.15 1.16
Ramachandran zone distribution (%) 92.1, 7.5, 0.1, 92.5, 7.2, 0.1, 91.8, 7.9, 0.3, 92.1, 7.6, 0.3, 92.0, 7.6, 0.3,
0.3 0.1 0 0 0.1
PDB Accession code 2vjl 2vjk 2vjm 2vjo 2vjn
(includes amino acid part of residue 169) ** (includes CoA/formyl part of covalent complex at residue 169)









that the two active sites in the YfdW dimer can "communicate" so that only a single active site

can catalyze the reaction at a given time ("half-sites" reactivity) (141). As a result, if CoA-

derivatives bind to a free CoA site in a YfdW/substrate complex (or catalytic intermediate), then

the enzyme undergoes a conformational change that precludes the formation of critical

intermediates (18, 124) or product release at the other site. A more interesting observation was

that YfdW is inhibited by elevated levels of oxalate, a kinetic behavior that is not seen for the

Oxalobacter formyl-CoA transferase. This inhibition was hypothesized to arise from oxalate

binding at a second "non-productive" site defined (in part) by the Gln-48 side chain in YfdW.

Such binding is precluded by the presence of a tryptophan residue in FRC, and replacement of

Trp-48 by glutamine to give the W48Q FRC variant yields an enzyme for which oxalate

inhibition is observed. Hence, it seems that replacing the indole side chain by that of glutamine

"opens" a hole in the FRC active site into which oxalate can bind in a non-productive

conformation. This mutation also results in altered conformational preferences of a tetraglycine

loop that is known to be important for catalytic function (18, 123, 124, 197), implying that

altered active site dynamical motions may play a role in modulating kinetic properties (24, 72).

The high Ki determined for oxalate in YfdW inhibition seems to preclude any physiological

importance for this behavior. With the identification of YfdW as a formyl-CoA:oxalate CoA

transferase, questions are raised concerning the extent and importance of oxalate-related

metabolism in Escherichia coli, especially because this work demonstrates that YfdU is a ThDP-

dependent oxalyl-CoA decarboxylase. Although Escherichia coli has been implicated in the

biomineralization processes leading to formation of calcium oxalate crystals (41), recent

measurements suggest that Escherichia coli does not degrade oxalate in media containing this

compound at 5 mM concentration (247). The experiments, however, did not systematically vary











regions can be monitored fluorescently to characterize folding intermediate populations as seen


in experiments on p-lactamase (96) and the trp repressor (166).


-10-



-12-
--


a -14-
0)
a-

? -16-
E
0 0
0)
18-



-20-


20 40 60 80 100

Temperature (OC)


Figure. 5-3. Melting curve for FRC monitored by CD at 290 nm.


Due to the irreversible nature of FRC dimerization, it seems likely that chaperones play an


important in the expression of the active enzyme. If this is true, both E. coli and 0. formigenes,


must both express this chaperone. A simple immuniprecipitation experiment in conjuction with


proteolytic MS will likely identify this theoretical protein. As both FRC and YfdW can be


expressed with polyhistidine fusion tags, commercially available anti-His antibodies can be used


to coprecipitate the transferases with any proteins associated in their synthesis.


I



'I


a%









conditions or protein buffer for this complex contained no chloride ions and it is likely that the

ion was bound during expression or purification of the mutant and remained due to the lack of a

glutamine side chain occupying the site.

G259A, G260A, and G261A FRC Loop Mutants

Three FRC variants, where Gly-259, Gly-260, and Gly-261 were mutated into alanine

residues, were prepared to investigate the importance of the glycine loop. The mutations were

expected to impact the loop movement, because the peptide geometries of the Gly-259 and Gly-

260 residues are positioned in the disallowed region of the Ramachandran plot for alanine. As


4.0 20 08
g 15 0.6

S5 02 -
3.0 0.0
0 20 40 60 80 0 20 40 60 80
2.5 xate], mM [Oxalate], mM
E
2 2.0

i 1.5

1.0


-0.03 -0.01 0.01


0.03 0.05 0.07 0.09 0.11
1/[Formyl-CoA], pM-1


0.13 0.15


Figure. 2-11. Substrate inhibition of the G261A variant by oxalate against varied formyl-CoA.
Initial velocity plot of initial velocities against varied [formyl-CoA] (9.9 mM 78.4
piM) at 7.5 (e), 30.5 (o), and 77.0 mM [oxalate] with 45.3 nM enzyme. Data were
fitted to the Lineweaver-Burk equation. Apparent inhibition constants for substrate
inhibition by oxalate, Kic = 4 mM and Kiu = 73 mM, were determined from the replots
of slopes and intercepts.









237. Stewart, C. S., S. H. Duncan, and D. R. Cave. 2004. Oxalobacterformigenes and its
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244. Thomas, P. D., H. Mi, and S. Lewis. 2007. Ontology annotation: mapping genomic
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246. Turano, A., W. Furey, J. Pletcher, M. Sax, D. Pike, and R. Kluger. 1982. Synthesis
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Figure. 3.18. Active-site structure in the W48Q FRC variant. A, superimposition of apo-FRC
with the tetraglycine loop in its open (white) and closed (green) conformations and
the W48Q FRC variant (pink). B, superimposition of apo-YfdW cyann) with the open
conformation of the tetraglycine loop, the YfdW-acetyl-CoA-oxalate ternary
complex with the tetraglycine loop in its closed conformation (blue), and the W48Q
FRC variant with the tetraglycine loop in its closed conformation (pink). In both
panels, the catalytic residue, Asp-169, and side chains important in controlling the
conformational properties of the tetraglycine loop are displayed as stick models. Met-
44 in the W48Q FRC variant is modeled in two conformations, and the carbonyl
group of acetyl-CoA also adopts two conformations in the structure of the YfdW-
acetyl-CoA-oxalate ternary complex (97). Taken from Toyota 2008 (245).

the incubation conditions and so it is possible that conditions exist under which Escherichia coli

can metabolize exogenous oxalate. On this point, it should be noted that the YhjX gene product

has been annotated as a possible formate:oxalate antiporter based on 25% sequence identity to

Oxalobacterformigenes OxlT, which has been extensively characterized (108, 255). In a recent

transcriptomic profiling study, YhjX has been identified as a transporter upregulated by rapid

cellular acidification (pH 5.5) in Escherichia coli (129). Work is therefore needed to establish if

Escherichia coli can mediate oxalate degradation, especially when in low pH environments. It is

therefore interesting that the coupled action of YfdW and YfdU results in the consumption of a









site allowing loop B to open, then disruption may allow oxalate to again bind out of turn in the

second site. The competitive and uncompetitive components of CoA inhibition, 2 [iM and 41

rIM, respectively, are the lowest seen for any FRC variant and these results also support the

critical nature of the loop.

Table 2-4. Summary of kinetic constants for wild-type FRC and mutants
kcat/KM(F-CoA) kcat/KM(oxalate)
kcat (s-') KM(F-CoA) ([tM) (s-M^-) KM(oxalate) (mM) (s-M^)1)
FRC 5.3 0.1 2.0 + 0.3 2.7 0.4 x 106 3.9 0.3 1.4 0.1 x 103
G259A 1.9 0.1 4.7 0.8 4.1 0.6 x 105 12.1 0.5 160 7
G260A 0.23 0.02 18 3 1.3 0.2 x 104 18.0 1.6 12 1
G261A 1.65 0.01 26.6 0.9 6.2 0.2 x 104 0.47 0.08 3.5 0.2 x 103
Q17A 0.12 0.1 3.3 0.5 3.6 0.6 x 104 13.2 0.6 8.7 0.9

Table 2-5. Summary of the inhibition constants and patterns for wild-type FRC and mutants.
([tM) FRC Q17A G258A G259A G260A G261A
CoASH competitive mixed-type mixed-type mixed-type mixed-type
K,, 16.7 0.7 16.0 0.6 6.0 1.0 55 19 2 1
K, -- 100 14 460 129 290 5 41+ 1

Hydroxylamine Trapping of G261A Variant

The proposed mechanism for formyl-CoA dependent hydroxylamine inactivation requires

that the nucleophilic hydroxylamine attacks the carbonyl of Asp-169 in the putative formyl-

aspartyl anhydride. Chemically, it makes better sense for the attack to occur at the formyl group.

An explanation is that the enzyme active site protects the formyl carbonyl from attack;

interference with tetraglycine loop may allow addition to the less hindered formyl group. If this

is the case, a reduction in effective inactivation is expected. Thus, the G261A variant was tested

Table 2-6. Formyl-CoA dependent inactivation of G261A (0.27 [iM) by hydroxylamine
[Formyl-CoA] G261A
(lM) Residual Activity (%)
0 100 8
77 30 10
260 30 14









fixed, varied formyl-CoA concentrations and various concentrations of the appropriate acid

acceptor. Linear fits to the replots of (KM/Vmax)app and (1/Vmax)app against [formyl-CoA] were then

used to estimate KM and Kia (216). All curve fitting was performed with KaleidaGraph 3.5

(Synergy Software, Reading, PA).

Determination of the Specific Activity of FRC and YfdW with Alternate Substrates

The specific activities of FRC with alternate substrates were determined by incubating 8.8

nM enzyme (83 ng FRC in 200 pL) in 60 mM potassium phosphate, pH 6.7, 125 mM in the CoA

acceptor, and 80 i[M in the CoA donor at 300 C. In the case of YfdW, 11.2 nM (108 ng YfdW in

200 IL) was used with 75 mM acceptor and 350 |[M CoA donor. Specific activities for YfdW

with succinate or oxalate were determined from initial velocity experiments. Substrates were

regarded as having no activity when no products were detected in reactions that were run for 60

min.

Crystallization and Structure Determination of the W48F and W48Q FRC Variants

Crystallization and analysis of FRC variants was carried out by Dr. Catrine L. Berthold at

the Karolinska Institutet, Stockholm, Sweden. Crystallization of the FRC mutants was

performed by the vapor diffusion method in 24-well plates where hanging drops of 2 ptL protein

solution and 2 ptL well solution were set up to equilibrate against 1 mL well solution at 293 K. A

protein solution containing 7.5 mg/mL of the desired mutant in 50 mM MES buffer, pH 6.2 with

additional 10% glycerol was used when screening for optimal conditions for crystallization. The

W48Q variant of FRC was crystallized against a well solution of 1.35 M sodium citrate and 0.1

M HEPES buffer, pH 7.2-7.4, resulting in crystals of the tetragonal space group 14. These

crystals were protected in a cryosolution of three parts well solution mixed with one part 100%

ethylene glycol before being flash-frozen in liquid nitrogen. For the W48F FRC mutant a well









26. Boynton, Z. L., G. N. Bennett, and F. B. Rudolph. 1994. Intracellular Concentrations
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28. Britz, M. L., and R. G. Wilkinson. 1982. Leucine dissimilation to isovaleric and
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29. Brunger, A. T., P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, R. W. Grosse-
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reaction of citramalate lyase. Eur J Biochem 64:263-7.

31. Buckel, W. 1992. Unusual Dehydrations in Anaerobic-Bacteria. Fems Microbiology
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32. Buckel, W., and H. A. Barker. 1974. 2 Pathways of Glutamate Fermentation by
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33. Buckel, W., and A. Bobi. 1976. The enzyme complex citramalate lyase from
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34. Buckel, W., V. Buschmeier, and H. Eggerer. 1971. [The action mechanism of citrate
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35. Buckel, W., U. Dorn, and R. Semmler. 1981. Glutaconate CoA-transferase from
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36. Burns, K L., L. T. Gelbaum, M. C. Sullards, D. E. Bostwick, and S. W. May. 2005.
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37. Caldwell, J., A. J. Hutt, and S. Fournelgigleux. 1988. The Metabolic Chiral Inversion
and Dispositional Enantioselectivity of the 2-Arylpropionic Acids and Their Biological
Consequences. Biochemical Pharmacology 37:105-114.

38. Campieri, C., M. Campieri, V. Bertuzzi, E. Swennen, D. Matteuzzi, S. Stefoni, F.
Pirovano, C. Centi, S. Ulisse, G. Famularo, and C. De Simone. 2001. Reduction of
oxaluria after an oral course of lactic acid bacteria at high concentration. Kidney Int
60:1097-105.









exist as a ap heterohexamer (44 and 46 kDa subunits). Similar enzymes in Pseudomonas sp.

and Micrococcus sp. activate itaconate to itaconyl-CoA which is converted to acetyl-CoA and

pyruvate in a series of steps (49, 50).

Acetate CoA-transferase (AA-CoA transferase, atoDA; EC 2.8.3.8)

Acyl-CoA:acetate CoA-transferase activity was detected as early as 1968 in E. coli (250)

and is associated with short fatty acid, e.g. butanoate and propanoate, metabolism. The

transferase and a CoA lyase, both members of the ato operon, are upregulated when E. coli is

grown on acetoacetate (184). Two subunits of molecular mass 26 kDa (atoD) and 24-26 kDa

(atoA) compose the a434 hetero-octameric protein (231). In an experiment similar to that of

Solomon and Jencks (229), the catalytic glutamate residue on the 0 -subunit was detected by

NaBH4 treatment after incubation with acyl-CoA (231). The apparent KM for acetyl-CoA with

acetoacetate and KM for acetoacetyl-CoA with acetate were 0.26 mM and 35 riM, respectively.

Butyrate-acetoacetate CoA-transferase (EC 2.8.3.9)

Butryate-acetoacetate CoA transferase was first purified from the acetoacetate degradation

operon (ato) in constitutive E. coli (232), then later from lysine-fermenting Clostridium SB4 (12)

and solventogenic (acetone) Clostridium acetobutylicum ATCC 824 (261). The enzyme is

important in detoxification of the medium from acetate and butyrate fermentation products.

Enzymes from all three organisms were reported to comprise a434 hetero-octameric quaternary

structure with subunits of about 23 and 25 26 kDa each.

Acetyl-CoA: glutaconate CoA-transferase (GCT; EC 2.8.3.12)

Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F.

fusiformis metabolize glutamate through the hydroxyglutarate pathway involving many

transformations carried out at the CoA-ester level (31, 32). Like propionate CoA transferase,

glutaconate CoA transferase does not have the (S)ENG motif. Recombinant proteins from









where CoA and is transferred from a donor acid (A) to an acceptor acid (B). Mechanisms of this

type can be divided into those which proceed through a ternary complex, ordered or random, and

those which pass through a substituted mechanism, i.e. ping-pong mechanism.

Family I CoA Transferases

Family I CoA transferases, specifically in EC 2.8.3.5-6 and 8-9, share two signature

motifs: signature 1 (PS01273) is found in the N-terminal region of the a-subunit and may be

involved in CoA binding. The second consensus sequence motif (PS01274) (S)ENG, where E is

the active-site glutamate, is found in the N-terminal region of the P-subunit (162, 183). Family I

CoA transferases, i.e. glutaconate-CoA transferase (35), involve an enzyme-CoA thioester

intermediate and follow a substituted mechanism which can be identified by the set of initial

velocity plots where i/velocity vs. the reciprocal of one substrate yields a pattern of parallel lines

(Figure 1-3). Evidence for the formation of the two anhydride intermediates, in addition to the

enzyme-CoA intermediate, has been obtained from model reactions with citramalate lyase (30).


20-

16- X

S12" "12

< 8 1 8


4 l yAcetyl-CoAY'(mM-1)
10 20

0 2 4 6 8 10
LGlutaconatel- (mM-')

Figure. 1-3. Ping-pong kinetics of Family I glutaconate-CoA transferase (GCT) from
Acidaminococcusfermentan. Taken from Buckel 1981 (35).









170. Minkler, P. E., V. E. Anderson, N. C. Maiti, J. Kerner, and C. L. Hoppel. 2004.
Isolation and identification of two isomeric forms of malonyl-coenzyme A in commercial
malonyl-coenzyme A. Anal Biochem 328:203-9.

171. Mishra, P. K., and D. G. Drueckhammer. 2000. Coenzyme A Analogues and
Derivatives: Synthesis and Applications as Mechanistic Probes of Coenzyme A Ester-
Utilizing Enzymes. Chem Rev 100:3283-3310.

172. Mitchell, W. M. 1968. Hydrolysis at arginylproline in polypeptides by
clostridiopeptidase B. Science 162:374-5.

173. Mosbacher, T. G., A. Bechthold, and G. E. Schulz. 2005. Structure and function of the
antibiotic resistance-mediating methyltransferase AviRb from Streptomyces
viridochromogenes. J Mol Biol 345:535-45.

174. Murshudov, G. N., A. A. Vagin, and E. J. Dodson. 1997. Refinement of
macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol
Crystallogr 53:240-55.

175. Murshudov, G. N., A. A. Vagin, and E. J. Dodson. 1997. Refinement of
macromolecular structures by the maximum-likelihood method. Acta Crystallographica
Section D-Biological Crystallography 53:240-255.

176. Nachmansohn, D., and M. Berman. 1946. Studies on Choline Acetylase .3. On the
Preparation of the Coenzyme and Its Effect on the Enzyme. Journal of Biological
Chemistry 165:551-563.

177. Nachmansohn, D., and A. L. Machado. 1943. The formation of acetylcholine. A new
enzyme 'choline acetylase'. J. Neurophysiol 6:397-403.

178. Nakai, T., N. Nakagawa, N. Maoka, R. Masui, S. Kuramitsu, and N. Kamiya. 2004.
Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-
oxo acid dehydrogenase (El) from Thermus thermophilus HB8, as revealed by X-ray
crystallography. J Mol Biol 337:1011-33.

179. Nureki, O., K. Watanabe, S. Fukai, R. Ishii, Y. Endo, H. Hori, and S. Yokoyama.
2004. Deep knot structure for construction of active site and cofactor binding site of
tRNA modification enzyme. Structure 12:593-602.

180. O'Brien, P. J., and D. Herschlag. 1999. Catalytic promiscuity and the evolution of new
enzymatic activities. Chemistry & Biology 6:R91-R105.

181. Pace, C. N., F. Vajdos, L. Fee, G. Grimsley, and T. Gray. 1995. How to measure and
predict the molar absorption coefficient of a protein. Protein Sci 4:2411-23.

182. Pao, S. S., I. T. Paulsen, and M. H. Saier, Jr. 1998. Major facilitator superfamily.
Microbiol Mol Biol Rev 62:1-34.









binding domain ofDeinococcus radiodurnas phytochrome (254); and figure-of-eight knots in the

crystal structure of the plant protein acetohydroxy acid isomeroreductase (243).

FRC is similar to the disulfide-linked examples above with the exception that its two

monomers are non-covalently linked through each other. A preliminary melting curve monitored

by CD and fluorescence shows that the dimer denatures at 57 C (Figure 5-3) in a process that

appears to be irreversible (data not shown). Proteolytic MS data (Figure 2-14) suggest that the

central dimer interface which includes a-helix-9 from both monomers is exceptionally resistant

to enzymatic digestion. An FRC heterodimer, comprising an active monomer and an inactive

monomer where Asp-169 has been replaced with alanine, was successfully overexpressed in E.

coli. The mechanism by which E. coli and 0. formigenes (and all other organisms that employ

this interlocked dimer scaffold) form the active dimer in vivo is an interesting problem and

deserves careful study.

Monitoring of the fluorescence of native tryptophan residues is a method that can be used

to examine FRC folding(204). Trp-109 is found on a-helix-6 in the large domain of FRC. It is

located 10 A and 24 A from surface residues Ser-389 and Glu-394, respectively, both found on

the linker region near the C-terminus of FRC. These residues are candidates for mutation to

cysteine residues for labelling with IAEDANS; they should provide excellent reporters on

unfolding FRC. Another good experiment will use ANS, or 8-anilino-l-naphthalenesulfonic

acid (see Figure 5-4), an environment-sensitive fluorophore which is virtually nonfluorescent in

water, but can be used as a probe for hydrophobicity-in nonpolar environments it emits a blue

fluorescence with quantum yield -0.70 (238). Thus, as FRC is treated with increasing

concentrations of chaotropic agents, increased binding of ANS with exposed hydrophobic










Table 5-1:


Cysteine residues in wild type FRC. The MS column indicates whether the cysteine
diseriue-containing nentides have been ietfdinM exrrm ts~


protein, proteolyse, and analyse by mass spectrometric methods. Table 5-1 also summarizes the

total sequence coverage of mass spectrometric analysis of digests of FRC to date.

Tryptophan has been used as a FRET donor in conjunction with ANS or IAEDANS

acceptors (Ro = 22 A)(219). IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-

sulfonic acid, covalently attached to engineered cysteine residues has been used for FRET

studies (93). FRC has 8 native tryptophan residues: Trp-48, 109, 156, 265, 272, 289, 301, and



OH
0 =S=
11 H 0 NH O=S=O


Figure. 5-4. IAEDANS and ANS.


Conservation Location Interacts MS Environment Mutant
Cys-22 Conserved in Helix a6 Helix a9 V8DE Large domain; buried, A
"true FRCs": hydrophobic
A,G, T
Cys65 R, S(YfdW), H Loop possible no Large domain; S
or C (P5- a7) H-bond partially buried
with His379
(0311)
Cys145 C, A, S, or G Helix a7 Sheet 07 no Large domain; buried, A
hydrophobic
C) Highly Sheet 07 Near Helix no Small domain; near A
conserved: F or a16 surface, but buried,
Y hydrophobic
Cys293 Conserved in Helix Helix a13 V8DE Small domain, buried, A
"true FRCs": a12 hydrophobic
A, S, R, M, L,
or V
Cys347 Conserved: A, Sheet 09 Helix a16 Trypsin Small domain, buried, A
V, or Y ____hydrophobic










L acidophilus
MCR M tuberculosis
BbsF T aromatica
s Polaromonas1251
ao B bronchiseptica RB50
100 B bronchiseptica 1308
3 B parapertussis1188

T volcanium
N crassa

a B parapertussisl230
100 M magnetotacticuml 221
C tetani
R coelicolor

100 M magnetotacticum1194
io R eutropha1194

10 R metallidurans

O formigenes
B japonicum
10 R palustris
67 1o B fungorum

97 Polaromonasl248
7 S flexneri
53
1 I E coli K12
65
E coli o157H7
9 Polaromonas1278

n100 1M magnetotacticuml251
-- Reutrophal251
CaiB E coli
1o BaiF C scindens
HadA C difficile
1io FIdA C sporogenes

BbsE T aromatica
0.1 changes

Figure.C-2. Phylogram of Family III CoA transferases from pair-wise sequence alignment of
amino acid sequences.









aq. HAc (10 [L). The amount of the appropriate thioester product was then quantitated by

injection of these samples onto a C18 analytical column (Dynamax Microsorb 60-8 C18, 250 x

4.6 mm reverse-phase analytical column equilibrated with 86% Buffer A (25 mM NaOAc, pH

4.5) and 14% Buffer B (Buffer A containing 20% CH3CN)) at a flow rate of 1 mL/min.

Immediately after injection the proportion of Buffer B was increased to 6% over 210 s, then to

100% for 90 s before the wash using 98% Buffer A and 2% Buffer B. CoA-containing species

were observed by monitoring absorbance at 260 nm, and their concentrations were determined

by integrating the peak areas and comparison with those for known amounts of authentic

material. These measurements were calibrated using independent determinations of formyl-CoA

concentration using (i) a hydroxylamine-based colorimetric assay (227) and (ii) the oxalate

concentration in hydrolyzed and nonhydrolyzed samples of oxalyl-CoA as measured with a

standard detection kit (Sigma). No formation of CoA ester products was observed in control

experiments when the enzyme or either substrate was omitted from the mixture.

In kinetic assays of FRC, His-tagged FRC and the two FRC variant enzymes, a similar

HPLC-based procedure was followed except that assays contained either FRC (41 ng), His-

tagged FRC (46 ng), W48F (41 ng) or W48Q (43 ng). Reactions were quenched in 10% aq. HAc

(10 pL) and aliquots were eluted initially with 96% Buffer A (25 mM NaOAc, pH 4.5) and 4%

Buffer B (Buffer A containing 40% CH3CN) at a flow rate of 1 mL/min. The amount of buffer B

was then increased to 11% over 210 s and then to 100% Buffer B for 90 s, after which it was

returned to 4%.

Determination of Steady-State Kinetic Constants

Kinetic constants were obtained by curve-fitting to the following equations for sequential

bi-bi kinetics (Eqn. 1), substrate inhibition (Eqn. 2), competitive inhibition (Eqn. 3) and mixed-

type inhibition (Eqn. 4) (47)):









Pro-159 corresponds to Arg-146 in MCR and both residues are found in a small, solvent-

accessible loop connected to a-helix-9. Clostripain, a cysteine protease from Clostridium

hystolytica, was chosen as an alternative to trypsin as it cleaves Arg-Pro peptide bonds (172).

The peptide was expected to retain a positive charge upon ionization and should be a good

candidate for MALDI-TOF MS. However, the protein was not overexpressed under the normal

expression and purification conditions.

Half-Sites versus Independent Active Sites Reactivity

Half-sites reactivity is an extreme limit of negative cooperativity (see Seydoux for a review

(220))-ligand-binding induces structural changes and alterations in subunit interactions lower

enzyme affinity for the substrate in a second, otherwise equivalent, active site. Half-sites

reactivity is common in nature, examples include glyceraldehye-3-phosphate dehydrogenase

(48), thymidylate synthase (121), and the pyruvate dehydrogenase complex, where a "proton

wire" is proposed to mediate half-sites reactivity (86). Family I CoA transferases exhibit this

form of cooperativity, e.g. SCOT (156) and acetyl-CoA transferase from E. coli (233), mass

spectrometric analysis of NaBH4-treated protein, and use of the 1,N6-etheno-CoA, a fluorescent

CoA analogue, demonstrated that in both cases, only one monomer was active at a time. Based

on the asymmetry of the FRC structure with CoA bound, it has been suggested that the dimer

might exhibit half-sites reactivity as well (124). A construct in the Novagen pET-Duet vector

containing two cloning sites was therefore prepared allowing co-expression of FRC with the

D169S inactive mutant. Constructs with a poly-histidine tag on FRC or the D169S mutant

allowed the purification of heterodimers and histidine-tagged homodimers. If dimer formation is

statistical, a 1:2:1 ratio of histidine-homodimer: histidine-heterodimer: heterodimer can be

expected. Table 2-7 shows the predicted specific activities for both independent and half-sites









Table 3-6. Data collection and refinement statistics for the W48F and W48Q FRC mutants.

Data Collection W48Q FRC W48F FRC
Beamline ID14ehl (ESRF) ID23eh2 (ESRF)
Space Group C2 14
Unit cell (A) 214.2, 98.9, 152.5 152.7, 152.7, 99.45
(0) 90, 135.3, 90 90, 90, 90
Molecules in asymmetric unit 4 2
Resolution (A) 1.8 (1.9-1.8)a 1.95 (2.06-1.95)
Rsym (%) 6.3 (40.8) 13.0(53.2)
Mean (I/o(I)) 12.4 (2.3) 8.8 (2.7)
Completeness (%) 92.0 (68.7) (97.9) (99.5)
Wilson B-factor 22.6 17.8
Refinement W48Q FRC W48F FRC
Resolution range 30-1.8 30-1.95
R factor/Rfree (%) 18.2/21.1 16.7/20.0
Atoms modeled 14725 7608
Number of residues 1708 854
Number of waters 1309 936
Mean B-factor model (A2) 26.1 20.1
RMS deviation, bonds (A) 0.008 0.009
RMS deviation, angles (0) 1.08 1.11
Ramachandran zone distribution (%) 92.0 / 7.5 / 0.5 / 0 91.7 / 7.9 / 0.1 / 0.3
PDB deposition ID 2vjq 2vjp
Values given in parentheses represent those of the highest resolution shell.

proximal methionine residue (Met-44) (Figure 3-18). For the W48Q FRC variant to bind oxalate

in the site with the tetraglycine loop in a "closed" conformation, Gln-48 and Met-44 would both

have to change rotamer conformation. In YfdW, the methionine position is occupied by a smaller

valine residue.

Formyl-CoA Hydrolysis in the Presence and Absence of FRC, D169S, and YfdW

In contrast to FRC and the D169A variant of FRC, YfdW does not appear to mediate the

hydrolysis of formyl-CoA when pseudo-first order rate constants for the hydrolysis of formyl-

CoA were determined. The half-lives for formyl-CoA hydrolysis in the presence of FRC,

D169A, and YfdW were 51, 58, and 198 minutes, respectively. Formyl-CoA hydrolysis has

been reported with a half-life of 150 minutes at pH 6.7 and 300C (124).









277 K into 1 mM HC1 using a prepacked NAP-5 column (Amersham Biosciences). The protein

elution of 1 ml was mixed with an equal volume of 98% acetonitrile and 2% formic acid. Data

were immediately acquired in positive mode on a QTOF ULTIMA API instrument (Waters

Corp., Milford, MA) equipped with the standard Z-spray source with a capillary voltage of 1.5

kV. The instrument was calibrated between 300 and 1400 m/z with myoglobin prior to the run.

The sample was introduced with a metal-coated borosilicate glass capillary needle (Proxeon

Biosystems A/S, Odense, Denmark). Data were collected over a mass range between 300 and

2500 m/z and with a scan time of 1 s for about 5 min. The spectra were combined and

deconvoluted to zero charged ions with MaxEnt 1 in the Masslynx software (Waters Corp.,

Milford, MA).

Half-Sites (pET-Duet) Constructs

Wild-type FRC and D169S mutant sequences were cloned from pET-9a constructs (197).

Forward primers included a clamp region and restriction site terminating in the start codon for

the gene. The reverse primers comprised a clamp region, restriction site, and an in-frame stop

codon: 5'-FRC BamH] 5'-AGG AGA TAT AGG ATC CGA TGA CTA AAC CAT TAG ATG

GAA TTA ATG TGC, 3'-FRC HindlII (stop) 5'- ACA GGT AGT TTG AAG CTT AGA CTT,

5'-Ndel 5'-AGG AGA TAT ACA TAT GAC TAA ACC ATT AGA TGG AAT TAA TGT GC

3'-D169S A7ol(stop) 5'-ACA GGT AGT TTG ACT CGA GAG ACT T. The amplified products

were subjected to restriction enzyme digest with BamH] and Hindlll for the D169S PCR product

and Ndel and Xhol restriction enzymes for wt-FRC. The resulting D169S and FRC fragments

were isolated and ligated one at a time into the pETDuet-1 multiple cloning sites 1 (with N-

terminal His6 tag) and 2 (no fusion tag) to generate the DuetHisDW and DuetHisWD constructs.

JM109 competent cells (Stratagene) were transformed with the resulting plasmids, screened for

correct insert size, and confirmed by DNA sequencing.









143. Laker, M. F., A. F. Hofmann, and B. J. Meeuse. 1980. Spectrophotometric
determination of urinary oxalate with oxalate oxidase prepared from moss. Clin Chem
26:827-30.

144. Laskowski, R. A., M. W. Macarthur, D. S. Moss, and J. M. Thornton. 1993.
Procheck a Program to Check the Stereochemical Quality of Protein Structures. Journal
of Applied Crystallography 26:283-291.

145. Leeper, F. J., D. Hawksley, S. Mann, C. P. Melero, and M. D. H. Wood. 2005.
Studies on thiamine diphosphate-dependent enzymes. Biochemical Society Transactions
33:772-775.

146. Leslie, A. G. W. 1992. Jnt CCP4/ESF-EACBM Newsl. Protein Crystallogr. 26.

147. Leslie, A. G. W. 1997. MOSFLM, version 5.40. MRC Laboratory of Molecular Biology,
Cambridge, United Kingdom.

148. Leuthner, B., and J. Heider. 2000. Anaerobic toluene catabolism of Thauera
aromatica: the bbs operon codes for enzymes of 0 oxidation of the intermediate
benzylsuccinate. J Bacteriol 182:272-7.

149. Leutwein, C., and J. Heider. 2001. Succinyl-CoA:(R)-benzylsuccinate CoA-transferase:
an enzyme of the anaerobic toluene catabolic pathway in denitrifying bacteria. J Bacteriol
183:4288-95.

150. Liang, C. Z., and K. Mislow. 1995. Topological Features of Protein Structures Knots
and Links. Journal of the American Chemical Society 117:4201-4213.

151. Lindqvist, Y., G. Schneider, U. Ermler, and M. Sundstrom. 1992. Three-dimensional
structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution.
Embo J 11:2373-9.

152. Lipmann, F. 1945. Acetylation of Sulfanilamide by Liver Homogenates and Extracts.
Journal of Biological Chemistry 160:173-190.

153. Lipmann, F., N. O. Kaplan, G. D. Novelli, and L. C. Tuttle. 1950. Isolation of
Coenzyme-A. Journal of Biological Chemistry 186:235-243.

154. Lipmann, F., N. O. Kaplan, G. D. Novelli, L. C. Tuttle, and B. M. Guirard. 1947.
Coenzyme for Acetylation, a Pantothenic Acid Derivative. Journal of Biological
Chemistry 167:869-870.

155. Lipmann, F., N. O. Kaplan, and G. K. Novelli. 1947. Chemistry and Distribution of the
Coenzyme for Acetylation (Coenzyme-a). Federation Proceedings 6:272-272.

156. Lloyd, A. J., and P. M. Shoolingin-Jordan. 2001. Dimeric pig heart succinate-
coenzyme A transferase uses only one subunit to support catalysis. Biochemistry
40:2455-67.










intermediate structure, the Ca-OH and 4'-NH of ThDP form a close contact, with a hydrogen

bond distance of 2.55 A. The Ca-OH also makes a hydrogen bond to Tyr-120 (Figure 4-3B). At

the approximate positions of the substrate carboxyl oxygen atoms in the oxalyl-CoA complex,

two water molecules (W2 and W3 in Figure 4-3B) are bound. W3 also interacts with Ser-553 and

Tyr-483. W2 makes a hydrogen bond to the main chain nitrogen of Ile-34 and is ideally

positioned (2.7 A) to have donated a proton to the Ca atom, and further, proton transfer to the a-

carbanion is likely to have occurred. Thus, the structure represents the covalently bound product

(vida infra).

Structure of the Formyl-CoA Complex

The product complex [6] was formed by soaking crystals of ThDP-bound OXC in a

solution of formyl-CoA (Figures 4-3C and 4-4C). No structural changes in the protein

framework are observed in OXC completed with formyl-CoA when compared with the

9-
8- *

7

6 -

E 5-

3* *
4-

0

1 0 2 4 6
time, min
0
0 100 200 300 400 500
[Oxalyl-CoA], pM

Figure. 4-6. Initial velocity plot of S553A OXC variant. Oxalyl-CoA concentration was
varied form 10 500 iM and enzyme concentration was 9 nM. Inset, progress curve
for the formation of formyl-CoA over time.









where nTr is the number of tryptophans, nTyr is the number of tyrsosine residues, and ncystine is

the number of disulfide bonds in the protein in question. This method yields values with

standard percent deviation of 3.836 when compared to a literature set of concentrations

determined by dry weight method, amino acid analysis, Kjeldahl nitrogen determination, or the

Edelhoch method for 80 proteins.

While the general equation above does a good job of predicting 280, the Gill and von

Hippel method is slightly more accurate, accounting for the slight change in absorption of buried

amino acid residues, and involves measuring A280 in denaturing 6 M guanidinium hydrochloride.

This e2(6MGuHC1) can then be used to calculate the e (buffer), which can then be used to easily

and non-destructively determine concentration of samples in storage buffer. The following

equation uses values reported by Pace:

s280 (M-1 cm-) = (nTr)(5,685) + (nTyr)(1,285) + (ncystine)(125)

Aliquots of equal volumes of FRC in storage buffer were lyphophilized and subsequently

resuspended in either 100 [tL of FRC storage buffer (100 mM potassium phosphate, pH 6.5 with

300 mM NaC1) or storage buffer with 6 M GuHC1. The absorbances at 280 and 333 nm were

collected. The 280(6MGuHC) was corrected for the effects of light scattering by subtracting 1.929

x A333 and used to calculate the concentration in 6 M GuHC1. This concentration was used to

calculate the (280(buffer) 58, 202 M1 cm1.

Assay for Coenyzme A Esters

Concentrations of formyl-, oxalyl-, and succinyl-CoA were determined with the single-

point HPLC assay developed by J6nsson (123, 124). CoA ester separation was achieved by C18

reversed-phase HPLC (Dynamax Microsorb 60-8 C18, 250 x 4.6 mm or Varian Pursuit XRs C18

150 x 4.6 mm) with a singlewavelength detector at 260 nm. Varian Galaxie Chromatography

Data System software version 1.9.3.2 was used for data analysis.









subunits of citrate lyase and citramalate lyase are so similar that active hybrid enzyme complexes

have been formed from the a- and P-subunits of citramalate lyase and the ACP (-subunit from

citrate lyase (62).

Family III CoA Transferases

Previous work, including site-directed mutagenesis, steady-state kinetics, and product

inhibition studies on recombinant FRC from Oxalobacterformigenes, the representative enzyme

of Family III CoA transferases, have demonstrated that Asp-169 is a vital catalytic residue and

that the reaction proceeds through a ternary complex where formyl-CoA binds first followed by

oxalate followed by release of format prior to oxalyl-CoA in an ordered sequential mechanism

(124). Thus, unlike the Family I ping-pong kinetic mechanism, format must remain in the

active site until chemistry is complete. However, this does not exclude the possible formation of

an enzyme-CoA thioester intermediate like that of glutaconate-CoA transferase during catalysis

(35, 217). Indeed, an X-ray crystal structure in the study showed electron density consistent with

an oxalyl-aspartyl mixed anhydride in the active site, evidence that just such a covalent enzyme-

substrate intermediate may exist along the reaction pathway. Theoretical reaction mechanisms

are shown in Figure 1-6.; Mechanism 1 in Figure 1-6 is a viable proposal conditional that

format remains in the active site. Two other likely mechanistic pathways exist: Mechanism 2,

the direct attack of the formyl-CoA carbonyl by oxalate, in a mechanism analogous to that of

Family II transferases, followed by the CoA thiolate attacking oxalate; and, Mechanism 3

proposed by J6nsson et al. (124) where nucleophilic attack of the formyl carbonyl by Asp-169 is

followed by attack of oxalate on the resultant aspartyl-formyl mixed anhydride to form an

aspartyl-oxalyl mixed anhydride intermediate which is finally attacked by CoA, which has

remained in the active site generating the final product.









Results


Kinetic Characterization of YfdW

The initial experiments examined whether Escherichia coli YfdW could catalyze the synthesis of

oxalyl-CoA from formyl-CoA and oxalate, as inferred on the basis of structural genomics (92,

97). Incubating formyl-CoA and oxalate with YfdW in phosphate buffer, pH 6.7, did indeed

result in the appearance of oxalyl-CoA, and the amount of this product could be quantified by

direct HPLC measurement (Figure 2-17). YfdW differed from FRC in that stronger quenching

conditions were required. As seen in Figure 3-4, when attempts were made to stop the YfdW

catalyzed reaction with 10% HAc, YfdW continued to form oxalyl-CoA even when incubated on

ice. Increasing the concentration of HAc to 20%, however, clearly abolished YfdW activity, but

did not increase oxalyl-CoA hydrolysis. In addition, more stringent wash conditions for

syringes, the HPLC injection port, and column were necessary to prevent carryover activity.


6.0

5.0

E 4.0

0
3.0


O
x 2.0 0 0

1.0

0.0 -
0 20 40 60 80 100
t, min

Figure. 3-4. Quench conditions for YfdW. Reactions were quenched and mixtures were
inbubated with either FRC or YfdW. Oxalyl-CoA concentration was monitored over
time. YfdW with 10% HAc incubated at 0 oC(e), YfdW with 20% HAc at 32 C (o),
and FRC with 10% HAc at 32 C (m).









substrate specificity, and (ii) the ability of oxalate to exhibit substrate inhibition only in the case

of YfdW. Structural studies on FRC had, however, revealed the importance of a tetraglycine

segment in stabilizing a putative reaction intermediate (124), and conformational changes in this

FRC and the Trp-48 FRC mutants. loop appeared correlated with the orientation of the Trp-48

side chain in FRC (197). Super-imposition of the crystal structures for the two CoA transferases

Table 3-3. Steady-state parameters for the formyl-CoA/oxalate transferase activities of YfdW,
Enzyme Formyl-CoA Oxalate
kcat (s-1) KM(app) (gM) kcat/KM(app) (mM-'s-') KM(app) (mM) kcat/KM(app) (mM's-'1)
His-YfdW 130 17 352 4 370 0.51 + 0.03 255
WT FRC 5.3 0.1 2.0 + 0.3 2650 3.9 0.3 1.36
His-FRC 5.5 + 0.4 4.7 + 1.6 1200 1.2 + 0.3 4.58
W48F FRC 17.1 0.2 0.7 0.4 24430 1.5 0.3 11.4
W48Q FRC 5.8 + 0.3 2.7 0.9 2148 0.43 0.03 13.5

Table 3-4. Summary of the inhibition constants and patterns for His-YfdW, FRC, His-FRC, and
and variants.
(jiM) FRC His-FRC His-YfdW W48F W48Q
CoASH competitive competitive mixed-type mixed-type competitive
K,, 16.7 0.7 9 7 218 21 11 5 55 19
K,, -- -- 213 16 35 6 290 5

Table 3-5. Steady-state parameters for the formyl-CoA/succinate transferase activities of YfdW,
FRC and the Trp-48 FRC mutants.
Enzyme Formyl-CoA Succinate
kcat KM(app) kcat/KM(app) KM(app) kcat/KM(app)
(S-1) (gM) (mm-ls-1) (mM) (mm-ls-1)
His-YfdW 5.3 + 0.4 180 + 14 29.4 80 40 0.07
WTFRC 149 13 16 2 9312 0.32 0.03 465
W48F FRC 42 6 12 6 3500 0.015 0.005 2800
W48Q FRC 17.9 + 0.5 6.7 + 0.9 2672 0.07 + 0.01 256

showed that this tryptophan residue was replaced by glutamine in YfdW (Figure 3-16).

Moreover, for YfdW, an oxalate molecule was seen to bind to a "closed" conformation of this

tetraglycine loop (corresponding to residues 246GGGGQ250 in YfdW) although the observed

glutamine side chain rotamer was the same as seen for Trp-48 in FRC when the cognate loop

segment was in its "open" conformation (46). Thus, it was investigated whether site-specific

mutagenesis of Trp-48 in FRC might yield variant enzymes exhibiting modified kinetic behavior









Acidaminococcusfermentans have been overexpressed in E. coli and the active protein exists as

a404 hetero-octamer where the two subunits have molecular masses of 35.5 kDa and 29 kDa

respectively (35, 162). Initial velocity studies showed that the enzyme uses a ping-pong kinetic

mechanism (35) and peaks of m/z consistent with the enzyme-CoA intermediate have been

detected by MALDI-TOF mass spectrometry of enzyme incubated with only glutaryl-CoA (217).

Glu-54 of the P-subunit was identified as the catalytic amino acid residue by reducing the

enzyme-CoA thioester with sodium boro[3H]hydride and detecting 2-amino-5-hydroxy[5-

3H]valeric acid in a peptide generated by trypsin proteolysis (35, 162). Site-directed mutagenesis

experiments where Glu-p54 was replaced with alanine or asparagine completely abolished all

activity while conversion to glutamine retained 1% of wild type activity (164). Intriguingly,

when the 3E54Q variant was incubated with substrates for 40 hours at 370 C nearly wild-type

activity was restored. The enzyme has been converted from a CoA transferase to a thioester

hydrolase by replacing Glu-54 with aspartate (163). Oxygen exchange between [802]-acetate,

glutaconate CoA transferase, and glutaryl-CoA was further proof of the Family I mechanism

(217) (see Figure 2-1). In addition, the 3E54D hydrolase variant did not undergo 180 uptake into

the aspartate carboxylate. The first X-ray crystal structure (PDB 1POI) of a CoA transferase was

solved for GCT (119).

Family II CoA Transferases

Family II transferases involve a ternary complex and yield a pattern of intersecting lines in

the double-reciprocal plots when either substrate concentration is varied (see Figure 1-5). The

transferase a-subunit of citrate or citramalate lyase complexes facilitate the direct transfer of

CoA from acetate to citrate or citramalate without any covalent bond forming between enzyme

and substrate (34, 63). The CoA cofactor is covalently bound to the both enzymes by a










Table 3-7. Formyl-CoA hydrolysis in the presence of FRC, YfdW, and D169A variant of FRC.
Conditions ti/, min

Formyl-CoA 153
Formyl-CoA + FRC (328 nM) 51
Formyl-CoA + YfdW (28 nM) 198
Formyl-CoA + D169A (254 nM) 58



4.5

4.0

3.5 *

3.0 -

E 2.5- 0

2.0- 0* --
4
1.5- 2

1.0 -
0 1000 2000
0.5 [ADP], pM

0.0
0 200 400 600 800 1000 1200
[Oxalyl-CoA], pM


Figure. 3-16. Initial velocity plot of His-YfdU activity with varied oxalyl-CoA in the absence of
added ADP (e) and in the presence of 150 [M (o) and 1500 [M ADP (m).

Expression, Purification, and Enzyme Activity of OXC Homologue HisYfdU

The yfdU gene in E. coli is the homologue of oxc in Oxalobacterformigenes and the

theoretical partner of YfdW. In an attempt to understand the physiological role of the

YfdW/YfdU enzyme pair in E. coli, a construct containing the yfdU gene product cloned from

genomic DNA was generated. The protein was expressed, purified by nickel affinity

chromatography, and assayed by HPLC. The His-tagged YfdU fusion has a KM of 180 pM and

kcat of 15 s-1, and a turnover number of 8.6 x 104 M ^s^. The corresponding values for wild-type










Table 2-9. Formyl-CoA HPLC method
Time, min Buffer B, %
0.00 6
0.15
10.00 11
10.10 95
14.00 95
14.10 6
16.00 6


Flow, mL/min
1.0


Wait (close)


12 20 2006 1 54 03 PM11.DATA 800 MIB Channel 4


0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Min
Figure. 2-19. Representative chromatogram for separation of CoA (tR = 9.10 min) and formyl-
CoA (tR = 10.10 min). Note the 2'-phosphoryalated iso-formyl-CoA at around 11.2
min.


Table 2-10. Succinyl-CoA HPLC method
Time, min Buffer B, %
0.00 6
0.50
11.50 14
11.60 95
14.00 95
14.10 6
18.00 6


Flow, mL/min
1.0


Wait (close)









Formyl-CoA transferase (FRC)

Formyl-CoA transferase catalyzes the transfer of CoA from formyl-CoA to oxalate to yield

oxalyl-CoA and format (9, 124). FRC is the best-studied member of the Family III CoA

transferases and will be discussed later in detail (see page 37).

Oxalyl-CoA decarboxylase (OXC)

OXC is a thiamine-dependent non-oxidative decarboxylase. The native enzyme was first

purified from 0. formigenes by Baetz et al; the 260 kDa homotetramer was reported to comprise

four 65 kDa monomers (10). Steady-state kinetics studies demonstrated that specificity constant

for OXC is about 3.8 x 106 M^s-1, KM(oxalyl-CoA) is 23 jIM, and kcat is 88 s-1 (15). Adventitious

ADP was found tightly bound in the 1.8 A X-ray crystal structure in the same report. Analysis

showed that micromolar concentrations of ADP were required for maximal enzymatic activity,

whereas ATP had no effect. Considering the vital role that OXC plays in oxalate metabolism,

this finding strongly suggests that ADP-dependent activation of OXC in 0. formigenes is

physiologically relevant.

Coenzyme A

History of Coenzyme A

Coenzyme A (CoA), is a cofactor essential to all living organisms, involved in over 100

different metabolic reactions, and purported to be utilized by about 4% of all known enzymes.

The discovery of CoA is historically linked to the elucidation of the mechanism by which

saturated fatty acids are utilized in living organisms and, as such, Franz Knoop's famous 1904

use of a labelled phenyl group to show that fatty acids are degraded by successive removal of

two-carbon units (139) was to become the starting point for both understanding 3-oxidation and

identifying CoA. In 1942 Feodor Lynen demonstrated that after respiring yeast had exhausted

their supply of substrates, a lag or induction period was required before the process of oxidizing










1.8 -
0.8
1.6 E 0.7-)


0.3

S0 10 20 30
[CoA], pM


S0.8

0.6

0.4

0.2


-0.50 0.)0 0.50 1.00 1.50
-0.2 -
1/[F-CoA], pM1


Figure. 2-2. Double-reciprocal plot for the inhibition of FRC by free CoA against varied
[formyl-CoA] at constant saturating [oxalate] = 77 mM showing lines fitted to the
data by linear-regression methods. CoA concentrations were 1.5 [iM (e), 11.5 [iM
(o), and 24.3 iM (m). Ki(CoA) of 16 .7 + 0.7 IM was determined from the replot of
KMapp/Vapp VS. [CoA] (ili,'l I).

The resulting intermediates from the formyl-CoA and oxalyl-CoA soaks are thus highly similar

and the dimeric structures superimpose with an r.m.s. deviation of 0.3 A over 851 Ccu atoms. An

intriguing feature is that in both complexes, the two subunits of the dimer adopt different active

site conformations with the pantetheine arm of the CoA molecule bound in different orientations

(Figures 2-4 and 2-6). Several residues show different conformations in the two subunits of the

dimer. Tyr-139 is centrally positioned in the active site and moves with the side chain hydroxyl

group shifted approximately 3 A, allowing the two different orientations of the pantetheine

moiety. Lys-137 is positioned on the same side of the CoA molecule and shows a shift of 4.5 A
































Figure. 2-4. Stereoview of the overlay of the two active site conformations of the P-aspartyl-
CoA thioester complex. The "resting" conformation A, shown in grey with one
bound chloride ion in orange, was observed in subunit A with an open glycine loop.
The other "activated "conformation in cyan shows two bound chloride ions (green)
and subunit B with a closed glycine loop. Note the two rotamer conformations of
Q17. Taken from Berthold 2008 (18).

In both complexes Val-16 is positioned in the disallowed or generously allowed part of the

Ramachandran plot in both monomers which was also observed in the complex of FRC with

bound CoA reported earlier (197). Inspection of the structures reveals that Val-16 has a strained

conformation in order to fit the CoA moiety. In the P-aspartyl-CoA thioester complex, Glu-140

is in the disfavored part of the Ramachandran plot in subunit B, which can be explained by the

structure adopted by the adjacent residue Tyr-139, enforced by the different conformation of the

CoA-moiety in that subunit. The B-factors show a clear difference in the region of the small

domain comprising the two loops 230-247 and 282-347 between the two subunits (Figure 2-3).











0.20

0.18 0.045
0035
0.16 0025
0015
0.14 0.005
0 100 200 300
2 0.12 |Ac-CoA], M

S0.10

. 0.08


0.04

0.02

0.00
0.000


0.005 0.010
1/[F-CoA], pM1


0.015


Figure. 3-9. Double-reciprocal plot for the inhibition of YfdW by acetyl-CoA against varied
[formyl-CoA] at constant saturating [oxalate] = 2.5 mM and constant [CoA] = 51.6
pM. Ac-CoA concentrations were 0 pM (e), 53.81 pM (o), 217.8 PM (m). Kiu, 94 +
2 pM, was determined from the replot of 1/ Vapp vs. [CoA] (in'e I). There was no
effect on KMapp/Vapp within experimental error. Lines were modeled with the
Michaelis-Menten equation modified for uncompetitive inhibition.


12
11
3.0 -
0.
02.5
7 0.4- -
0 D 20 40
. 2.0 [Ac-CoA], pM


1.5


0.5 1.0 1.5
1/[F-CoA], pM"1


2.0 2.5


Figure. 3-10. Double-reciprocal plot for the inhibition of FRC by acetyl-CoA against varied
[formyl-CoA] at constant saturating [oxalate] = 77 mM and constant [CoA] = 1.5 pM
with lines fitted to the data by linear-regression methods. Acetyl-CoA concentrations
were 0 pM (e), 12.1 pM (o), and 36.4 pM (m). Ki(Ac-CoA) of 56 6 pM was
determined from the replot of Kmapp/Vapp vs. [acetyl-CoA] (inei' i).









65. Dimroth, P., R. Loyal, and H. Eggerer. 1977. Characterization of the isolated
transferase subunit of citrate lyase as a CoA-Transferase. Evidence against a covalent
enzyme-substrate intermediate. Eur J Biochem 80:479-88.

66. Doane, L. T., M. Liebman, and D. R. Caldwell. 1989. Microbial Oxalate Degradation -
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68. Dunathan, H. C. 1971. Stereochemical aspects of pyridoxal phosphate catalysis. Adv
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69. Duncan, S. H., A. J. Richardson, P. Kaul, R. P. Holmes, M. J. Allison, and C. S.
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70. Edelhoch, H. 1967. Spectroscopic determination of tryptophan and tyrosine in proteins.
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71. Edwards, M. R., M. Singh, and P. K Tubbs. 1973. A simple purification of
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72. Eisenmesser, E. Z., O. Millet, W. Labeikovsky, D. M. Korzhnev, M. Wolf-Watz, D.
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73. Elsden, S. R., and M. G. Hilton. 1978. Volatile acid production from threonine, valine,
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74. Elssner, T., C. Engemann, K. Baumgart, and H. P. Kleber. 2001. Involvement of
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75. Elssner, T., L. Hennig, H. Frauendorf, D. Haferburg, and H. P. Kleber. 2000.
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76. Emsley, P., and K Cowtan. 2004. Coot: model-building tools for molecular graphics.
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involved in carnitine metabolism of Proteus sp. Arch Microbiol 183:176-89.











Table 1-1. Summary of Family III CoA Transferases. Structural data are summarized for the known Family III CoA transferases.
AA refers to the number of amino acids in the monomer and %FRC is sequence identity with FRC.


Name
BaiF (266)


BbsEF (149)


a-methylacyl-CoA racemase


X-ray
Structure


Quaternary Subunit MW
Structure (kDa)
-- 47.5


%
AA FRC
426 27


44 and 45 410,
409


Description
cholyl-CoA hydrolase; proposed CoA
transferase

succinyl-CoA: (R)-benzylsuccinate CoA
transferase


y-butyrobetainyl-CoA: (R)-camitine CoA
transferase


(E)-cinnamoyl-CoA: (R)-phenyllactate
CoA transferase


formyl-CoA: oxalate CoA transferase


(R)-2-hydroxyisocaproyl-CoA: (E)-2-
isocaprenoate CoA transferase


multiple
ligands
(see text)
apo, Ac-
CoA


YfdW (97, 245) formyl-CoA transferase


homodimer


homodimer


Organism/Pathway
anaerobic bile acid
transformation


23, 28 first step of anaerobic
toluene catabolism in
Thauera aromatica


405 24 anaerobic carnitine
metabolism in E. coli and
Proteus sp.


46 405 25 Stickland-fermentation in
Clostridia (sporigenes and
difficile)


428 100 oxalate metabolism in
Oxalobacterformigenes


25 leucine fermentation in
Clostridum difficile


39 (89) 360 23 bile acid synthesis in
Mycobacterium tuberculosis

48.3 415 60 E. coli


apo, CoA homodimer



homodimer,
heterotrimeric
complex with
FldABC
multiple homodimer
ligands
(see text)
homodimer


CaiB (79)


FldA (137)
(PLCT)


FRC (124)


HadA (137)


MCR (210)
(Amacr)









157. Lohkamp, B., Emsley, P., and Cowtan, K 2005. CCP4 Newsletter 42:Contribution 7.

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Der Chemie-Justus Liebig 574:1-32.

160. Ma, Z., S. Gong, H. Richard, D. L. Tucker, T. Conway, and J. W. Foster. 2003.
GadE (YhiE) activates glutamate decarboxylase-dependent acid resistance in Escherichia
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161. Machius, M., R. M. Wynn, J. L. Chuang, J. Li, R. Kluger, D. Yu, D. R. Tomchick,
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reactivity in human branched-chain alpha-ketoacid dehydrogenase. Structure 14:287-98.

162. Mack, M., K. Bendrat, O. Zelder, E. Eckel, D. Linder, and W. Buckel. 1994.
Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning
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51.

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Acidaminococcusfermentans into an acyl-CoA hydrolase by site-directed mutagenesis.
Febs Letters 405:209-212.

164. Mack, M., and W. Buckel. 1995. Identification of glutamate beta 54 as the covalent-
catalytic residue in the active site of glutaconate CoA-transferase from Acidaminococcus
fermentans. FEBS Lett 357:145-8.

165. Mallam, A. L., and S. E. Jackson. 2005. Folding studies on a knotted protein. J Mol
Biol 346:1409-21.

166. Mann, C. J., and C. R. Matthews. 1993. Structure and stability of an early folding
intermediate ofEscherichia coli trp aporepressor measured by far-UV stopped-flow
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90.

167. Mann, S., C. P. Melero, D. Hawksley, and F. J. Leeper. 2004. Inhibition of thiamin
diphosphate dependent enzymes by 3-deazathiamin diphosphate. Organic &
Biomolecular Chemistry 2:1732-1741.

168. Masuda, N., and G. M. Church. 2002. Escherichia coli gene expression responsive to
levels of the response regulator EvgA. J Bacteriol 184:6225-34.

169. Masuda, N., and G. M. Church. 2003. Regulatory network of acid resistance genes in
Escherichia coli. Mol Microbiol 48:699-712.









210. Savolainen, K., P. Bhaumik, W. Schmitz, T. J. Kotti, E. Conzelmann, R. K.
Wierenga, and J. K Hiltunen. 2005. a-Methylacyl-CoA racemase from Mycobacterium
tuberculosis. Mutational and structural characterization of the active site and the fold. J
Biol Chem 280:12611-20.

211. Schink, B. 1985. Mechanisms and Kinetics of Succinate and Propionate Degradation in
Anoxic Fresh-Water Sediments and Sewage-Sludge. Journal of General Microbiology
131:643-650.

212. Schmitz, W., C. Albers, R. Fingerhut, and E. Conzelmann. 1995. Purification and
characterization of an a-methylacyl-CoA racemase from human liver. Eur J Biochem
231:815-22.

213. Schmitz, W., R. Fingerhut, and E. Conzelmann. 1994. Purification and properties of
an a-methylacyl-CoA racemase from rat liver. Eur J Biochem 222:313-23.

214. Schweiger, G., and W. Buckel. 1984. On the dehydration of (R)-lactate in the
fermentation of alanine to propionate by Clostridium propionicum. FEBS Lett 171:79-84.

215. Seal, S. N., and S. P. Sen. 1970. Photosynthetic Production of Oxalic Acid in Oxalis-
Corniculata. Plant and Cell Physiology 11:119-&.

216. Segel, I. H. 1993. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and
Steady-State Enzyme Systems, New ed. Wiley-Interscience.

217. Selmer, T., and W. Buckel. 1999. Oxygen exchange between acetate and the catalytic
glutamate residue in glutaconate CoA-transferase from Acidaminococcusfermentans -
Implications for the mechanism of CoA-ester hydrolysis. Journal of Biological Chemistry
274:20772-20778.

218. Selmer, T., A. Willanzheimer, and M. Hetzel. 2002. Propionate CoA-transferase from
Clostridium propionicum. Cloning of gene and identification of glutamate 324 at the
active site. Eur J Biochem 269:372-80.

219. Selvin, P. 1995. Fluorescence Resonance Energy Transfer, p. 300-34. In K. Sauer (ed.),
Methods in Enzymology, vol. 246. Academic Press, Inc., San Diego.

220. Seydoux, F., O. P. Malhotra, and S. A. Bernhard. 1974. Half-site reactivity. CRC Crit
Rev Biochem 2:227-57.

221. Sharp, J. A., and M. R. Edwards. 1978. Purification and properties of succinyl-
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65.

222. Shaw, W. V., D. G. Brenner, and I. A. Murray. 1985. Regulation of antibiotic
resistance in bacteria: the chloramphenicol acetyltransferase system. Curr Top Cell Regul
26:455-68.









The (E)-cinnamoyl-CoA:(R)-phenyllactate CoA transferase from Clostridium sporogenes was

not inactivated by hydroxylamine and retained 50% activity when treated with NaBH4 (60).

Activity of succinyl-CoA:(R)-benzylsuccinate CoA transferase from Thauera aromatica was

also unaffected by hydroxylamine but could be reduced to 3.5% in the presence of

benzylsuccinyl-CoA and 10 mM NaBH4 (149). The effect of both of these inhibitors on formyl-

CoA transferase preincubated with formyl-CoA at different concentrations was examined.

FRC incubated with oxalate and formyl-CoA was subsequently treated with

hydroxylamine. As any activated acyl groups are expected to be trapped as hydroxylamine

adducts, oxalyl- and formyl-acyl as well as acyl-thioester intermediates in the transferase

reaction should be trapped. The enzyme showed a reduced activity after removing all small

molecules (i.e. excess CoA and hydroxylamine) by gel filtration. Table 2-2 shows that the

addition of 77 iM formyl-CoA followed by hydroxylamine reduces the activity about 88%. The

same experiment performed without addition of oxalate also displayed a trapping effect by

hydroxylamine (Table 2-3). A clear dependence on formyl-CoA concentration was discovered

for the inactivation of FRC by hydroxylamine.

Trapping experiments with sodium borohydride to reduce possible thioester intermediates

in FRC also led to reduced activity (Table 2-3). The resulting alcohol from the borohydride

reduction has not been identified.

Comparison with Previous FRC Complexes

Previously determined structures of wild type FRC include the apoenzyme structure (pdb

code: lp5h) an inhibitory complex with CoA bound in the active site (pdb code: lp5r) and a

structure where co-crystallization with oxalyl-CoA resulted in a crystal structure where CoA is

bound in both subunits, but where one subunit also contains the aspartyl-oxalyl mixed anhydride

(pdb code: lt4c) Superimposition of these three existing structures with the P-aspartyl-CoA









and 4-4A). Tyr- 483 is in a strained conformation in the disallowed part of the Ramachandran

plot and Ile-34 is followed by the conserved Pro-35, the amide of which adopts a cis

conformation (98). Consistently, in all determined structures of OXC, a water molecule (W1) is

observed, bound between the side chains of Tyr-120 and Glu-121 and the carbonyl oxygen atom

of Ile-34. The substrate Ca-carbonyl oxygen atom makes hydrogen bonds to Tyr-120 and Wl,

bridging to Glu-121 and the cis-Pro-35 loop. The substrate is thus perfectly positioned for attack

by the activated cofactor with a distance of approximately 3 A between the substrate Ca atom

and C2 of the ThDP thiazolium ring. The structure of the CoA complex containing ThDP, solved

to 2.2 A resolution, is virtually identical (rmsd, 0.181 A for 559 Ca atoms) to the substrate

complex; the structured C-terminus also folds over CoA.

Structure of a Trapped Covalent Intermediate

A transiently accumulated covalent intermediate [5], formed after attack of the C2 of the ThDP

thiazolium ring on the substrate Ca atom and after decarboxylation, was trapped in


Figure. 4-4. Annealed composite omit maps calculated for the structures shown around the
active site: in A, the oxalyl-CoA complex [2]; B, the postdecarboxylation
intermediate complex [5]; and C, the product complex [6]. The contour level is lC.
Residue labels can be seen in Figure 4-3. Taken from Berthold 2007 (17).









first identified in a Family I transferase in 1968 by electrophoresis and chromatography studies

with isotope-labeled borohydride (229) and was recently trapped in a crystallographic study,

giving the first structural proof of its existence (196). The smaller group of Family II CoA-

transferases catalyzes a partial reaction in the citrate and citramalate lyase complexes. These

reactions do not include covalent enzyme intermediates, and the transfer of a dephospho-CoA,

which is covalently bound to an acyl carrier protein (ACP) in the enzyme complex, is carried out

through a ternary complex where a mixed anhydride is formed between the two acids during the

transition state (Figure 2-1, Mechanism 3) (33, 63).

During initial studies of the Family III CoA-transferases, steady state kinetics showed that

the reaction is not consistent with a ping-pong mechanism as in the Family I CoA-transferases.

The mechanism was instead interpreted to proceed through a ternary complex, where both

formyl-CoA and oxalate need to be bound to the enzyme before catalysis (60, 124, 149). The

crystal structure of an aspartyl-oxalyl mixed anhydride led to the suggestion that the reaction was

initiated in the ternary complex with both substrates by the formation of an aspartyl-formyl

anhydride and CoA-S-. The CoA-S- was then kept bound in the active site as a spectator while

oxalate replaced format, before attacking the aspartyl-oxalyl anhydride yielding oxalyl-CoA

(124). A freeze-trapped crystal structure reveals that the enzyme-P-aspartyl-CoA thioester

intermediate is also formed during catalysis by formyl-CoA transferase, a finding leading to

reassessment of the catalytic mechanism of Family III CoA-transferases. The mechanistic

investigation is complemented with the crystal structure of a trapped aspartylformyl anhydride

similar to the previously characterized aspartyloxalyl anhydride complex (124) and two mutant

protein structures, where one contains the complex with P-aspartyl-CoA and oxalate. Central to

the catalyzed reaction is a glycine-rich loop that adopts two different conformations controlling









chloramphenicol or kanamycin by acetylation in resistant bacteria (194, 195, 222). In 3-

oxidation of fatty acids, CoA is purported to stabilize the negative charge on the a-carbon for

dehydrogenation and addition of water across the double bond (252).

In simple terms, the pantetheine and ADP moieties can be considered the structural

components responsible for enzyme interaction and aligning the reactive sulfur atom. In a study

by Jencks et al. it was demonstrated that the pantoic acid and ADP domains were critical for

transition state stabilization and contributed significantly to kcat/KM (259). However, other

studies have shown that in some cases the ADP moiety contributes little to binding and catalysis

(55, 83),

Coenzyme A Transferases

Coenzyme A transferases, initially termed thiophorases or transphorases, catalyze the

reversible transfer of coenzyme A from a thioester donor to a free acid. CoA transferases are

found in all Eubacteria and Eukaryota (putative genes are found in Archaea) and play important

roles in amino acid catabolism (12, 21, 32, 39, 218), ketone-body metabolism (22, 236), aromatic

(132) and chloro-aromatic degradation (91), acetone-butanol fermentation (4, 122), and fatty acid

fermentation (140). There are currently 16 enzymes listed as CoA transferases (EC 2.8.3;

IUBMB, 2005); these can be further separated into three CoA Transferase Families based on

function, structure, and sequence similarity (102).

In addition to other properties, the three Families are separated on the basis of kinetic

mechanism. All three share a common group transfer mechanism summarized by the following

equations:

A+B P + Q or ACoA + B t A+ BCoA









phosphodiester linkage from Ser-14 to 2'-(5"-phosphoribosyl)-3'-dephospho-CoA (19, 61, 200,

226) (see Figure 1-4).

Citrate CoA-transferase (EC 2.8.3.10)

Citrate lyase (EC 4.1.3.6) catalyses the reversible cleavage of citrate to yield oxaloacetate

and acetyl-CoA used in fatty acid synthesis. Citrate CoA transferase is the largest subunit (a) of

the three-subunit citrate lyase complex. Most biochemical characterization has been carried out

by the enzyme purified from Klebsiella pneumoniae (formerly Klebsiella aerogenes). Neither

initial velocity studies performed with varied citrate concentrations and varied fixed acetyl-CoA

concentrations nor with varied citryl-CoA and varied fixed acetate showed the parallel line

pattern expected in the double-reciprocal plots of ping-pong kinetics (65).

NH2

0 0 /0\ /o N NH

\ 0







Figure. 1-4. Prosthetic cofactor of citrate lyase 2'-(5"-phosphoribosyl)-3'-dephospho-CoA is
bound to Ser-14 of the ACP subunit by the ribose 5'- phosphate linkage.

Further evidence for no enzyme-CoA intermediate includes: no isolable enzyme-CoA

intermediate upon incubation of citrate CoA transferase with acetyl-CoA; no enzyme inhibition,

nor acetyl-CoA hydrolysis, were detected when an enzyme/acetyl-CoA mixture was treated with

excess borohydride; and, no increase in radiolabel was detected in acetate measured after the

enzyme and [14C]-acetyl-CoA were incubated together (65). Citrate blocks the reacetylation of

the deacetylated lyase with acetic anhydride which has been shown to be an intermediate

substrate analogue for citramalate lyase (30, 65).
substrate analogue for citramalate lyase (30, 65).













- xl 7


FRC MTKPLDGINVLDFTHVQAGPACTQ4MMGFLGANVIKIERR 39
YfdW SYYHHHHHHLESTSLYKKAGLMSTPLQGIKVLDFTGVQSGPSCTQMLAWFGADVIKIERP 60
MCR MAGPLSGLRVVELAGIGPGPHAAMILGDLGADVVRIDRP 39
CaiB MDHLPMPKFGPLAGLRVVFSGIEIAGPFAGQMFAEWGAEVIWIENV 46
** *. *. ** ** *. *:


NM44 Y, 8 Y59 F r
FRC GSGDMTIR QDKPNVDS LYFTMFNCNKRSIELDMKTPEGKELLEQMIKKADVMVENFGP 99
YfdW GVGDVT IPDIDALYFTMLNSNKRSIELNTKTAEGKEVMEKLIREADILVENFHP 120
MCR SSV .... ...... .DGISRDAMLRNRRIVTADLKSDQGLELALKL IAKADVLIEGYRP 86
CaiB ....... ......... YPQLSRRNLHALSLNIFKDEGREAFLKLMETTDIF IEASKG 99
: *: *: :*


r Y 139
FRC GALD RMGFTWEY I QELNPRVILASVKGYAEGHANEHLKVYENVAQCSGGAAATTGFWDGP 159
YfdW GAIDHMGFTWEHIQEINPRLIFGSIKGFDECSPYVNVKAYENVAQAAGGAASTTGFWDGP 180
MCR GVTERLGLGPEECAKVNDRLIYARMTGWGQTGPRSQQAGHDINYISLNGILHAIGRGDER 146
CaiB PAFARRGITDEVLWQHNPKLVIAHLSGFGQYGTEEYTNLPAYNTIAQAFSGYL IQNGDVD 158
:: *


DM 9 M200
FRC PTVSGAAL SNSG. MHLMIGILAALEMRHKTGRGQKVAVAMQDAVLNLVRIKLRDQQR 217
YfdW .PLVSAAAL SNTG.MHLLIGLLAALLHREKTGRGQRVTMSMQDAVLNLCRVKLRDQQR 238
MCR .PVPPLNLV FGGGSMFLLVGILAALWERQSSGKGQVVDAAMVDGSSVLIQMMWAMRAT 205
CaiB QPMPAFPYT FSG. LTATTAALAALHKVRETGKGESIDIAM1YEVMLRMGQYFMMDYFN 217
**** ** *
linker region
--- -------------.
r25 -C261
FRC LERTGILAEYPQAQPNFAFDRDGNPLSFDNITSVPRGPGWMLKCKGWETDAD 277
YfdW LDKLGYLEEYPQY.PNGTFG ........... DAVPRGGN PGWILKCKGWETDPN 286
MCR GMWTDTRGANML.................................YDTYECAD...... 231
CaiB GGEM............ CPRMSKGD.. PY ............ CGCGLYKCAD ...... 243


*


- mono-l


FRC S, YVYF. TIAANMWPQICDMIDKPEWKDDPAYNTFEGRVD .....KLMDIFSFIETKFA 329
YfdW A..YIYF.TIQEQNWENTCKAIGKPEWITDPAYSTAHARQP.....HIFDIFAEIEKYTV 338
MCR .GRYVAVGAIEPQFYAAMLAGLG.....LDAAELPPQNDRA....RWPELRALLTEAFA 280
CaiB ..GYTVMELVGITQIEECFKDIGLAHLLGTPEIPEGTQLIHRIECPYGPLVEEKLDAWLA 301




FRC DKDKFEVTEWAAQYGIPCGPVMSMKELAHDPSLQKVGTVVEVVDEIRGNHLTVGAPFKFS 389
YfdW TIDKHEAVAYLTQFDIPCAPVLSMKE I SLDPSLRQS GSVVEVEQPLRGKYLTVGCPMKFS 398
MCR SHDRDHWGAVFANSDACVTPVLAFGEVHNEPHI IERNTFYEA..... NGGWQPMPAPRFS 335
CaiB THTIAEVKERFAELNIACAKVLTVPELESNPQYVARESITQWQTMDGRTCKGPNIMPKFK 361
*. *




FRC GFQPE.ITRAPLLGEHTDEVLKELGLDDAKIKEL... HAKQVV 428 100%
YfdW AFTPD.IKAAPLLGEHTAAVLQELGYSDDEIAAMKQNHAI 437 61%
MCR RTASSQPRPPAATID.IEAVLTDWDG 360 23%
CaiB NNPGQIWRGMPSHGMDTAAILKNIGYSENDIQE LVSKGLAKVED 405 18%
+ ** *









intermediate, ideally positioned for transfer of a proton to a Ca-carbanion intermediate, and

provides an explanation for the observation that no single mutation of active site residues (except

Glu-56) abolishes catalysis. Replacement of residues Tyr-120 or Glu-121 with alanine has an

effect on kcat due to the importance in firmly positioning the water molecule, Wl. W1 also makes

a hydrogen bond to the Ile-34 carbonyl oxygen with the adjacent Pro-35 in the cis-conformation,

an interaction that is crucial for positioning the Ile-34 NH group (Figure 4-3). The Ile-34 NH

group is involved in binding substrate, and most importantly, the water molecule W2, involved

in transfer to the a-carbanion intermediate. Tyr-483 and Ser-553 also participate in positioning

W2 via W3, and there is also a significant reduction in the turnover number when these residues

are replaced by site-directed mutagenesis. The short distance between water molecules W2 and

W3 (2.5 A) suggests that W2 might have some hydroxide ion character, which would be

consistent with a proton having been transferred to the a-carbanion. Thus it appears that the

covalently bound product is present, indicating that, in the catalytic cycle, product release from

the cofactor is the slowest step of the reaction catalyzed by OXC. There is also a close contact

between Ca-OH and 4'-NH2, 2.55 A, which confirms participation of the 4'-amino group of

ThDP in proton transfers to and from the substrate carbonyl oxygen and stabilization of the

intermediate. In OXC, the enamine/a-carbanion before protonation may be nonplanar and thus

has more a-carbanion character than would a planar enamine. A planar enamine would not have

the proton donor, W2, optimally positioned for proton transfer, and the Ca carbon would be less

basic. The difference from other enzymes in which a planar enamine state has been observed (82,

178, 262) may be explained by the fact that the latter oxidative enzymes require a second

"acceptor" substrate. To allow the intermediate to proceed via the energetically more stable

enamine might then be a way for these enzymes to protect the intermediate from protonation of









OXALATE-DEGRADING ENZYMES OF
Oxalobacterformigenes AND Escherichia coli
















By

CORY G. TOYOTA


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008









thioester intermediate. In addition, flexible loops in both enzymes, the C-terminal peptide loop

of OXC and the tetraglycine loop (25GGGGQ262) of FRC, have been identified as critical to

catalysis and substrate specificity.

Structural genomics studies have shown that YfdW, encoded by the gene yfdW from an

operon that appears to enhance the ability of Escherichia coli MG1655 to survive under acidic

conditions, is structurally homologous to FRC. This work confirms that YfdW is a formyl-CoA

transferase that appears to be more stringent than FRC in employing formyl-CoA and oxalate as

substrates. Replacing Trp-48 in the FRC active site with the glutamine residue that occupies an

equivalent position in the E. coli protein shows that Trp-48 precludes oxalate binding to a site

that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48

yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble

that seen for YfdW. Finally, in addition to demonstrating the value of utilizing structural

homology in assigning protein function, this work suggests that the yfdW and yfdU genes in E.

coli may be be involved in conferring oxalate-dependent acid resistance to the bacterium.









models and the experimental specific activities assayed with saturating formyl-CoA and oxalate.

It appears that the active sites of FRC work independently of each other.

Table 2-7. Predicted and experimental activities of half-sites constructs.
S.A. (U/mg) % wild type Half-sites (%) Independent (%)
WT-FRC 6.5 0.4 100 6
DuetHisWT/D169S 4.0 + 0.4 62 + 6 100 66
DuetHisD169S/WT 1.8 0.2 28 + 2 66 33

Discussion

The reaction catalyzed by both Family I and III of CoA-transferases includes the formation

of aspartyl- (Family III) or glutamyl- (Family I) mixed anhydride intermediates with the

oxyacids, as well as covalent thioester intermediates to the CoA moiety (Figures 2-1 and 2-15).

A distinction between the two families is that the Family I enzymes catalyze a classical ping-

pong reaction while the kinetics of Family III enzymes differ; release of donor oxyacid is not

observed prior to binding of the acceptor oxyacid. This leaves two possibilities, either the

requirement of a ternary complex for catalysis, or the completion of the reaction before any

product can be released. It can be settled from the kinetic trapping experiments and crystal

structures presented above that hydrolysis of both formyl-CoA and oxalyl-CoA as well as

formation of the mixed anhydride can be accomplished in FRC in the absence of acceptor

carboxylic acid. Thus, the reaction does not need the formation of a ternary complex to proceed

and the most probable interpretation of the kinetic data is that the leaving oxyacid remains

bound in the enzyme and is released together with the acceptor thioester. Based on all available

data a new proposal for the FRC reaction mechanism is presented in Figure 2-15. The glycine

loop (258GGGGQ261) plays a central role during catalysis in FRC, and together with Gln-17, it

protects the different intermediates from hydrolysis. The X-ray data suggest that upon binding of

formyl-CoA, the CoA carrier adopts the resting conformation observed in most structures









Propionate CoA transferase (EC 2.8.3.1)

The first reports of catalysis of the reversible transfer of CoA to various fatty acids was in

cell-free extracts from Clostridium kluyveri (234), but propionate CoA transferase from C.

propionicum was the first purified and reported to have a tetrameric quaternary structure

comprising four identical 67 kDa subunits (214). Propionate CoA transferase is an important

enzyme in the nonrandomizing alanine fermentation pathway of C. propionicum: alanine is

converted to ammonia and pyruvate which is reduced to (R)-lactate, activated as (R)-lactoyl-

CoA, and subsequently reduced to propionate. Propionate CoA transferase activates lactate as

the CoA thioester using propionyl-CoA as the CoA donor (214). The enzyme is also implicated

in the methylmalonyl-CoA pathway for the propionate-oxidizing metabolic pathway of

Pelotomaculum thermopropionicum (140, 211). Glu-324 was identified as the active site

carboxylate by MALDI-TOF MS of enzyme incubated with priopionyl-CoA, labelled with either

borohydride (-14 Da) or hydroxylamine (+15 Da), and subsequently proteolytically digested

individually by chymotrypsin, endoprotease-AspN, endoprotease-GluC, or trypsin (218).

Further, propionate CoA transferase lacks the (S)ENG consensus motif shared by Family I CoA

transferases.

Succinyl-CoA:oxalate CoA transferase (EC 2.8.3.2)

Quayle has done extensive study on the facultative autotroph Pseudomonas oxalaticus,

now called Cupriavidus oxalaticus (249), and its metabolism of oxalate and format (191-193).

Limited characterization of cell lysates suggested the presence of a CoA transferase that

reversibly transfers CoA between oxalate and succinate.

Acetyl-CoA:malonate CoA-transferase (EC 2.8.3.3)

Bacterial growth on malonate generates the end products acetate and CO2 (for a review,

see Dimroth (64)). The malonate decarboxylase complex in Pseudomonas ovalis comprises five










L acidophilus
MCR M tuberculosis
BbsF T aromatica
55 Polaromonas1251

,8 B bronchiseptica RB50
1io' B bronchiseptica 1308
73 B parapertussis1188

T volcanium
N crassa
B parapertussisl230
1io M magnetotacticum 1221

C tetani
R coelicolor

-100 M magnetotacticum1194
R eutropha1194
10
R metallidurans

O formigenes
B japonicum
i10 R palustris

67 i IB fungorum
97 Polaromonas1248
6 S flexneri
53
ion E coli K12

E coli o157H7
98 Polaromonasl278

ion M magnetotacticum1251
n2R eutrophal251

CaiB E coli
10 BaiF C scindens
HadA C difficile
100 FIdA C sporogenes

BbsE T aromatica
0.1 changes

Figure. 5-2. Phylogram of FRC, putative formyl-CoA transferases, and known Family III CoA
transferases from pair-wise sequence alignment of polypeptide sequences.










expected the mutated enzyme is impaired. For the G260A mutant, the KM value for oxalate

increases almost 5 times and kcat/KM is 75 times reduced (Table 2-4). The 2.0 A resolution crystal

structure of the G260A mutant, showed clear strain in the loop, which could not adopt the same

conformation as in the wild type enzyme in the closed form (Figure 2-12). The mutation

containing loops were modeled in the most probable conformation based on the electron density

in an omit map, although difference density around the loops shows them to be partly disordered.


-181
101

150

120

90

60

30

Psi 0

-30

-60

-90

-120

-150

-18 I


-30 0
Phi


30 60 90 120 150


Gly-B259


S GlcyA2S0


30 60 90 120 150


Figure. 2-12. Ramachandran plot showing loop glycine residues (258GGGG261). Plot generated
in Swiss-PDB Viewer V3.7 from the apo-enzyme structure (lp5r). Chain A is in the
closed conformation and chain B is in the open conformation. The black regions
represent the generously allowed regions.


SGlyB60


S80

150

120

90

60

30

0

-30

-60

-930

-120

-150

180
180











3
.N -" N RR

N N- H2
I
1',4'-iminopyrimidine H



O R2

ThSCoA
ThDP-4'NH2 0


N N R1

4N NH2H
4'-aminopyrimidine


R =p-hydroxyethyldiphosphate
R2=4'-imino-2-methyl-5-pyrimid


(1") ylide


0-


HO


CO2

R2 N


HOS
'-4'NH SCoA
enamine


ThDP-4'NH


Figure. 4-1. Scheme of OXC mechanism for ThDP-dependent oxalyl-CoA decarboxylation. 1,
ThDP-ylide; 2, oxalyl-CoA; 3, pre-decarboxylation intermediate; 4, a-carbanion/
enamine intermediate; 5, formyl-CoA-ThDP covalent complex; and 6, formyl-CoA.
Crystal structures of OXC with oxalyl-CoA bound, OXC with formyl-CoA, and
covalent intermediate are highlighted.


(3


ThDF


-carbanion


Glu56-COOE


Glu. -COOH


vv









CHAPTER 5
SUMMARY

Kinetic Mechanism of Family III CoA Transferases

Family III CoA transferases differ from Family I transferases both in sequence similarity

and in kinetic mechanism. Hydroxylamine and borohydride trapping experiments, as well as

more direct evidence from MS and crystallographic data, have demonstrated that, despite their

divergent kinetic mechanisms, both Families mediate the transferase reaction through a covalent

enzyme CoA thioester intermediate. The formyl-CoA transferases, a subgroup of Family III

CoA transferases, rely on a conserved flexible loop comprising four glycine residues to protect

labile reaction intermediates and contribute to substrate specificity. Analysis of two formyl-CoA

transferase homologs from E. coli and 0. formigenes demonstrates that modification of the

residues near the glycine loop, but outside of the active site, confers 100-fold increase for oxalate

affinity and replacement of these residues with alanine severely impairs the ability of the enzyme

to catalyze the formation of oxalyl-CoA from formyl-CoA

FRC and OXC from E. coli

This work has clearly demonstrated that YfdW and YfdU from E. coli are a formyl-CoA

transferase and an oxalyl-CoA decarboxylase, respectively; these enzymes are functionally

homologous to FRC and OXC from 0. formigenes. The submillimolar KM values for formyl-

CoA and oxalyl-CoA are an order of magnitude larger than for the 0. formigenes' enzymes, but

are similar to each other as is expected with a complementary enzyme pair. The turnover

number for oxalate with YfdW is 100 times higher than for the 0. formigenes enzyme. Thus,

despite the critical nature of the FRC reaction, YfdW is more stringent for using formyl-CoA and

oxalate than is FRC. In an X-ray structure (lpt8), oxalate is seen bound outside of the active site










In subunit B this region is much more flexible, and in the P-aspartyl-CoA thioester complex

obtained from formyl-CoA, residues 286-316 have no interpretable electron density and are

modeled with zero occupancy. Inspection of the crystal packing reveals that the corresponding

region of subunit A forms crystal contacts with the adjacent molecule while this region in

subunit B is freely exposed to solvent.

Inhibition of FRC by Chloride Ions and Glyoxalate

The identification of chloride ions bound in the active sites was followed up by kinetic

measurements showing that chloride has an inhibitory effect on the transferase activity in FRC.

Chloride is a weak competitive inhibitor against oxalate with Kic of 3 2 mM (Figure 2-7).

Glycolate may act as an oxalate analogue and be a good tool for ascertaining the oxalate binding

1.1 1.oo

1.0 -- O M
0.9 O-
S0.0 -


E 0.7 0.00 20.00 40.00
S[KCI], mM
3- 0.6 -

0.5

0.4 -

0.3


0.1

0.0 1
-0.20 0.00 0.20 0.40 0.60 0.80 1.00

1/[Oxalate], mM-1


Figure. 2-7. Double-reciprocal plot of competitive Cl- inhibition of FRC against varied
[oxalate] (1.0 77.0 mM) at 21.4 iM [formyl-CoA] and 10 iM [CoA] with 8.8 nM
enzyme. KCl concentrations were 5 mM (e), 15 mM (o), and 30 mM (m). Kio = 3
2 mM was determined from the replot of KMapp/Vapp vs. [KCl] (ilii'e I). Taken from
Berthold 2008 (18).








Vm a [B]
v = [B (Eqn. 1)

4 [A]) [A]

v Vmax[S] (Eqn. 2)
KM +[S]+ [S]2


Ks
v Vmax[S] (Eqn. 3)
K ^+ [I] j [S

Km + [S]
v= max[S] (Eqn. 4)

[KcI) \KK,


In these equations, Ka, KMA and KMB represent the dissociation of the first substrate to bind

to the enzyme (formyl-CoA) and the KM values for formyl-CoA and oxalate, respectively, K,s is

the substrate inhibition constant of oxalate against varied formyl-CoA concentration, and K~c and

K,, are the inhibition constants for competitive and uncompetitive mechanisms, respectively.

Patterns of intersecting lines in double-reciprocal plots (supporting information) were used to

ascertain the mode of inhibition, and hence the correct equations for use in evaluating the

inhibition constants (47, 216) K,c and K,, and the formyl-CoA KM were determined by fitting

initial velocity plots of CoA inhibition directly with the Michaelis-Menten equation for mixed-

type (Eqn. 3) or competitive inhibition (Eqn. 4). KM values for oxalate and succinate and Kia

could then be determined by fitting initial velocity plots using an ordered bi-bi equation when the

second substrate (oxalate or succinate) was varied (Eqn. 1). In the case of YfdW, attaining

saturating formyl-CoA concentrations proved to be impractical. In this case, apparent KM and

Vmax values for either oxalate or succinate were obtained by fitting to the initial velocity plots at









and 2-6B). Binding of format at this site can result in hydrogen bonds to both the pantetheine

arm and the main chain amide of Gln-262 (Figure 2-9). The activated CoA conformation creates

a cavity below the /-aspartyl-CoA thioester with connection to the surface, where oxalate can

enter and bind in the second anion site identified in subunit B of the -aspartyl-CoA thioester

complex (C12B in Figures 2-4 and 2-6B). Manual modeling of oxalate at this site results in strong

hydrogen bonds to the amides of Gln-17 and Ala-18 and minor shifts would place also His-15

and Asn-96 within hydrogen bonding distances, ensuing bonds to all four oxygens of oxalate

(Figure 2-9). With a favorable orientation, and a distance of approximately 3.7 A to Cy of Asp-

169, oxalate is perfectly aligned for a nucleophilic attack at the /-aspartyl-CoA thioester [D].

The second mixed anhydride, the aspartyl-oxalyl anhydride results, and CoAS- shifts back

to its resting conformation. The final attack by CoAS- at the oxalyl moiety regenerates the

aspartate together with oxalyl-CoA [E]. Finally, in the product leaving step, opening of the

glycine loop allows release of the acceptor thioester together with format.

Experimental Methods

Site-Directed Mutagenesis and Protein Production

The Q17A, G259A, G260A, and G261A variants were prepared by QuikChange site-

directed mutagenesis (Stratagene) with the FRC gene in the pET-9a vector (Novagen, San

Diego, CA) with the following primers: 5'-Q17A 5'-GCT TGA CTT TAC CCA CGT CGC

GGC AGG TCC TGC CTG TAC ACA GAT GAT GGG, 3'-Q17A 3'-CCC ATC ATC TGT

GTA CAG GCA GGA CCT GCC GCG ACG TGG GTA AAG TCA AGC, 5'-G259A 5'- GGT

GCG GGC GGC CAG CCA GGC TGG, 3'-G259A 3'- GCC CGC ACC TGC GTT ACC ACC

ACG TGG, 5'-G260A 5'- GGC GCG GGC CAG CCA GGC TGG ATG CTG, 3'-G260A 3'-

GCC CGC









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

LIST OF TABLES .............. ...... ........................ ...............

L IST O F F IG U R E S .... ........ ............. ........................... .....................................11

LIST OF ABBREVIATION S .............. ...................................... .... .................. 14

A B S T R A C T ............ ................... ............................................................ 16

CHAPTER

1 INTRODUCTION ................... ......... .. ...... ... ...... ....... 18

O xalic A cid ............... .. ..........................................................18
Oxalobacter formigenes .................................................. ........ ................ 18
M medical Relevance in Hum ans ......................................................... ............... 19
M etabolism of Oxalobacterformigenes....................................................................... 19
Key Metabolic Proteins of Oxalobacterformigenes.....................................................20
Oxalate-2:form ate-1 antiporter (OxlT).................................. ........................ 21
Form yl-CoA transferase (FRC).................. .......... ......................... ...... ....... 22
Oxalyl-CoA decarboxylase (OXC) ........................................ ....... ............... 22
C o en zy m e A ..................................................................................................................... 2 2
H history of C oenzym e A .......................................................................... ................... 22
C oenzym e A P ool in B bacteria ........................................ ...................... .....................23
R activity of C oenzym e A ........................................... .................. ............... 24
C oenzym e A T ransferases ............................................................................ ....................25
F am ily I C oA T ransferases...................................................................... ..................26
Propionate CoA transferase (EC 2.8.3.1) ................ ..... ............. 27
Succinyl-CoA:oxalate CoA transferase (EC 2.8.3.2)............... ...............27
Acetyl-CoA:malonate CoA-transferase (EC 2.8.3.3)............... ...............27
Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5) ...........................28
3-Oxoadipate CoA -transferase (EC 2.8.3.6) ............................................................29
Succinyl-CoA:citramalate CoA-transferase (SmtAB; EC 2.8.3.7)..........................29
Acetate CoA-transferase (AA-CoA transferase, atoDA; EC 2.8.3.8)...................30
Butyrate-acetoacetate CoA-transferase (EC 2.8.3.9) ............................................30
Acetyl-CoA: glutaconate CoA-transferase (GCT; EC 2.8.3.12)............................30
F am ily II C oA T ransferases ................................................................... ...................3 1
Citrate CoA-transferase (EC 2.8.3.10) ...........................................32
Citramalate CoA-transferase (EC 2.8.3.11) ........... ................... ................33
Fam ily III CoA Transferases ................................................ .................. ............... 34
Formyl-CoA transferase (FRC; EC 2.8.3.16) .................................. ............... 35
B aiF .......................................... .......... ....... .................... ..... ......... ........ 3 6
Succinyl-CoA:(R)-benzylsuccinate CoA-transferase (BbsEF; EC 2.8.3.15)...........37









39. Cardon, B. P., and H. A. Barker. 1946. Two New Amino-Acid-Fermenting Bacteria,
Clostridium propionicum and Diplococcus glycinophilus. J Bacteriol 52:629-634.

40. Chang, A. K., P. F. Nixon, and R. G. Duggleby. 2000. Effects of deletions at the
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42. Chica, R. A., N. Doucet, and J. N. Pelletier. 2005. Semi-rational approaches to
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43. Chohnan, S., K. Akagi, and Y. Takamura. 2003. Functions of malonate decarboxylase
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where water molecules were assigned and checked. The quality of the final structures were

validated using PROCHECK (144)and WinCoot (157) and annealed omit maps calculated in

CNS (29) were used to confirm the conformations in the active sites. All images of protein

molecules were generated using PYMOL (59).

Synthesis of [lO4]-Oxalate

Normalized H21O8 (95% enrichment) was obtained from Cambridge Isotopes, Inc.

(Andover, MA). The 180-enriched oxalic acid was prepared by dissolving approximately 6.5 mg

of (COOH)2-2H20 in 500 [iL of H2180 in a literature procedure (6). The sample was sealed in an

ampule and lyophilized after storage at room temperature for 5 weeks. The 180 content of the

oxalic acid (79%) was determined by LC-MS (Mass Spectrometry Laboratory, University of

Florida). Oxalate was resuspended in water and the pH brought to 7 with the addition of solid

KOH. Oxalate concentration was determined by oxalate decarboxylase enzymatic assay (143).

Isotope (80)-Labelling Experiment

180-labelling of FRC Asp-169 was attempted by incubating 12.4 [tg of wild-type

recombinant FRC with 236 pM formyl-CoA and 19.8 mM 79% enriched [s184]-oxalate for 1 and

2 minutes at 300 C. The unquenched reaction mixtures were immediately buffer exchanged into

300 mM sodium chloride in 25 mM sodium phosphate, pH 6.2 on Amicon Microcon

concentration devices to a final volume of about 50 tL. Half was subsequently reincubated with

236 pM formyl-CoA and 77 mM [1604]-oxalate for 2 minutes.

Peptide Generation by Proteolysis

The trapped samples (theoretically 12 [tg of protein for hydroxylamine experiments or 6.2

[tg of FRC each for the 1sO-labelling experiments) were buffer exchanged into about 50 [tL each

of 50 mM NH4HCO3, pH 8.5 or 50 mM sodium phosphate, pH 7.5 with Amicon Microcon

centrifugal filter devices (Millipore). Concentrators were prepared by storing in 4% Tween 20









CHAPTER 3
FORMYL-COA TRANSFERASE (YFDW) FROM ESCHERICHIA COLI2

Introduction

With the completion of genome sequences for several strains of Escherichia coli (23, 101,

186, 256), attention has turned to the annotation of proteins encoded by specific genes of

unknown function (244). Deletion studies have shown that the yfdXWUVE operon (Figure 3-3),

in which the yfdX gene is under the control of the EvgAS regulatory system (169), encodes

proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions

(168). Although the molecular mechanisms underlying this phenotypic behaviour remain to be

elucidated, the proteins encoded by the yfdW and yfdU genes in this operon (YfdW and YfdU,

respectively) are homologous to the formyl-CoA transferase (FRC) (2, 29, 45) and the oxalyl-

CoA decarboxylase (OXC) (10, 15) present in the obligate anaerobe Oxalobacterformigenes

(237). FRC and OXC are essential for the survival of Oxalobacter in that they mediate the

conversion of oxalate into format and CO2 in a coupled catalytic cycle (Figure 3-1). In

combination with an oxalate:formate antiporter (OxlT) (15), this cycle is thought to maintain


0 A 00,, V0,rk_ + 0oK
O O

H s,,CoA 0+ O FR SCoAO H
O O

H', -CO2
OXC

Figure. 3-1. Coupled enzymes of oxalate catabolism in 0. formigenes.






2 Reproduced in part with permission from Journal of Bacteriology, Vol. 190 (12), Toyota, C. G., Berthold, C. L.,
Gruez, A., Jonsson, S., Lindqvist, Y., Cambillau, C., and Richards, N. G. Pages 2556-2564. Copyright 2008 Journal
of Bacteriology









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APPENDIX A
PRIMERS USED FOR MUTAGENESIS AND CLONING

Primers for pET-28b constructs

5'-Ndel FRC 5'-AGG AGA TAT ACA TAT GAC TAA ACC ATT AGA TGG AAT TAA TGT GC
3'-FRC BamHl(stop) 5'-AAG TCT GGA TCC TCA AAC TAC CTG T
5'-Ncol FRC 5'-AAG GAG CCA TGG AGA TGA CTA AAC CGT TAG ATG
3'-FRC Xhol(stop) 5'-CTG ACC TCG AGA ACT ACC TGC TTG C
5'-BamH1FRC 5'-AGG AGA TAT AGG ATC CGA TGA CTA AAC CAT TAG ATG GAA TTA ATG TGC
3'-FRC Xhol 5'-CCC AGA AAG TCT GAC CTC GAG AAC TAC CTG TTT TGC ATG C

Primers for Duet constructs

5'-FRC BamH1 5'-AGG AGA TAT AGG ATC CGA TGA CTA AAC CAT TAG ATG GAA TTA ATG TGC
3'-FRC HindIII(stop) 5'- ACA GGT AGT TTG AAG CTT AGA CTT
3'-FRC Xhol(stop) 5'- ACA GGT AGT TTG ACT CGA GAG ACT T

QUICKCHANGE primers for FRC variants

5'-Q17I 5'-GTC ATT GCA GGT CCT GCC TGT ACA CAG-3'
3'-Q17I 5'-TGC AAT GAC GTG GGT AAA GTC AAG CAC-3'
5'-Q17A 5'-GCT TGA CTT TAC CCA CGT CGC GGC AGG TCC TGC CTG TAC ACA GAT GAT GGG-3'
3'-Q17A 5'-CCC ATC ATC TGT GTA CAG GCA GGA CCT GCC GCG ACG TGG GTA AAG TCA AGC-3'
5'-W48F 5'-GAT ATG ACT CGT GGA TTC CTG CAG GAC AAA CC-3'
3'-W48F 5'-GGT TTG TCC TGC AGG AAT CCA CGA GTC ATA TC-3'
5'-W48Q 5'-GAT ATG ACT CGT GGA CAG CTG CAG GAC AAA CC-3'
3'-W48Q 5'-GGT TTG TCC TGC AGC TGT CCA CGA GTC ATA TC-3'
5'-P159R 5'-CCG GTT TCT GGG ATG GTC GTC CAA CCG TTT CCG GC-3'
3'-P159R 5'-GCC GGA AAC GGT TGG ACG ACC ATC CCA GAA ACC GG-3'
5'-G258A 5'-GGT GGT AAC GCA GCG GGC GGC GGC C-3'
3'-G258A 5'-GGC CGC CGC CCG CTG CGT TAC CAC C-3'
5'-G259A 5'-GGT GCG GGC GGC CAG CCA GGC TGG-3'
3'-G259A 5'-GCC CGC ACC TGC GTT ACC ACC ACG TGG-3'
5'-G260A 5'-GGC GCG GGC CAG CCA GGC TGG ATG CTG-3'
3'-G260A 5'-GCC CGC GCC ACC TGC GTT ACC ACC ACG-3'
5'-G261A 5' GGT GGC GGC GCG CAG CCA GGC TGG
3'-G261A 5' GCC GCC GCC CGC TGC GTT ACC ACC

Nest primers for YfdW and YfdU

5'-YfdVUW 5'-CGC CTG GCC GGT GTT GGC GTA ATG G-3'
3'-YfdVUW 5'-CCC TGT TTG CCC GAG TAA TAG ATA CAA ATA GAG CCG C
5'-YfdW 5'-AGG TAT TCA TAT GTC AAC TCC ACT TCA AGG AAT TAA AGT TCT CGA TTT C-3'
3'-YfdW 5'-GGG AGC AAG CTT CCC CCG TTA ATA TCA GAT GGC G -3'
5'-YfdU 5'-CGA GGT TAT TAC ATA TGT CAG ATC AAC TTC AAA TGA CAG ATG G-3'
3'-YfdU 5'-CTC ACC ATC GCA TAA TGA GTT AAG CTT AGG AGA CGA TGT CAG-3'

Nested primers for YhjX

5'-YhjX nest 5'-GCC GTT TTT CCC CAG GCA TAA AGT GC-3'
3'-YhjX nest 5'-GCC CAG TAG CTC GCG GC-3'
5'-YhjX 5'-GCA GGA ATA CTC ATA TGA CAC CTT CAA ATT ATC AGC GTA CCC GC-3'
3'-YhjX 5'-CCA GTA GCT CGA AGC TTA GCA TTA AAG GGA GCC-3'









completely aligned, the negative Ca charge cannot be as effectively delocalized into the

thiazolium ring, the basicity of Ca is increased, and the subsequent step of the reaction when Ca

is protonated is enhanced. This is likely to be true in this case as well, because it allows the

proton donor (W2) to come close to Ca (2.7 A). The close Ca-OH-to-4'-NH2 contact and out-of-

plane distortion was observed in the predecarboxylation intermediates of PDH-E1 (5) and POX

(262). The authors of these studies claimed that the strain was a contributing force for the

decarboxylation step and that it would be relaxed upon enamine formation. The strained out-of-

plane distortion might persist also in the postdecarboxylation intermediate of OXC, and could

therefore act as a contributing force throughout the reaction.

Formyl-CoA Release

The reason for the relative stability of the covalent product intermediate might be the close

contact to the 4'-amino group of ThDP. The strained conformation, with the Ca out of plane with

the thiazolium ring and the positive charge on the thiazolium ring, might be the factors leading to

cleavage of the C2-Ca bond and product release from the cofactor.

Conclusions on Catalysis in Simple Decarboxylating ThDP Enzymes

Common to OXC and many other ThDP-dependent enzymes catalyzing a decarboxylation,

mutations of active site residues, other than the conserved glutamate needed for activation of the

cofactor, hardly ever lead to abolished activity (Table 4-2) (88, 125) showing that only the

cofactor itself is essential for catalysis. In agreement with the postulated mechanism for OXC in

Figure 4-1 and the structure of the intermediate complex presented here, it has been shown by

CD data on several ThDP enzymes (126, 176), and directly observed by NMR (134), that the 4'-

N atom of the pyrimidine moiety performs most of the acid-base reactions involving the

substrate Ca-carbonyl oxygen atom in the reaction sequence by conversion between the 4'-

amino and the 1',4'-imino form of the cofactor. OXC and other related simple decarboxylating









thioester intermediate structures results in r.m.s. deviations of 0.4-0.5 A over 854 Ca atoms for

the dimer. The differences between the structures are mainly found in the flexible segments of

the small domain in subunit B (residue 230-247 and 282-347) and among the active site residues

that shift orientations in the two active sites. The orientation of the CoA moiety observed in

subunit B of the P-aspartyl-CoA thioester complex (Figure 2-4 and 2-6B) has not been observed

before and most probably represents a new state in the catalytic cycle. This conformation will be

referred to as the "activated" conformation of CoA while the conformation in subunit A is

described as the "resting" conformation.

Aspartyl-Formyl Anhydride Complex

A structure of FRC containing the aspartyl-formyl anhydride complex was obtained in the

absence of chloride ions and oxalate upon flash-freezing a crystal 10 min after addition of

formyl-CoA. At a resolution of 1.87 A, subunit A of the dimer was interpreted to contain the

covalent P-aspartyl-CoA thioester and subunit B the P-aspartyl-formyl anhydride and free CoA

(Figure 8B). The formyl group of the aspartyl-formyl anhydride was modeled at occupancy 0.6

to best fit the observed electron density. In the subunit containing the trapped mixed anhydride,

the active site is nicely shielded by the glycine loop which adopts the closed conformation. The

other subunit has an open glycine loop and noise in the electron density map indicates

flexibility/disorder in the active site, especially in the region of the glycine loop and Tyr-139.

The CoA moieties are found in the resting conformation in both subunits. Superposition of the

aspartyl-formyl anhydride and aspartyl-oxalyl anhydride (pdb code: lt4c) complex structures

results in an r.m.s. deviation of 0.4 A over 427 Ca atoms of the monomer (Figure 8A). The

structures show very small changes in the enzyme core and the active sites are highly similar,

while the flexible solvent exposed areas display some differences.











family as the crystal structures of the formyl-CoA transferase ortholog in Escherichia coli coded


by the yfdW gene (97) and the close homolog (-butyrobetaine-CoA:carnitine CoA transferase


(196, 235) were determined. The Family I CoA-transferases, including mostly enzymes


involved in fatty acid metabolism, have a well established mechanism described in Figure 2-1


(Mechanism 1). The formation of covalent intermediates involving a glutamate residue of the


enzyme results in a classical ping-pong mechanism with exchanging substrate product glutamyl-


acyl anhydrides and y-glutamyl-CoA thioesters (217, 229). The y-glutamyl- CoA thioester was


,CoA
0
08H HC SI- GOA

Glusa -O L

Family I Mechanism









Family II Mechanism


0
OA C CHa
GlulS OS, CoA'

0
o
EQO-,-Y--
OH









0o
-- OH
ACP-CoAS o' OH

>HC
HC


oG
Glu / kO


00
00 C0 0
ACP-CoA.S O
OH


CoA

HO


GIuCNO rOSCoA
t0
HO

00


0



QOOCH3


Proposed Family III Mechanism


O CoA



00
0 .c


0
OA O H 0 H



CO O S
SC2
co?


Figure. 2-1. Summary of kinetic mechanisms for the three known CoA transferase Families.
Mechanism 1 is the ping-pong scheme shown for GCT. The enzyme-CoA covalent
intermediate is highlighted. Mechanism 2 is the transferase reaction with acetyl-CoA
and citrate in citrate lyase. Mechanism 3 is the 2004 proposed kinetic mechanism for
Family III CoA transferases from (124). Putative tetrahedral intermediates have been
omitted to save space.










HPLC gradient methods

Separation methods were optimized for the above C18 columns. Analysis with the

Dynamax Microsorb column was achieved with previously described HPLC methods (123, 124).

Methods for separation of CoA esters with the Varian Pursuit XRs C18 column and varyied

gradients of Buffer A and Buffer B are summarized below. Buffer A was 50 mM sodium

acetate, pH 4.7 and Buffer B was 50 mM sodium acetate with 90% CH3CN, pH 4.5.

Table 2-8. Oxalyl-CoA HPLC method
Time, min Buffer B, % Flow, mL/min
0.00 5 1.0
0.50 Wait (close)
6.00 9
6.10 95
9.00 95
9.10 5
11.00 5



YfdW 7 30 2007 10 16 15 AM3.DATA 800 MIB Channel 4

160,000

140,000

120,000

100,000

1 80,000

60,000

40,00 o

20,00(


0 1 2 3 4 5 6 7 8 9
Mm
Figure. 2-18. Representative chromatogram for separation of oxalyl-CoA (tR = 5.2 min).









94 2 aM. In contrast, acetyl-CoA is a competitive inhibitor of FRC with respect to formyl-

CoA, exhibiting a Ki, value of 56 6 aM at a fixed CoA concentration of 1.5 aM (Figure 3-10).

Size-Exclusion Chromatography Measurements.

Size exclusion chromatography of YfdW resulted in a MW value of 85.5 kDa (Figure 3-

11). Thetheoretical mass of the dimer is 96.6 kDa (monomer 48.3 kDa), suggesting that the

active conformation is dimeric, similar to that of FRC and consistent with crystal structure data.

The slightly low MW may be a result of a tight overall quaternary structure and is also similar to

the results seen for FRC (124).


0.2
VeNVo


Figure. 3-11. Size-exclusion chromatography data used to estimate the molecular mass of
catalytically active YfdW. Retention coefficients (KD) for the molecular weight
standards, used to calibrate the column, are shown by filled circles (*) and the open
circle (o) represents the experimentally determined KD of recombinant YfdW.









oxalate. It was not until 1980 that Dawson and coworkers isolated an anaerobic, gram negative

rod with oxalate-catabolic activity (57). Similar isolates from sheep rumena, pig ceca, and

human feces compose a unique group and have been designated with the new genus and species

Oxalobacterformigenes (1).

Medical Relevance in Humans

Oxalic acid is both an organic acid and a strong chelating agent for divalent cations such as

Ca+2 (110). In addition to the potential lethal toxicity in humans and animals at high

concentrations, hyperoxaluria leads to the formation of calcium oxalate stones, the most

prevalent form of kidney stone (105). Several oxalate-degrading bacteria, including

Bifidobacterium infants (38), Eubacterium lentum (117), Enterococcus faecalis (111),

Clostridium oxalicum (58), Lactobacillus spp. (38, 247), Oxalobacterformigenes (1, 56),

Oxalobacter vibrioformis (58), and Streptococcus thermophilus (38), have been identified and

are possible candidates for probiotic treatment of hyperoxaluria in man. 0. formigenes is by far

the best studied and its presence has been linked to normal urinary oxalate excretion and its

absence coincides with an increased incidence of hyperoxaluric patients (66, 224). Treatment of

patients suffering from primary hyperoxularia with oral doses of 0. formigenes (frozen paste or

enteric-coated capsules) has been successful at lowering urinary excretion levels, but has had

mixed long term results-intestinal colonization appears to be mainly of a transient nature (69,

113, 114). In addition to intraluminal oxalate-degrading activity, some product of, or O.

formigenes itself, interacts with colonic mucosa to stimulate oxalate secretion, effectively

lowering urinary oxalate levels (99).

Metabolism of Oxalobacterformigenes

In their 1985 study, Allison et al. reported that oxalate is the sole growth substrate for 0.

formigenes, but that a small amount of acetate (0.5 mM) is also required (1). Further, for every









CHAPTER 1
INTRODUCTION

Oxalic Acid

Oxalic acid is the simplest organic diacid. It is both a relatively strong organic acid, pKa1

of 1.23 and pKa2 of 4.19, and a strong chelator of divalent cations forming mostly insoluble

oxalates (110). Rapid heating can cause degradation into CO2, CO, and H20 and oxalic acid can

be oxidized by permanganate to CO2 and water. Oxalic acid is used in household cleaners, as a

mordant for dyeing, wood stripper, and as a miticide. It is synthesized industrially by heating

NaOH and CO under pressure. Oxalate biosynthesis in plants is linked primarily to cleavage of

ascorbate and oxidation of byproducts of photorespiration, glycolate and glyoxalate, by glycolate

oxidase (198, 215). Plants are proposed to utilize oxalate in calcium regulation, heavy metal

detoxification, and, in the form of calcium oxalate, as protection. Calcium oxalate is thought to

be a source of hydrogen peroxide for cereal defense and a thick layer of calcium oxalte armours

spruce from insect infestation (85). Oxalate in mammals comes partly from dietary sources, like

rhubarb and spinach, and partly from metabolic processes in the body, e.g. a byproduct of amino

acid degradation (112). Man has no endogenous oxalate metabolism.

Oxalobacterformigenes

Sheep grazing in the western United States are susceptible to oxalate poisoning through

consumption of oxalate-containing plants like Halogeton (120). Efforts to understand oxalate

resistance in ruminants led to researcher awareness of ruminal microbes with oxalate degrading

activity. In a study of two crossbred sheep and one 600 kg fistulated Holstein cow it was

reported that degradation activity was increased if the animals were adapted to increased levels

of dietary oxalate (2). Isolation of the bacterium responsible proved difficult; the same study

reported that of the 99 pure bacterial isolates collected from adapted sheep none degraded










SA230-247


Monomer


FRC Dimer

Figure. 2-3. Two formyl-CoA transferase monomers displayed separately and in the dimer.
The figure displays the structure of the P-aspartyl-CoA thioester derived from oxalyl-
CoA. The Ca trace and CoA molecules (shown as stick models) are coloured by B-
factor, with blue representing the lowest B-factor and red the highest. The arrow
indicates the central hole in Monomer B. Modified from Berthold 2008 (18).

at the side chain amino group. Residues Arg-38 and His-15 also adopt different side chain

conformations in order to adapt to the two CoA conformations. Finally, Gln-17 takes on two

different rotamer conformations, with a position behind Asp-169 in subunit A and above the

thioester bond in subunit B. As was observed already for the apo-enzyme the side chain

conformation of Trp-48 is flipped 900 between the two monomers and the glycine loop

(258GGGGQ261) then assumes the open and closed conformations in subunits A and B,

respectively (Figure 2-4).

Neither format nor oxalate was detected in the active site. Interestingly, subunit A

contains density interpreted as one chloride ion bound behind the active site residue Asp-169, at

a position occupied by residue Gln-17 in the other subunit, and subunit B has density interpreted

as two chloride ions bound, one on each side of the pantetheine arm of CoA, where one chloride

ion is interacting with the closed glycine loop and the other with the main chain amides of Gln-

17 and Ala-18 (Figure 2-4).









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

OXALATE-DEGRADING ENYZMES OF
Oxalobacterformigenes AND Escherichia coli

By

Cory G. Toyota

August 2008

Chair: Nigel G. J. Richards
Major: Chemistry

Oxalate is a toxic organic diacid found in a variety of foods; build up of oxalate in the gut

is linked to the formation of calcium oxalate kidney stones. Humans have no innate mechanism

for metabolizing oxalate, so enzymes that catalyze the degradation of oxalate may represent a

form of therapy. Oxalobacterformigenes and Escherichia coli may influence oxalate

homeostasis in man. The focus of this work is on understanding the kinetic mechanisms and

metabolic roles of the proteins involved in oxalate metabolism in these organisms. Two

enzymes are essential to 0. formigenes metabolism: formyl-CoA transferase (FRC) and oxalyl-

CoA decarboxylase (OXC). FRC catalyzes the transfer of CoA from formyl-CoA to oxalate to

yield one molecule each of format and oxalyl-CoA. The thioester product is subsequently

decarboxylated by OXC in a proton-consuming reaction that is essential in energy generation for

the organism. In addition to recombinant wild-type and His-tagged fusion proteins, a series of

active site and truncation mutants were prepared for steady state kinetic analysis.

Hydroxylamine and borohydride trapping experiments in conjunction with high resolution X-ray

crystallographic freeze trapping experiments have outlined the complete kinetic mechanisms for

both OXC and FRC. This work has demonstrated that despite differing in kinetic mechanism

both Family I and Family III CoA transferase reactions proceed through an enzyme-CoA










POX A L plantarum


B animals


E coli K12 YfdU
81

O formigenes


B japonicum


M tuberculosis
100
53

M bovis


BFD P putida


UO A flavus


PDH El E coli


PDC Z mobilis


yPDC S cerevisiae


9s PDH El S cerevisiae

69
AHAS S cereviseae


V vinifera


POX B L plantarum
0.1 substitutions/site

Figure.C-3. Phylogram of ThDP-dependent decarboxylases from pair-wise sequence
alignment of nucleotide sequences.































Figure. 2-9. Stereoview of oxalate and format modeled into the FRC active site. Oxalate and
format are modeled into the anion binding sites occupied by chloride ions in subunit
B of the 0-aspartyl-CoA thioester complex. Pockets are calculated with a probe
radius of 1.4 A and are displayed in a surface representation. Both of the pockets
have a connecting channel to the surface. The glycine loop in the closed
conformation protects the thioester from an attack from above. Hydrogen bonds are
indicated by dashed yellow lines and the red dashed line shows where nucleophilic
attack will take place. Taken from Berthold 2008 (18).

Q17A FRC Mutant and Oxalate Binding

The active site residue Gln-17, positioned in the close proximity of Asp-169, has two

distinct rotamer conformations corresponding to the two different active site conformations in

the P-aspartyl-CoA thioester complexes (Figure 2-4). Replacement of this residue by an

alanine results in severely impaired activity, with a 45-fold reduced kcat (Table 2-4). A crystal

structure of the Q17A mutant was solved to 2.2 A resolution from a crystal incubated with

formyl-CoA and oxalate. The enzyme variant still could bind formyl-CoA and the reaction

proceeded until formation of the P-aspartyl-CoA thioester, which was observed in the resting

conformation in both active sites of the dimer. Interestingly, the glycine loop displayed the open









ThDP enzymes achieve a significant fraction of their catalytic power from setting up the central

a-carbanion intermediate for proton transfer to Ca (269). In the case of OXC, the proton is

derived from a bound water molecule in the active site. The cis-Pro loop containing Ile-34 has a

central role in the OXC active site by positioning the substrate, the product, and the water

molecule, W2, donating the proton to the a-carbanion intermediate. The conformation of the

loop is maintained by a hydrogen bond network involving the invariant water molecule, W1, in

the active site. There have been several studies reporting on communication between the active

sites through a proton-conducting channel resulting in alternating site reactivity (66, 86, 125,

127). In OXC, there is no proton conduction channel and no evidence for alternating site

reactivity in the structures of OXC. Although the electron density of ligands is often better

defined in one subunit than in the other due to different occupancies, in no case are different

species visible in the active sites. Transketolase has also been reported to lack alternating site

reactivity (82).

Experimental Methods

Protein Expression, Mutagenesis, and Purification

OXC was produced recombinantly in E. coli as described previously (15, 16). Site-

specific OXC variants were produced by the QuikChange site-directed mutagenesis method

(Stratagene) or the overlap extension method (109), and were expressed and purified according

to protocols used for the wild-type enzyme (15, 16). Briefly, this procedure includes affinity

chromatography on a Blue-sepharose fast flow affinity column followed by desalting on

Sephadex G-25 size-exclusion column, and further purification by QHP anion-exchange

chromatography. The C-terminal truncation variant lost the capacity to bind to the affinity

column, but could be retained and purified to homogeneity by ion-exchange chromatography.

The OXC apoenzyme was prepared by dialysis overnight against 50 mM Tris (2-amino-2-









oxalyl-CoA was measured by separating the quenched reaction mixtures by reverse-phase

chromatography, monitoring the absorbance at 260 nm, and integrating the area under the peak.

The effects of contaminating CoA were controlled by first determining the inhibitory effect of

CoA against varied concentrations of formyl-CoA. Kinetic constants Vmax, KiCoA, and KM(formyl-

CoA) were then used to fit initial velocity plots of varied oxalate concentration at constant formyl-

CoA concentrations to determine the apparent KM(oxalate) and Kia.

The inhibition of FRC by chloride ions was determined at different oxalate concentrations

at saturating concentration of formyl-CoA (21.4 iM) and 10 iM CoA with KC1 concentrations

of 5, 15, and 30 mM.

Hydroxylamine and Sodium Borohydride Trapping Experiments

The experiments were carried out in a reaction volume of 500 tL, containing 6.2 tg of

recombinant wild-type FRC in 60 mM potassium phosphate buffer, pH 6.7, and 77 mM oxalate.

The reaction was started by the addition of 173 |M formyl-CoA and was allowed to run for 10

seconds before treatment with 15 mM hydroxylamine at pH 7 for 30 seconds at 30 oC. Small

molecules were immediately removed from the reaction solution by size exclusion

chromatography (5 mL G-25) after which the residual specific activity of FRC was assayed using

the normal HPLC point assay. The trapping experiment was repeated in a reaction mixture in

absence of oxalate with FRC or G261A incubated for 30 seconds at 30 oC with varied

concentrations of formyl-CoA (0.14 to 260 iM). As before, the reaction mixture was separated

by gel filtration chromatography 30 seconds after the addition of hydroxylamine. Residual

activity of the protein treated only with NH20H was also assayed.

Borohydride trapping experiments were carried out as above with the exception that the

reaction was trapped with the addition ofNaBH4 (1M NaBH4 in 1M NaOH) to a final











APPENDIX C
DENDOGRAMS


BaiF C scindens
T volcanium
C tetani
HadA C difficile

FIdA C sporogenes


N crassa
SBbsE T aromatica
SMCR M tuberculosis
BbsF T aromatica
SPolaromonas 1251
B bronchiseptica 1308
B parapertussis1188
M magnetotacticuml221
B bronchiseptica RB50
B parapertussisi230
L acidophilus
10 S flexneri
E coli K12
E coli o157H7
-- formigenes
-R coelicolor


100o M magnetotacticum1194
R metallidurans
Polaromonasl278
o10 R eutrophal 194

R eutrophal 251
SM magnetotacticumi251

1o 0B japonicum
in R palustris
B fungorum
Polaromonasl 248
CaiB E coli


-- 0.05 changes

Figure.C-l. Phylogram of Family III CoA transferases from pair-wise sequence alignment of
nucleotide sequences.


lo1


74
8



50
100


100


64

73


80














0 1.0
u 0.3

02 2 0.8
0.00 0.05 0D10
1/[F-CoA], pWMI .-

S0.6



10-6- 0.4
0.4
0.2
0.00 0.05 0.10 .2
Il[F-CoA], IMi1

0.0 ,
-1.00 -0.50 0.00 0.50 1.00

1/[oxalate], mM1


Figure. 3-8: Double reciprocal plot of initial velocities of HisFRC with varied [oxalate] (2.5 -
75 mM) at 10.1 (e), 30.3 (o), and 80.8 pM (m) [formyl-CoA].

the observed difference in kcat values for YfdW and FRC (3-3) cannot be attributed to this

structural modification.

The crystallographic observation of a YfdW/acetyl-CoA/oxalate ternary complex (97)

suggested that YfdW might be inhibited by acetyl-CoA, and therefore the steady state kinetic

behavior of the enzyme in the presence of this compound was assayed (Figure 3-9). As in earlier

experiments, the concentration of free CoA was maintained at a fixed value (52 PM) as formyl-

CoA was varied. Given that acetyl-CoA binds to the CoA site in the YfdW crystal structure (97),

it was assumed that acetyl-CoA and CoA were mutually exclusive inhibitors at a given active

site. This permitted the separation of their contributions to the overall rate equation (78), and

acetyl-CoA proved to be an uncompetitive inhibitor of formyl-CoA, with a Kiu value of









B-2 Summary of all inhibition constants and patterns for wild-type FRC and variants.........142

B-3 Steady-state parameters for the formyl-CoA/succinate transferase activities of YfdW,
FRC and the Trp-48 FRC mutants. ................. .. ......... ......................... 143

B-4 Steady-state parameters for OXC ............................................................................. 143










Table B-3. Steady-state parameters for the formyl-CoA/succinate transferase activities of YfdW,
FRC and the Trp-48 FRC mutants.


Enzyme


kcat (s-1)


His-YfdW
WT FRC
W48F FRC
W480 FRC


5.3 + 0.4
149 + 13
42 6
17.9 0.5


Formyl-CoA
KM(app) kcat/KM(app)
(gM) (mM-ls-)
180 + 14 29.4
16 2 9312
12 6 3500
6.7 + 0.9 2672


Table B-4. Steady-state parameters for OXC
Enzyme KM, iM
OXC (15) 23 3.5
E56A --
Y120F 43 + 9
Y120A 60+ 14 0.
E121Q 18 4 3
E121A 41 8 0.
Y483F 40+ 11 1
Y483A 24 + 7 1
S553A 21 5 1
R555A 66 8
553-565
HisYfdU 180 39


kcat, s 1
88 4


Succinate
KM(app) kcat/KM(app)
(mM) (mM-s-1)
80 40 0.07
0.32 + 0.03 465
0.015 + 0.005 2800
0.07 + 0.01 256


%WT
100
0
8.2
0.3
3.8
0.1
1.9
1.6
15
96
0.001


7.2 + 0.6
26 + 0.03
.3 + 0.3
1 + 0.01
.7+ 0.2
.4 0.1
13 1.5
85 4

15 1


kcat/KM, s-'M-1
3.8 x 106

1.7 x105
4.1 x 103
1.8 x 105
2.4 x 103
4.1x 104
5.6x 104
6.2 x 105
1.3 x 106

8.6x 104


Kia
(gM)
30 + 19
0.5 + 0.4
12 8
9+4




Full Text

PAGE 1

1 OXALATE-DEGRADING ENZYMES OF Oxalobacter formigenes AND Escherichia coli By CORY G. TOYOTA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Cory G. Toyota

PAGE 3

3 To Stefn Jnsson (1972-2007)

PAGE 4

4 ACKNOWLEDGMENTS There are far too m any people to thank for thei r support and help these past five years: Lori; my parents and family; Bubba; the Richards Group past and present, especially Patricia, Sue, Jemy, and Larissa; Mark Settles; John a nd Tracy; Jessica Light; Be th-Anne; Reid Bishop; Ylva Lindqvist; and my Committee, especially Drs. Lyons and Horenstein for their advice, so I would just like to kindly thank Catrine Berthold, St. Jude, and Nigel Richards, in that order.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................11 LIST OF ABBREVIATIONS........................................................................................................ 14 ABSTRACT...................................................................................................................................16 CHAP TER 1 INTRODUCTION..................................................................................................................18 Oxalic Acid.................................................................................................................... .........18 Oxalobacter formigenes ..........................................................................................................18 Medical Relevance in Humans........................................................................................ 19 Metabolism of Oxalobacter formigenes ..........................................................................19 Key Metabolic Proteins of Oxalobacter formigenes .......................................................20 Oxalate-2:formate-1 antiporter (OxlT)....................................................................... 21 Formyl-CoA transferase (FRC)................................................................................ 22 Oxalyl-CoA decarboxylase (OXC)..........................................................................22 Coenzyme A...........................................................................................................................22 History of Coenzyme A...................................................................................................22 Coenzyme A Pool in Bacteria......................................................................................... 23 Reactivity of Coenzyme A.............................................................................................. 24 Coenzyme A Transferases...................................................................................................... 25 Family I CoA Transferases.............................................................................................. 26 Propionate CoA transferase (EC 2.8.3.1)................................................................. 27 Succinyl-CoA:oxalate CoA transferase (EC 2.8.3.2)...............................................27 Acetyl-CoA:malonate CoA-transferase (EC 2.8.3.3)............................................... 27 Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5) .............................28 3-Oxoadipate CoA-transferase (EC 2.8.3.6)............................................................29 Succinyl-CoA:citramalate CoA-tr ansferase (Sm tAB; EC 2.8.3.7).......................... 29 Acetate CoA-transferase (AA-CoA transferase, atoDA; EC 2.8.3.8) ...................... 30 Butyrate-acetoacetate Co A-transferase (EC 2.8.3.9) ...............................................30 Acetyl-CoA: glutaconate CoA-transferase (GCT; EC 2.8.3.12).............................. 30 Family II CoA Transferases............................................................................................ 31 Citrate CoA-transferase (EC 2.8.3.10)..................................................................... 32 Citramalate CoA-transferase (EC 2.8.3.11)............................................................. 33 Family III CoA Transferases........................................................................................... 34 Formyl-CoA transferase (FRC; EC 2.8.3.16).......................................................... 35 BaiF..........................................................................................................................36 Succinyl-CoA:( R )-benzylsuccinate C oA-tran sferase (BbsEF; EC 2.8.3.15)........... 37

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6 Crotonobetainyl/ -butyrobetainyl-C oA:carnitin e CoA-transferase (CaiB)............. 38 Cinnamoyl-CoA: phenyllactate CoAtransferase (FldA; EC 2.8.3.17) ................... 38 2-Hydroxyisocaproate CoA transferase (HadA)......................................................39 -Methyl-CoA racemase (MCR and Amacr)........................................................... 40 Conserved Structure of Family III CoA Transferases..................................................... 40 Research Objectives............................................................................................................ ....43 2 CATALYTIC MECHANISM OF FORMYL-COA TRANSFERASE.................................. 46 Introduction................................................................................................................... ..........46 Results.....................................................................................................................................49 Wild-Type FRC Activity................................................................................................. 49 Enzyme-Aspartyl-CoA Thioester Com plexes............................................................. 49 Inhibition of FRC by Chloride Ions and Glyoxalate.......................................................55 Hydroxylamine and Sodium Borohydride Trapping....................................................... 56 Comparison with Previous FRC Complexes................................................................... 57 Aspartyl-Formyl Anhydride Complex............................................................................ 58 Q17A FRC Mutant and Oxalate Binding........................................................................ 60 G259A, G260A, and G261A FRC Loop Mutants........................................................... 62 Hydroxylamine Trapping of G261A Variant.................................................................. 65 Mass Spectrometry Analysis of Proteolysed FRC.......................................................... 66 Engineering Trypsin-Friendly FRC................................................................................. 67 Half-Sites versus Independent Active Sites Reactivity................................................... 68 Discussion...............................................................................................................................69 Experimental Methods............................................................................................................71 Site-Directed Mutagenesi s and Protein Production ......................................................... 71 Determination of Protein Concen tration by the Edelhoch Method. ................................ 73 Assay for Coenyzme A Esters......................................................................................... 74 HPLC gradient methods...........................................................................................75 Assay for coenzyme A concentration....................................................................... 77 Enzyme Kinetic and Inhibition Studies........................................................................... 77 Hydroxylamine and Sodium Borohyd ride Trapping Experim ents.................................. 78 Crystallization and Freeze-Trapping Experiments.......................................................... 79 Data collection, Structure Determination, and Refinement............................................. 80 Synethesis of [18O4]-Oxalate...........................................................................................81 Isotope Labelling Experiment.........................................................................................81 Peptide Generation by Proteolysis................................................................................... 81 Peptide Generation by Proteolysis (with GuHCl)........................................................... 82 Mass Spectrometric Analysis.......................................................................................... 82 Half-Sites (pET-Duet) Constructs................................................................................... 83 3 FORMYL-COA TRANSFERASE (YfdW) FROM ES CHERICHIA COLI ..........................84 Introduction................................................................................................................... ..........84 Results.....................................................................................................................................86 Kinetic Characterization of YfdW...................................................................................86 Size-Exclusion Chromatography Measurements............................................................ 93

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7 Alternate Substrate Studies..............................................................................................94 Kinetic and Structural Characterization of the W48F and W 48Q FRC Variants............ 96 Formyl-CoA Hydrolysis in the Presence and Absence of FRC, D169S, a nd YfdW.... 100 Expression, Purification, and Enzyme Activity of OXC Hom ologue HisYfdU........... 101 Discussion.............................................................................................................................102 Experimental Methods..........................................................................................................106 Materials........................................................................................................................106 Expression and Purificati on of His-Tagged YfdW .......................................................106 Expression and Purificati on of His-Tagged FRC .......................................................... 107 Expression and Purifica tion of FRC Variants ............................................................... 107 Size-Exclusion Chromatography Measurements.......................................................... 108 Confirmation of Quench Conditions............................................................................. 108 Steady-State Kinetic Assays..........................................................................................108 Determination of Steady-State Kinetic Constants......................................................... 109 Determination of the Specific Activity of FRC a nd YfdW with Alternate Substrates. 111 Crystallization and Structure Determinati on of the W 48F and W48Q FRC Variants.. 111 Formyl-CoA Hydrolysis in the Presence and Absence of FRC, D169S, a nd YfdW.... 112 Cloning, Expression, and Purificatio n of HisYfdU and HisYfdW ...............................112 Activity of HisYfdU......................................................................................................113 4 OXALYL-COA DECARBOXYLASE................................................................................ 114 Introduction................................................................................................................... ........114 Results...................................................................................................................................115 Structure of OXC with dzThDP.................................................................................... 115 Structure of the Oxalyl-CoA Complex and the C oA Complex..................................... 117 Structure of a Trapped Covalent Intermediate.............................................................. 119 Structure of the Formyl-CoA Complex......................................................................... 122 Kinetic Validation of Active Site Re sidues Deduced from the OXC Crystal Structures...................................................................................................................123 Discussion.............................................................................................................................124 Organization of C-Terminus Upon Substrate Binding..................................................124 Substrate Alignment for Ylide Attack........................................................................... 125 Postdecarboxylation Intermediate................................................................................. 125 Formyl-CoA Release..................................................................................................... 128 Conclusions on Catalysis in Simple Decarboxylating ThDP Enzymes........................ 128 Experimental Methods..........................................................................................................129 Protein Expression, Mutagenesis, and Purification.......................................................129 Enzyme Assay............................................................................................................... 130 Crystallization and Complex Formation.......................................................................130 Data Collection and Structure Determination............................................................... 131 5 SUMMARY........................................................................................................................ ..133 Kinetic Mechanism of Family III CoA Transferases........................................................... 133 Formyl-CoA Transferas e (FRC) and OXC from E. coli ......................................................133 Future Work..........................................................................................................................134

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8 Folding...........................................................................................................................134 Dynamics.......................................................................................................................138 APPENDIX A PRIMERS USED FOR MUTAGENESIS AND CLONING.............................................. 141 B SUMMARY OF KINETIC CONSTANTS......................................................................... 142 C DENDOGRAMS..................................................................................................................144 LIST OF REFERENCES.............................................................................................................148 BIOGRAPHICAL SKETCH.......................................................................................................169

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9 LIST OF TABLES Table page 1-1 Summary of Family III CoA Transferases......................................................................... 42 2-1 Data collection and refinem ent statistics........................................................................... 53 2-2 Form yl-CoA dependence of inactiva tion of FRC by hydroxylamine trapping................. 56 2-3 Formyl-CoA dependence of inactiv ation of FRC by hydroxylam ine and borohydride trapping in the absence of oxalate...................................................................................... 56 2-4 Summary of kinetic constant s for wild-type FRC and m utants.........................................65 2-5 Summary of the inhibiti on constants and patterns fo r wild-type FRC and mutants. ......... 65 2-6 Formyl-CoA dependent inactivation of G261A by hydroxylamine..................................65 2-7 Predicted and experimental ac tivities of half-s ites constructs. ..........................................69 2-8 Oxalyl-CoA HPLC method................................................................................................ 75 2-9 Formyl-CoA HPLC method............................................................................................... 76 2-10 Succinyl-CoA HPLC method............................................................................................ 76 3-1 FRC and YfdW substrate specifi city for alternate CoA acceptors.. .................................. 94 3-2 FRC and YfdW substrate specificity for alternate CoA donors with either f ormate or oxalate as the accep tor....................................................................................................... 94 3-3 Steady-state param eters for the formyl-CoA/oxalate transferase activities of YfdW....... 97 3-4 Summary of the inhibiti on constants and patterns for His-YfdW, FRC, Hi s-FRC, and and variants........................................................................................................................97 3-6 Data collection and refinement statis tics for the W48F and W48Q FRC mutants. ......... 100 3-7 Formyl-CoA hydrolysis in the presence of FRC, YfdW, and D 169A variant of FRC. ...101 4-1 Data collection and refineme nt statistics for OXC structures .......................................... 120 4-2 Summary of kinetic data fo r OXC, OXC variants, and His-YfdU. ................................. 124 5-1 Cysteine residues in wild type FRC................................................................................. 139 B-1 Summary of all kinetic constant s for wild-type FRC and variants .................................. 142

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10 B-2 Summary of all inhibition constants and patterns for w ild-type FRC and variants. ........142 B-3 Steady-state parameters for the formyl-CoA/succ inate transferase activities of YfdW, FRC and the Trp-48 FRC mutants................................................................................... 143 B-4 Steady-state parameters for OXC.................................................................................... 143

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11 LIST OF FIGURES Figure page 1-1 Cartoon representation of the key m etabolic enzymes of O. formigenes ..........................21 1-2 Coenzym e A..................................................................................................................... ..24 1-3 Ping-pong kinetics of Fam ily I glutac onate-CoA transferase (GCT) from Acidaminococcus fermentans .............................................................................................26 1-4 Prosthetic cofactor of citrate lyase .....................................................................................32 1-5 Double-reciprocal p lots of in itial velocity data consistent with an ordered mechanism in Family II citramalate-CoA transferase from Klebsiella aerogenes (64)....................... 33 1-6 Possible reaction m echanisms for FRC............................................................................. 35 1-7 Cartoon representations of Fa m ily III CoA transferases................................................... 41 1-8 Structure-based sequence alignm ent of Fam ily III CoA transferase family members...... 44 2-1 Summ ary of kinetic mechanisms for the three known CoA transferase Families............. 47 2-2 Double-reciprocal plot f or the inhibition of FRC by free CoA against varied [formylCoA] at constant saturating [oxalate]................................................................................50 2-3 Two for myl-CoA transferase monomers displayed separately and in the dimer............... 51 2-4 Stereoview of the overlay of the tw o active site confor mations of the -aspartyl-CoA thioester complex.............................................................................................................. .52 2-5 Mass spectrum of formyl -CoA transferase incuba ted with formyl-CoA...........................54 2-6 Electron density m aps of -aspartyl-CoA thioester and chloride ions..............................54 2-7 Double-reciprocal p lot of competitive Clinhibition of FRC against varied [oxalate].....55 2-8 Steroview overlay of FRC aspartyl-form yl and aspartyl-oxalyl anhydride active sites.... 59 2-9 Stereoview of oxalate and for mate modeled into the FRC active site............................... 60 2-10 Initial velocity plot of initial velocities of G261A variant agains t varied [oxalate] .......... 61 2-11 Substrate inhibition of the G261A varian t by oxalate against varied form yl-CoA........... 62 2-12 Ramachandran plot showing loop glycine residues (258GGGG261).................................... 63 2-13 Stereoview of G260A te traglycine loop............................................................................ 64

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12 2-14 Spectrum of FRC digested with glutamyl endopeptidase and analysed by MALDITOF mass spectrometry..................................................................................................... 66 2-15 Combined sequence coverage by mass spectrometric peptide analysis............................ 67 2-16 Proposed reaction m echanism for formyl-CoA transferase............................................... 70 2-17 Models and crystal structures showing assum ed important features in the active site...... 72 2-18 Representative chrom atogram for separation of oxalyl-CoA............................................75 2-19 Representative chrom atogram for se paration of CoA and formyl-CoA............................ 76 2-20 Representative chromatogram for separation of succinyl-CoA......................................... 77 3-1 Coupled enzymes of oxalate catabolism in O. formigenes ...............................................84 3-2 Superim position of apo-YfdW and apo-FRC dimer structures........................................ 85 3-3 Graphical representation of putativ e for myl-CoA transferase genes................................. 87 3-4 Quench conditions for Y fdW............................................................................................. 86 3-5 Double-reciprocal plot f or the inhibiti on of YfdW by free CoA against varied [formyl-CoA].....................................................................................................................88 3-6 Double reciprocal p lot of initial velocities of YfdW with varied [oxalate]....................... 89 3-7 Double reciprocal p lot of initial veloci ties of YfdW with varied [F-CoA]....................... 90 3-8 Double reciprocal p lot of initial velocities of HisFRC with varied [oxalate].................... 91 3-9 Double-reciprocal p lot for the inhibiti on of YfdW by acetyl-C oA against varied [formyl-CoA].....................................................................................................................92 3-10 Double-reciprocal p lot for the inhibiti on of FRC by acetyl-CoA against varied [formyl-CoA].....................................................................................................................92 3-11 Size-exclusion chrom atography data.................................................................................93 3-12 Double reciprocal p lot of initial velocities of YfdW with varied [succinate]....................95 3-13 Double reciprocal p lot of initial velocities of FRC with varied [succinate]......................96 3-14 Initial velocities m easured for YfdW as f unction of oxalate concentration at 73.3 M formyl-CoA........................................................................................................................99 3-15 Initial v elocities measured for the W48Q FRC mutant as function of oxalate concentration......................................................................................................................99

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13 3-16 Initial velocity plot of His-YfdU activity with varied oxalyl-C oA.................................. 101 3-17 Comparison of the active-site residues in YfdW and FRC.............................................. 103 3.18 Active-site structure in the W 48Q FRC variant............................................................... 105 4-1 Schem e of OXC mechanism for ThDP-d ependent oxalyl-CoA decarboxylation........... 116 4-2 OXC tetramer and active site. ..........................................................................................117 4-3 Three snapshots of OXC interm ediates........................................................................... 118 4-4 Annealed composite om it maps calculated for the structures shown around the active site........................................................................................................................... .........119 4-5 Stereov iew of postdecarboxylation intermediate complex.............................................. 121 4-6 Initial velocity plot of S553A OXC variant ..................................................................... 122 4-7 Stereoview of the aligned OXC structures....................................................................... 127 5-1 Pair-wise sequence alignment 10 a round the active sites of FRC and YfdW.............. 134 5-2 Phylogram of FRC, putative formyl-CoA transferases, and known Family III CoA transferases from pair-wise sequence alignment of polypeptide sequences.................... 135 5-4 IAEDANS and ANS. ....................................................................................................... 139 C-1 Phylogram of Family III CoA transferases from pair-wise sequence alignment of nucleotide sequences........................................................................................................ 144 C-2 Phylogram of Family III CoA transferases from pair-wise sequence alignment of amino acid sequences.......................................................................................................145 C-3 Phylogram of ThDP-dependent decarboxylas es from pair-wise sequence alignment of nucleotide sequences...................................................................................................146 C-4 Phylogram of ThDP-dependent decarboxylas es from pair-wise sequence alignment of amino acid sequences.................................................................................................. 147

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14 LIST OF ABBREVIATIONS ACP Acyl-carrier protein ADP Adenosine diphosphate ANS 8-Anilino-1-naphthalenesulfonic acid AR Acid resistance atoDA Acetate CoA-transferase ATP Adenosine triphosphate BaiF Putative bile acid induced CoA transferase BbsEF Succinyl-CoA:(R)-benzylsuccinate CoA-transferase Bis-Tris Propane 1,3-bis(tris( hydroxymethyl)methylamino)propane CaiB -butryobetaine-CoA:carnitine CoA transferase CD Circular dichroism CoA Coenzyme A DTNB Ellmans reagent; 5,5-dithio-bis(2-nitrobenzoic acid) DTT 1,4-dithio-DL-threitol dzThDP 3-deaza thiamine diphosphate F-CoA Formyl-CoA FldA Cinnamoyl-CoA: pheny llactate CoA-transferase FPLC Fast protein liquid chromatography FRC Formyl-CoA transferase from Oxalobacter formigenes FRET Frster resonance energy transfer GCT Glutaconyl-CoA:glutarate CoA transferase HAc Acetic Acid HadA 2-Hydroxyisocaproate CoA transferase HEPES 4-(2-hydroxyethyl)piper azine-1-ethanesulfonic acid

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15 IAEDANS 5-((((2-iodoacetyl)amino)ethyl)am ino) naphthalene-1-sulfonic acid MES 2-(N-morpholino)ethanesulfonic acid MFS Major facilitator superfamily MW Molecular weight NMR Nuclear Magnetic Resonance OXC Oxalyl-CoA transferase from Oxalobacter formigenes Ox-CoA Oxalyl-CoA OxlT Oxalate:formate antiporter from Oxalobacter formigenes PEG Polyethylene glycol RP-HPLC Reverse phase high-performance liquid chromatography SCOT Succinyl-CoA:3-ketoacid CoA transferase SmtAB Succinyl-CoA:citramalate CoA-transferase Suc-CoA Succinyl-CoA ThDP Thiamine diphosphate ThTDP Thiamine-2-thiazolone diphosphate YfdU OXC homolog encoded by yfdU gene in Escherichia coli YfdW FRC homolog encoded by yfdW gene in Escherichia coli

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16 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy OXALATE-DEGRADING ENYZMES OF Oxalobacter formigenes AND Escherichia coli By Cory G. Toyota August 2008 Chair: Nigel G. J. Richards Major: Chemistry Oxalate is a toxic organic diacid found in a va riety of foods; build up of oxalate in the gut is linked to the formation of calcium oxalate kidney stones. Humans ha ve no innate mechanism for metabolizing oxalate, so enzymes that cataly ze the degradation of oxalate may represent a form of therapy. Oxalobacter formigenes and Escherichia coli may influence oxalate homeostasis in man. The focus of this work is on understanding the kinetic mechanisms and metabolic roles of the proteins involved in oxalate metabolism in these organisms. Two enzymes are essential to O. formigenes metabolism: formyl-CoA transferase (FRC) and oxalylCoA decarboxylase (OXC). FRC catalyzes the transf er of CoA from formyl-CoA to oxalate to yield one molecule each of formate and oxalyl-CoA. The thioester product is subsequently decarboxylated by OXC in a proton-consuming reacti on that is essential in energy generation for the organism. In addition to recombinant wild-t ype and His-tagged fusion proteins, a series of active site and truncation muta nts were prepared for steady state kinetic analysis. Hydroxylamine and borohydride trap ping experiments in conjunction with high resolution X-ray crystallographic freeze trapping experiments have outlined the complete kinetic mechanisms for both OXC and FRC. This work has demonstrated that despite differing in kinetic mechanism both Family I and Family III CoA transferase reactions proceed through an enzyme-CoA

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17 thioester intermediate. In addition, flexible l oops in both enzymes, the C-terminal peptide loop of OXC and the te traglycine loop (258GGGGQ262) of FRC, have been id entified as critical to catalysis and substrate specificity. Structural genomics studies have shown that YfdW, encoded by the gene yfdW from an operon that appears to enhance the ability of Escherichia coli MG1655 to survive under acidic conditions, is structurally homol ogous to FRC. This work confirms that YfdW is a formyl-CoA transferase that appears to be more stringent than FRC in employing formyl-CoA and oxalate as substrates. Replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein shows that Trp-48 preclud es oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibi tion is modified to resemble that seen for YfdW. Finally, in addition to demonstrating the value of utilizing structural homology in assigning protein functi on, this work suggests that the yfdW and yfdU genes in E. coli may be be involved in confer ring oxalate-dependent acid re sistance to the bacterium.

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18 CHAPTER 1 INTRODUCTION Oxalic Acid Oxalic acid is the sim plest organic diacid. It is both a relatively strong organic acid, p Ka 1 of 1.23 and pKa 2 of 4.19, and a strong chelator of diva lent cations forming mostly insoluble oxalates (110). Rapid heating can cause degradation into CO2, CO, and H2O and oxalic acid can be oxidized by permanganate to CO2 and water. Oxalic acid is used in household cleaners, as a mordant for dyeing, wood stripper, and as a miticide. It is synthesized industrially by heating NaOH and CO under pressure. Oxal ate biosynthesis in plants is linked primarily to cleavage of ascorbate and oxidation of byproduc ts of photorespiration, glycolat e and glyoxalate, by glycolate oxidase (198, 215). Plants are proposed to utilize oxalate in calcium regulation, heavy metal detoxification, and, in the form of calcium oxalate as protection. Calcium oxalate is thought to be a source of hydrogen peroxide for cereal defense and a thick layer of calcium oxalte armours spruce from insect infestation (85) Oxalate in mammals comes par tly from dietary sources, like rhubarb and spinach, and partly from metabolic processes in the body, e.g. a byproduct of amino acid degradation (112). Man ha s no endogenous oxalate metabolism. Oxalobacter formigenes Sheep grazing in the western United States are susceptible to oxalate poisoning through consumption of oxalate-containing plants like Halogeton (120). Efforts to understand oxalate resistance in ruminants led to researcher awar eness of ruminal microbes with oxalate degrading activity. In a study of two cr ossbred sheep and one 600 kg fist ulated Holstein cow it was reported that degradation activity was increased if the animals were adapted to increased levels of dietary oxalate (2). Isolation of the bacterium respons ible proved difficult; the same study reported that of the 99 pure bacterial isolates collected from adapted sheep none degraded

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19 oxalate. It was not until 1980 th at Dawson and coworkers isolated an anaerobic, gram negative rod with oxalate-catabolic activ ity (57). Similar isolates fr om sheep rumena, pig ceca, and human feces compose a unique group and have been designated with the new genus and species Oxalobacter formigenes (1). Medical Relevance in Humans Oxalic acid is both an organic acid and a str ong chelating agent for diva lent cations such as Ca+2 (110). In addition to the potential lethal toxicity in humans and animals at high concentrations, hyperoxaluria l eads to the formation of calcium oxalate stones, the most prevalent form of kidney stone (105). Se veral oxalate-degrading bacteria, including Bifidobacterium infantis (38), Eubacterium lentum (117), Enterococcus faecalis (111), Clostridium oxalicum (58), Lactobacillus spp. (38, 247), Oxalobacter formigenes (1, 56), Oxalobacter vibrioformis (58), and Streptococcus thermophilus (38), have been identified and are possible candidates for probiotic treatment of hyper oxaluria in man. O. formigenes is by far the best studied and its presence has been linked to normal urinary oxalate excretion and its absence coincides with an incr eased incidence of hyperoxaluric patients (66, 224). Treatment of patients suffering from primary hyperoxularia with oral doses of O. formigenes (frozen paste or enteric-coated capsules) has been successful at lowering urinary excretion levels, but has had mixed long term resultsintestinal colonization app ears to be mainly of a transient nature (69, 113, 114). In addition to intraluminal oxala te-degrading activity, some product of, or O. formigenes itself, interacts with colo nic mucosa to stimulate oxalate secretion, effectively lowering urinary oxalate levels (99). Metabolism of Oxalobacter formigenes In their 1985 study, Allison et al reported that oxalate is the sole growth substrate for O. formigenes but that a sm all amount of acetate (0.5 mM) is also required (1). Further, for every

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20 mole of oxalate degraded, 1 mol of protons wa s also consumed with the concomitant production of about equimolar CO2 and 0.9 mol of formate. Carbon a ssimilation is mediated by conversion of oxalyl-CoA into 3-phosphoglycer ate by way of glycerate (51). Formate appears to be an end product of oxalate catabolism in O. formigenes rather than a substrate for NAD-linked formate dehydrogenase and source of cellular reduci ng equivalents (1). Careful studies with native membrane vesicles in reconstituted proteoliposomes have demonstrated that energy generation in O. formigenes is the result of both electrostatic and proton gradients created by the oxalate-2:formate-1 membrane-bound antiporter Oxlt (3). Key Metabolic Proteins of Oxalobacter formigenes O. formigenes depends on three identified proteins for the generation of metabolic energy from oxalate: an oxalate-2:formate-1 antiporter (OxlT), formyl-C oA transferase (FRC), and oxalyl-CoA decarboxylase (OXC). In the metabolic cycle, OxlT transports one molecule of divalent extracellular oxalate into the cell with the concomitant transfer of one molecule of monovalent formate out of the cytosol (3). Oxalat e is activated by transfer to coenzyme A from formyl-CoA in a reaction catalyzed by FRC. The thioester product is subsequently decarboxylated by OXC producing CO2, regenerating formyl-CoA for subsequent cycles, and consuming one equivalent of cytosolic protons (3 9). Anion exchange promotes a polarization of the membrane. The net extrusion of protons has the effect of increasing cytosolic pH and drives an F1F0ATP synthase with a proposed stoichiometry of 3H+/ATP. Decarboxylation of oxalate and the assumption that CO2 diffuses irreversibly from the cell ensures a near stoichiometric link between oxalate and the combination proton-motive and chemiosmotic gradient. The importance of these three protei ns is evident: together FRC and OXC make up 20% of the total cytosolic protein (9) and OxlT constitutes a major fraction (~10%) of the inner membrane proteins in O. formigenes (205).

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21 Oxalate-2:formate-1 antiporter (OxlT) OxlT, an oxalate:formate exchange protei n, is an inner-membrane bound protein with 12 transmembrane helices (205). OxlT transfers on e divalent oxalate ion across the membrane for every monovalent formate ion shuttled in with an estimated turnover number of about 1000 s-1 (205), a value that is about an order of magnitude higher than ot her secondary carri ers. OxlT, a member of the major facilitator superfamily (MFS) of transporters (182), has been the focus of extensive study: substrate anal ysis (255), topology analysis by fluorescence labelling (267), homology modelling (265), and kine tic studies of the protein in reconstituted liposomes (3, 205). Figure. 1-1. Cartoon representation of the key metabolic enzymes of O. formigenes OXC, oxalyl-CoA decarboxylase; FRC, formyl-CoA transferase; OxlT, formate:oxalate antiporter; and ATP synthase.

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22 Formyl-CoA transferase (FRC) Form yl-CoA transferase catalyzes the transfer of CoA from formyl-CoA to oxalate to yield oxalyl-CoA and formate (9, 124). FRC is the be st-studied member of the Family III CoA transferases and will be discussed later in detail (see page 37). Oxalyl-CoA decarboxylase (OXC) OXC is a thiam ine-dependent non-oxidative de carboxylase. The native enzyme was first purified from O. formigenes by Baetz et al; the 2 60 kDa homotetramer was reported to comprise four 65 kDa monomers (10). Steady-state kinetics studies demonstr ated that specificity constant for OXC is about 3.8 x 106 M-1s-1, KM(oxalyl-CoA) is 23 M, and kcat is 88 s-1 (15). Adventitious ADP was found tightly bound in the 1.8 X-ray crysta l structure in the same report. Analysis showed that micromolar concentrations of ADP were required for maximal enzymatic activity, whereas ATP had no effect. Considering the vita l role that OXC plays in oxalate metabolism, this finding strongly suggests that ADP-dependent activation of OXC in O. formigenes is physiologically relevant. Coenzyme A History of Coenzyme A Coenzym e A (CoA), is a cofactor essential to all living organisms, involved in over 100 different metabolic reactions, and purported to be utilized by a bout 4% of all known enzymes. The discovery of CoA is historically linked to the elucidation of the mechanism by which saturated fatty acids are utilized in living organisms and, as such, Franz Knoops famous 1904 use of a labelled phenyl group to show that fatty acids are degraded by successive removal of two-carbon units (139) was to become the starting point for both understanding -oxidation and identifying CoA. In 1942 Feodor Lynen demonstrat ed that after respirin g yeast had exhausted their supply of substrates, a lag or induction period was required before the process of oxidizing

PAGE 23

23 acetate could resume, and further, that the process could be facilitated by the addition of oxidizable substrates such as ethy l, propyl, or butyl alc ohol (158). This lead to the theory that acetate must be activated in some fashion in orde r to be used as a substrate and, indeed, Lynen was later able to identify acetyl-CoA from starved yeast (159). At the same time, several independent research groups confirmed that some form of active acetic acid was required for the enzymatic acetylation of bot h sulfanilamide (152) and choline (80, 176, 177) and it was theorized that the same water soluble activator was involved in all cases. Fritz Lipmann (154) was able to purify this activatorcoenzyme for acetylationand demonstrated that it comprised pantothenic acid, or vitamin B5, whose origin and nature had b een reported in the 1930s (263), and, upon acid hydrolysis, -alanine. The complete purification protocol, the presence of sulfur, as well as the identification of a pyrophosphate br idge to an adenylate group was reported in 1950 (153, 155). Snell and coworkers were able to show that the sulfur-c ontaining component of CoA is cysteamine, or 2-amino-ethanethiol, linke d to pantothenic acid by a peptide bond (228). Baddiley used a combination of analysis and synthesis to show that the pyrophosphate group binds to the 4-position of pantothenate (8). Kaplan demonstrated that the third phosphate is bound to the 3-position of the adenylyl-bound ribose (223). With the composition of CoA understood and the availability of isolated acet yl-CoA, researchers would now have the tools necessary to unravel the mysterie s of the processes involved in -oxidation. Coenzyme A Pool in Bacteria In vivo CoA pools have been determined for E. coli by three m ethods. Radioisotopic methods were employed to assay the total CoA pool in -alanine auxotrophs (panD2 ) grown on glucose-minimal medium. Concentrations were about 380 M with about 80% found as acetylCoA(118). The three other major species were CoA, 52 M; succinyl-CoA, 22 M; and malonyl-CoA, 2 M. Chohnan and coworker s employed their malonate decarboxylase-

PAGE 24

24 dependent acyl-CoA cycling method (242) to amplify CoA levels in anaerobic and aerobic E. coli They report that CoA thioester concen trations are 10 times higher under anaerobic conditions than aerobic and their value for the total CoA pool concentration agreed with the previous report (300 5 20 M) (45, 46). Boynton et al. have used reverse-phase HPLC methods to analyse CoA thioesters in Clostridium acetobutylicum (ATCC 824) (26). The total CoA pool ranged from about 1.0 1.2 mM. Figure. 1-2. Coenzyme A. CoA comprises an adenine ring and 3-phosphorylated ribose linked to a pantetheine domain by a pyrophosphate linker. Reactivity of Coenzyme A Coenzym e A comprises an adenine ring, 3-pho sphorylated ribose linked to a pantetheine domain by a pyrophosphate linker (F igure 1-2). CoA both carries and activates acyl groups as thioesters formed by reaction with the nucleophilic CoA thiol in two waysthe carbonyl is more electrophilic and the -carbon is more acid (p Ka~21) (171). Acyl groups can be transferred to another nucleophile, or the act ivated thioesters can undergo -carbon condensation, 1,4-addition, or reduction reactions at the th ioester carbonyl. Enzymatic reactio ns with acetyl-CoA involve either Claissen or aldol condens ations as with citrate synthase the gateway to the TCA cycle; acetyl-CoA carboxylase which catalyzes the synthe sis of malonyl-CoA, the starting point for fatty acid synthesis; or, the acyla tion of compounds, e.g. the inactiv ation of antibiotics such as

PAGE 25

25 chloramphenicol or kanamycin by acetylation in resistant bacteria (194, 195, 222). In oxidation of fatty acids, CoA is purported to stabilize the negative charge on the -carbon for dehydrogenation and addition of water across the double bond (252). In simple terms, the pantetheine and ADP mo ieties can be considered the structural components responsible for enzyme interaction a nd aligning the reactive sulfur atom. In a study by Jencks et al it was demonstrated that the pantoic ac id and ADP domains were critical for transition state stabilization and contributed significantly to kcat/ KM (259). However, other studies have shown that in some cases the ADP moiety contributes little to binding and catalysis (55, 83), Coenzyme A Transferases Coenzym e A transferases, ini tially termed thiophorases or transphorases, catalyze the reversible transfer of coenzyme A from a thioester donor to a fr ee acid. CoA transferases are found in all Eubacteria and Euka ryota (putative genes are found in Archaea) and play important roles in amino acid catabolism (12, 21, 32, 39, 218) ketone-body metabolism (22, 236), aromatic (132) and chloro-aromatic degrad ation (91), acetone-butanol ferm entation (4, 122), and fatty acid fermentation (140). There are currently 16 en zymes listed as CoA transferases (EC 2.8.3; IUBMB, 2005); these can be further separated into three CoA Transferase Families based on function, structure, and seque nce similarity (102). In addition to other properties, the three Families are separated on the basis of kinetic mechanism. All three share a common group transfer mechan ism summarized by the following equations: A + B P + Q or ACoA + B A+ BCoA

PAGE 26

26 where CoA and is transferred from a donor acid (A) to an acceptor acid (B). Mechanisms of this type can be divided into those which proceed through a ternary complex, ordered or random, and those which pass through a substituted m echanism, i.e. ping-pong mechanism. Family I CoA Transferases Fa mily I CoA transferases, specifically in EC 2.8.3.5-6 and 8-9, share two signature motifs: signature 1 (PS01273) is found in the N-terminal region of the -subunit and may be involved in CoA binding. The second consensus sequence motif (PS01274) (S)ENG, where E is the active-site glutamate, is found in the N-terminal region of the -subunit (162, 183). Family I CoA transferases, i.e. glutacona te-CoA transferase (35), invo lve an enzyme-CoA thioester intermediate and follow a substituted mechanism which can be identified by the set of initial velocity plots where 1/velocity vs. the reciprocal of one substrate yields a pattern of parallel lines (Figure 1-3). Evidence for the formation of the two anhydride intermediate s, in addition to the enzyme-CoA intermediate, has been obtained from model reactions with citramalate lyase (30). Figure. 1-3. Ping-pong kinetics of Family I glutaconate-CoA tran sferase (GCT) from Acidaminococcus fermentan Taken from Buckel 1981 (35).

PAGE 27

27 Propionate CoA transferase (EC 2.8.3.1) The first reports of catalysis of the reversible transfer of CoA to vari ous fatty acids was in cell-free extracts from Clostridium kluyveri (234), but propionate CoA transferase from C. propionicum was the first purified and reported to have a tetrameric quaternary structure comprising four identical 67 kDa subunits (214). Propionate CoA transfer ase is an important enzyme in the nonrandomizing alanine fermentation pathway of C. propionicum : alanine is converted to ammonia and pyruvate which is redu ced to (R)-lactate, ac tivated as (R)-lactoylCoA, and subsequently reduced to propionate. Propionate CoA transferase activates lactate as the CoA thioester using propionyl-CoA as the CoA donor (214). The enzyme is also implicated in the methylmalonyl-CoA pathway for the propionate-oxidizing metabolic pathway of Pelotomaculum thermopropionicum (140, 211). Glu-324 was identi fied as the active site carboxylate by MALDI-TOF MS of enzy me incubated with priopionylCoA, labelled with either borohydride (-14 Da) or hydroxylamine (+15 Da), a nd subsequently proteolytically digested individually by chymotrypsi n, endoprotease-AspN, endoprotease-GluC, or trypsin (218). Further, propionate CoA transferase lacks the (S)ENG consensus motif shared by Family I CoA transferases. Succinyl-CoA:oxalate CoA transferase (EC 2.8.3.2) Quayle has done extensive study on the facultative autotroph Pseudomonas oxalaticus now called Cupriavidus oxalaticus (249), and its m etabolism of oxalate and formate (191-193). Limited characterization of cell lysates suggest ed the presence of a CoA transferase that reversibly transfers CoA between oxalate and succinate. Acetyl-CoA:malonate CoA-transferase (EC 2.8.3.3) Bacterial growth on m alonate genera tes the end products acetate and CO2 (for a review, see Dimroth (64)). The malonate decarboxylase complex in Pseudomonas ovalis comprises five

PAGE 28

28 subunits ( ) (44). The 60 kDa -subunit has malonate-CoA transf erase activity (43); CoA is transferred from actetyl-CoA to form malonyl-C oA which is subsequently decarboxylated (100). Succinyl-CoA:3-ketoacid CoA tran sferase (SCOT; EC 2.8.3.5) SCOT activates acetoacetate by tran sferring CoA from succinyl-CoA (236) and is an essential enzyme in ketone-body metabolism. A cetoacetyl-CoA is catabolized to acetyl-CoA which can enter the TCA cycle or fatty-acid me tabolic pathway. Bacterial SCOT comprises and -subunits that form a heterodimer (54). Mammalian SCOT from sheep kidney and rat brain exist as homodimers (207, 221). Pig heart SCOT has been reported to exist as a homodimer (71, 106), as well as a homotetramer that dissociates slowly to homodimer in high potassium chloride (202). However, each monomer comp rises two domains corresponding to the and -subunits in other Family I CoA transferases (54, 183). Initi al velocity studies and treatment with sodium boro[3H]hydride show that SCOT catalyses a ping-pong reaction where an enzyme-CoA intermediate exists with CoA cova lently bound to the enzyme through the -carboxylate of a glutamate residue at each activ e site in the protein (22, 106, 207, 229). Isotope labelling experiments where SCOT was incubated with [18O4]-succinate and acetoacetyl-CoA showed reversible incorporation of 18O into some oxygen-containing gr oup on the enzyme (14). This residue in pig heart SCOT has been iden tified as Glu-305 by mass spectrometry and the adventitious autolytic reaction that occurs with the thioesters of -carboxylates through a 5oxyprolyl intermediate (115, 201, 264). Because of its relative stability (the rate constant for hydrolysis has been calculated as 0.10 min-1 at pH 8.1), the enzyme-CoA intermediate has been the target of several lines of research. El ectrospray mass spectrometr ic analysis of SCOT identified peaks consistent w ith free SCOT, enzyme-CoA interm ediate after incubation with either acetacetyl-CoA or succinyl-CoA, and th e primary alcohol expected upon treatment with sodium borohydride (156). More interestingly, further experiments provided compelling

PAGE 29

29 evidence for half-sites reactivity, where only on e active site per dimer was required for full activity. Jencks and coworkers have us ed various hydroxylamine and hydroxamic acid derivatives to probe the nature of the reaction mechanism (187). There are currently five solved X-ray crystal structures archiv ed in the RCSB Protein Data Bank: three of SCOT at 2.5 (1m3e), 2.4 (1o9l), and 1.7 (1ooy) resolution, respectively (13, 53), and two SCOT deletion variants where residue s 249-254, a region easily cleaved by proteolytic enzymes, were removed (1ope and 1ooz) (53). 3-Oxoadipate CoA-transferase (EC 2.8.3.6) Oxoadipate CoA transferase catalyses the transfer of CoA from succinyl-CoA to ketoadipate, and the product, -ketoadipyl-CoA is subsequently converted to acetyl-CoA and succinyl-CoA by a thiolase in th e degradation of aromatics (132) These activities were first isolated in the cell-free lysate of Pseudomonas fluorescens (132). Homogeneous proteins from both Pseudomonas and Acetinobacter have an 22 oligomeric structure as determined by both gel filtration and gene analysis (183, 268). The ge nes for the two subunits ( pcaI and pacJ ) are separated by only 8 base pairs in Pseudomonas putida and it is proposed that a gene fusion event may explain the homodimeric structure of mamm alian succinyl-CoA transferase (183). The transferase from Pseudomonas sp. strain B13 is involved in degradation of aromatics and chloroaromatics (131). KM values for 3-oxoadipate and su ccinyl-CoA were 0.4 and 0.2 mM, respectively, at a pH optimum of 8.4. The B13 tr ansferase is a heterotetramer of the type 22 with an overall size of 120 kDa and subunits with molecular masses of 32.9 and 27 kDa. Succinyl-CoA:citramalate CoA -transferase (SmtAB; EC 2.8.3.7) Sm tAB (87) is proposed to be involved in CO2 fixation by Chloroflexus aurantiacus, a thermophilic green nonsulfur bacterial phototroph. The enzyme has been purified; it appears to

PAGE 30

30 exist as a heterohexamer (44 and 46 kDa subunits). Similar enzymes in Pseudomonas sp. and Micrococcus sp. activate itaconate to itaconyl-CoA which is converted to acetyl-CoA and pyruvate in a series of steps (49, 50). Acetate CoA-transferase (AA-CoA transferase, atoDA; EC 2.8.3.8) Acyl-CoA:acetate CoA-transferase activ ity was detected as early as 1968 in E. coli (250) and is associated with short fatty acid, e.g. butanoate and propanoate, metabolism. The transferase and a CoA lyase, both members of the ato operon, are upregulated when E. coli is grown on acetoacetate (184). Two subunits of molecular mass 26 kDa (atoD) and 24-26 kDa (atoA) compose the 44 hetero-octameric protein (231). In an experiment similar to that of Solomon and Jencks (229), the catalytic glutamate residue on the subunit was detected by NaBH4 treatment after incubation with acyl-CoA (231). The apparent KM for acetyl-CoA with acetoacetate and KM for acetoacetyl-CoA with acetate were 0.26 mM and 35 M, respectively. Butyrate-acetoacetate CoA-transferase (EC 2.8.3.9) Butryate-acetoacetate Co A transferase was firs t purified from the acetoacetate degradation operon ( ato ) in constitutive E. coli (232), then later from lysine-fermenting Clostridium SB4 (12) and solventogenic (acetone) Clostridium acetobutylicum ATCC 824 (261). The enzyme is important in detoxification of the medium from acetate and butyrate fermentation products. Enzymes from all three organisms were reported to comprise 44 hetero-octameric quaternary structure with subunits of about 23 and 25 26 kDa each. Acetyl-CoA: glutaconate CoA -transferase (GC T; EC 2.8.3.12) Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F. fusiformis metabolize glutamate through the hydroxyglutarate pathway involving many transformations carried out at the CoA-ester le vel (31, 32). Like propi onate CoA transferase, glutaconate CoA transferase does not have th e (S)ENG motif. Recombinant proteins from

PAGE 31

31 Acidaminococcus fermentans have been overexpressed in E. coli and the active protein exists as 44 hetero-octamer where the two subunits have molecular masses of 35.5 kDa and 29 kDa respectively (35, 162). Initial velo city studies showed that the enzyme uses a ping-pong kinetic mechanism (35) and peaks of m/z consistent with the enzyme-CoA intermediate have been detected by MALDI-TOF mass spectro metry of enzyme incubated with only glutaryl-CoA (217). Glu-54 of the -subunit was identified as the catalyt ic amino acid residue by reducing the enzyme-CoA thioester with sodium boro[3H]hydride and detecti ng 2-amino-5-hydroxy[5-3H]valeric acid in a peptide generated by trypsin proteolysis (35, 162). Si te-directed mutagenesis experiments where Glu54 was replaced with alanine or aspa ragine completely abolished all activity while conversion to glutamine retained 1% of wild type activ ity (164). Intriguingly, when the E54Q variant was incubated with substrat es for 40 hours at 37 C nearly wild-type activity was restored. The enzyme has been converted from a CoA transferase to a thioester hydrolase by replacing Glu-54 with aspart ate (163). Oxygen exchange between [18O2]-acetate, glutaconate CoA transferase, and glutaryl-CoA was further proof of the Family I mechanism (217) (see Figure 2-1). In addition, the E54D hydrolase variant did not undergo 18O uptake into the aspartate carboxylate. The firs t X-ray crystal structure (PDB 1POI) of a CoA transferase was solved for GCT (119). Family II CoA Transferases Fam ily II transferases involve a ternary comple x and yield a pattern of intersecting lines in the double-reciprocal plots when e ither substrate concentration is varied (see Figure 1-5). The transferase -subunit of citrate or citramal ate lyase complexes facilitate the direct transfer of CoA from acetate to citrate or citramalate without any covale nt bond forming between enzyme and substrate (34, 63). The Co A cofactor is covalently boun d to the both enzymes by a

PAGE 32

32 phosphodiester linkage from Ser-14 to 2 -(5 -phosphoribosyl)-3 -dephospho-CoA (19, 61, 200, 226) (see Figure 1-4). Citrate CoA-transferase (EC 2.8.3.10) Citrate lyase (EC 4.1.3.6) catalyses the reversible cleavage of citrate to yield oxaloacetate and acety l-CoA used in fatty acid synthesis. Citrate CoA transferase is the largest subunit ( ) of the three-subunit citrate lyase co mplex. Most biochemical charac terization has been carried out by the enzyme purified from Klebsiella pneumoniae (formerly Klebsiella aerogenes). Neither initial velocity studies performed with varied ci trate concentrations and varied fixed acetyl-CoA concentrations nor with varied citryl-CoA and varied fixed ac etate showed the parallel line pattern expected in the double-reciprocal plots of ping-pong kinetics (65). Figure. 1-4. Prosthetic co factor of citrate lyase 2 -(5 -phosphoribosyl)-3 -dephospho-CoA is bound to Ser-14 of the ACP subunit by the ribose 5phosphate linkage. Further evidence for no enzyme-CoA intermed iate includes: no is olable enzyme-CoA intermediate upon incubation of citrate CoA tran sferase with acetyl-CoA; no enzyme inhibition, nor acetyl-CoA hydrolysis, were detected when an enzyme/acetyl-CoA mixture was treated with excess borohydride; and, no increase in radiolabel was detected in aceta te measured after the enzyme and [14C]-acetyl-CoA were incubated together (6 5). Citrate blocks the reacetylation of the deacetylated lyase with ac etic anhydride which has been shown to be an intermediate substrate analogue for citramalate lyase (30, 65).

PAGE 33

33 Citramalate CoA-transferase (EC 2.8.3.11) Citram alate lyase (EC 4.1.3.8) catalyses the cleav age of citramalate to pyruvate and acetate and comprises six copies each of three different proteins: -subunit, acyl-carrier protein (ACP); -subunit, the citramalate CoA transferase; and -subunit, citramalyl-CoA lyase (EC 3.1.2.16) (62). Citramalate CoA tr ansferase purified from Klebsiella aerogenes catalyses the transfer of the thio-acyl carrier protein from (3S)-citramalylthio-acyl carrier protein to acetate to generate citramalate and acetyl-thio-acyl carrier protein (65). The -subunit lyase catalyses the cleavage of citramalyl-CoA to pyruvate and acetyl-CoA in a Mg+2-dependent reaction. As seen in Figure 1-5, there is no indication that the kinetics follo w a ping-pong mechanism when either substrate is varied. Interestingly, the enzyme purified fr om the citrate lyase protein complex catalyses the acetyl-CoA:citramalate CoA tran sferase reaction more efficiently. Treatment with borohydride did not inactivate the transferase, and no enzyme-s ubstrate intermediate could be isolated. The Figure. 1-5. Double-reciprocal plots of initial velocity data consistent with an ordered mechanism in Family II citram alate-CoA transferase from Klebsiella aerogenes Taken from Dimroth 1977 (65).

PAGE 34

34 subunits of citrate lyase and citr amalate lyase are so similar that active hybrid enzyme complexes have been formed from the and -subunits of citramalat e lyase and the ACP -subunit from citrate lyase (62). Family III CoA Transferases Previous work, including site -directed m utagenesis, stea dy-state kinetics, and product inhibition studies on recombinant FRC from Oxalobacter formigenes the representative enzyme of Family III CoA transferases, have demonstrated that Asp-169 is a vital catalytic residue and that the reaction proceeds through a ternary complex where formyl-CoA binds first followed by oxalate followed by release of formate prior to oxalyl-CoA in an ordered sequential mechanism (124). Thus, unlike the Family I ping-pong kine tic mechanism, formate must remain in the active site until chemistry is complete. However, this does not exclude the possible formation of an enzyme-CoA thioester intermediate like that of glutaconate-CoA tran sferase during catalysis (35, 217). Indeed, an X-ray crystal structure in the study showed electron de nsity consistent with an oxalyl-aspartyl mixed anhydride in the active site evidence that just such a covalent enzymesubstrate intermediate may exist along the react ion pathway. Theoretical reaction mechanisms are shown in Figure 1-6.; Mechanism 1 in Figure 1-6 is a viable proposal conditional that formate remains in the active site. Two other li kely mechanistic pathways exist: Mechanism 2, the direct attack of the formyl-CoA carbonyl by oxalate, in a mechanis m analogous to that of Family II transferases, followed by the CoA thiolate attacking oxalate; and, Mechanism 3 proposed by Jnsson et al. (124) where nucleophilic attack of the formyl carbonyl by Asp-169 is followed by attack of oxalate on the resultant aspartyl-formyl mixed anhydride to form an aspartyl-oxalyl mixed anhydride intermediate wh ich is finally attacked by CoA, which has remained in the active site generating the final product.

PAGE 35

35 Figure. 1-6. Possible reaction mechanisms for FRC. Mechanism 1 corresponds to the Family III CoA transferase pathway that exhibits ping-pong kinetics. Mechanism 1A is the ordered sequential variation of Mechanism 1 where formate must remain in the active site until oxalyl-CoA is formed. Mechanism 2 is analogous to the enzyme stabilized direct-attack mechanism of Family II CoA transferases. Mechanism 3 is a third proposed mechanism that avoids the enzyme-CoA thioester intermediate of Mechanism 1 (124). Enzyme aspartyl-oxa lyl mixed anhydride intermediates in the reaction pathways are highlighted. Putati ve tetrahedral intermediates have been omitted to save space. Formyl-CoA transferase (FRC; EC 2.8.3.16) Form yl-CoA transferase activity wa s first suggested by studies of Pseudomonas oxalaticus (192) and limited kinetic characterization was carried out on native fo rmyl-CoA transferase (FRC) isolated from Oxalobacter formigenes (9). FRC has been detected in Enteroccus faecalis and identified by Western blotting using antibodies against frc from O. formigenes (111).

PAGE 36

36 The frc gene has since been cloned from O. formigenes; recombinant wild-type FRC has 428 amino acids, pI of 5.2, a calculated monomeric mass of 47.3 kDa, and exists in solution as a homodimer (225). Steady state kinetics a nd product inhibition studies by Jnsson et al. established that the enzyme exhibits an ordered sequential Bi-Bi kinetic mechanism where formyl-CoA binds first followed by oxalate (124). Subsequent to the chemistry step, formate is released first followed by oxalyl-CoA. FRC was in itially reported to be a monomer (9), but later crystal structure data suggeste d that the catalytic subunit is dimeric (197). Size-exclusion chromatography experiments have since confirmed that active FRC exists as the dimer (124). High resolution X-ray crystal structures of YfdW, a putative formyl -CoA transferase in E. coli have been published independently by two groups (92, 97). YfdW, identified by structural genomics as part of the effort to annotate the many unknown genes in E. coli (251), shares 63% identity with FRC (see Figure 1-8) and is proposed to be a formyl -CoA transferase (97). YfdW is the gene product of the yfdXWUVE operon, where the yfdX gene is under the control of the EvGAS two-component regulatory system (see Figur e 3-3), and has been implicated in acid resistance in E. coli (169). In addition, frc and oxc genes also appear to be involved in acid protection mechanisms in Lactobacillus acidophilus Variants with frc and oxc deletions challenged by exposure to pH 3.5 showed a reduced ability to surviv e compared to the wild type (7). Interest in possible probiotic treatments for oxalate stones has also prompted study of formyl-CoA transferase activity in L. acidophilus. Of the 60 Lactobacillus strains evaluated, several showed high oxalate degrading activity (247). BaiF Som e prokaryotes are able to metabolize bile acids utilizing the bile acid inducible ( bai ) operon (see the review by Ridlon et al (199)). BaiF, the protein product of the baiF gene, is

PAGE 37

37 proposed to mediate CoA transfer between severa l cholic acid derivatives, i.e. CoA from 3dehydro-4-cholenoic acid to cholic acid, 3-dehydro-4-chenodeoxycholenoic acid to chenodeoxycholic acid, or 3-dehydro-4-ursodeoxycholenoic acid to ursodeoxycholic acid. Baif has been isolated from the intestinal anaerobe Eubacterium sp. strain VPI 12708 (258). The recombinant protein has been overexpressed in E. coli (266). The KM and Vmax for the hydrolysis of cholyl-CoA were ca. 175 M and 374 mol/min mg, respectively. Although the 47.5 kDa protein has only been demonstrated to have bile acid-CoA hydrol ase activity, its gene sequence does not contain a signatu re motif found in many thioeste rases (266); it is proposed to be a CoA transferase and play an important role in ATP-independent recycling of CoA thioesters and, thus, conserving energy (102). Succinyl-CoA:( R )-ben zylsuccinate CoA-transferase (BbsEF; EC 2.8.3.15) BbsEF is involved in the anaerobic degrad ation pathway of aromatic compounds in bacteria (for a review on anaerobic degrad ation of aromatics s ee Heider (103)). Thauera aromatica metabolizes toluene as well as phenol, p-cresol, anthranilate, and phenylalanine (104). In toluene metabolism, toluene is oxidized to benzoyl-CoA and succinyl-CoA in a proposed six step pathway. In one important step, CoA is transferred from succinylCoA to benzylsuccinate to form benzylsuccinyl-CoA. BbsEF has been purified from T. aromatica grown on toluene (149). BbsE (44 kDa) and BbsF (46 kDa) the two subunits that compose the active 22 protein, are products of the the bbs operon ( -oxidation of benzyl-succinate ) (148). Double-reciprocal plots of the intial velocity kine tics are consistent with an ordered sequential mechanism. The KM values for the reverse reaction were 40 M for 2and 3-( R )-benzylsuccinyl-CoA and 160 M for succinate. BbseF could be inactivated in a be nzylsuccinyl-CoA concentration dependent manner with high concentrations (1 and 10 mM) NaBH4, but lower concentrations (0.1 mM) and hydroxylamine had no effect.

PAGE 38

38 Crotonobetainyl/ -butyrobetainyl-CoA:carnitin e CoA-transferase (CaiB) Carnitine is an organic molecule im portant in long-chain fatty acid transfer across the mitochondrial inner membrane in humans. E. coli is able to utilize carnitine as an osmoprotectant (128) and as a terminal elect ron acceptor under anaerobic conditions (138). Carnitine is metabolized to -butyrobetaine by a three-enzyme system comprising CaiA, CaiB, and CaidD (74, 75, 190). Carnitine is activated by the CoA transferase CaiB; carnitine is added to CoA from -butyrobetainyl-CoA to form carnitinyl-CoA and -butyrobetaine. Carnityl-CoA is reduced to -butyrobetainyl-CoA by CaiD, a dehydratase, and CaiA, reductase. The end result is a 2 electron reduction coupl ed to the regeneration of -butyrobetainyl-CoA which is used for the next cycle. CaiB also catal yzes transfer from crotonyl-CoA (74). A fourth enzyme, CaiC, is thought to prime the cycle by ATP-dependent formation of carnityl-CoA (102). Dimeric X-ray crystal structures of CaiB from E. coli, CaiB/CoA, and CaiB/CoA /Crotonyl-CoA complexes have been solved (196, 235) and these structures share high structural similarity with FRC structures. CaiB is missing the te traglycine loop of FRC and has a larger active site consistent with its larger substrates. The small and large domains app ear to close together upon substrate binding (196, 235). Cinnamoyl-CoA: phenyllactate CoA -transferase (FldA; EC 2.8.3.17) Phenyllactate CoA transferase is one of three eny zmes in the heterotrimeric phenyllactate dehydratase complex important in L-phenylalanine fermentation in strictly anaerobic Clostridium sporogenes (210). FldA forms activated phenyllactyl-CoA by transferring CoA from cinnamoylCoA. The remaining enzymes in the [4Fe-4S]2+-containing complex, FldB and FldC, remove water to regenerate cinnamoyl-CoA and (E)-cinnamate. FldA has a molecular weight of 46 kDa and when purified alone appears as a dimer (97 kDa) in gel filtra tion analysis (60). The lines on the double-reciprocal plot of 1/ v vs 1/[phenylpropionate] and 1/[c innamoyl-CoA] intersected in

PAGE 39

39 the 2nd quadrant consistent with an ordered sequential ki netic mechanism. Micromolar KM values for both substrates were reported. Fl dA activity was reduced 50% when incubated with cinnamoyl-CoA and treated with NaBH4, but hydroxylamine had no effect. 2-Hydroxyisocaproate CoA transferase (HadA) HadA is one five enzymes im portant in the leucine fermentation pathway of Clostridium difficile a gram positive, non-spore forming strict anaerobe implicated in antibiotic-related diarrhea and psueodmembranous colitis in man (25, 188, 230). In the fermentation pathway where leucine is both oxidized to 3-methylbut yrate and reduced to isocaproate (28, 73). Recombinant HadA has been cloned from C. difficile expressed, and purified by streptavidinaffinity chromatography (137). Variants where the cognate Asp-171 is changed by site-directed mutagenesis to either an N or A showed activ ity 2000 times less than the wild-type enzyme. This compares to the D169A FRC variant where activity was reduced ~13 00-fold, but conflicts with the D169E variant that was completely inactive (124) HadA showed up to 88% inactivation with NaBH4 and 94% with NH2OH when the enzyme was incubated with 200 M (R)-2-hydroxyisocaproyl-CoA, but no inactivation in the absence of substrate (137). Wild-type activity could be restored by incubating inac tivated HadA with 2hydroxyisocaproate and isocaprenoate for 20 hours at pH 8.0 and 25C. Fu rther, HadA could be activated by incubation with the two acids, to a maxima l activation of 120% of wild-type activity before returning to 100%. However, in contrast to GCT, the D171N and D171N variants could not be induced to recover from inactivation. MALD I-TOF mass spectrometric analys is of the tryptic peptide showed only m/z of the untreated enzyme suggesting that Asp-171 may act in an alternate manner than Asp-169 in FRC.

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40 -Methyl-CoA racemase (MCR and Amacr) Amacr, or -methyl-CoA racemase, catalyzes the racemization of the (S)and (R)enantiomers of a variety of -methyl-branched chain-Co A substrates in the -oxidation of fatty acids (107) bile acid synthesi s (206), and ibuprofen bioactiva tion (37). The protein has been purified from rat and human liver (212, 213). Although, the catalytic oligomeric form is unknown, the monomer has a molecular mass of 45 kDa. It is proposed to be a member of Family III CoA transferases due to sequence similarity with other members (102). X-ray crystal structures have been reported for th e homologous recombinant apoenzyme from Mycobacterium tuberculosis (MCR) (210) and for substrate-enzyme complexes with several substrates, including ibuprofenyl-, methylmyristoyl-, and acetyl-CoA, bound to the protein (20). On the basis of the active site geometry and kinetic results from several MCR variants pr epared by site-directed mutagenesis (210), the two cataly tic acid-base residues have been identified as His-126 and Asp156 (analogous to Asp-169 in FRC). KM and Vmax for the release of 3H from [2-3H]pristanoylCoA were 41 M and 214 mol/min mg, respec tively. Thus, it appears that MCR (and the mammalian Amacr enzymes) with its high sequence and structural similarity has taken the FRC scaffold and evolved a new enzymatic activity. Conserved Structure of Family III CoA Transferases Since Heider categorized the CoA transferases based on sequence sim ilarity (102), X-ray crystallography studies have revealed that Family III CoA transferases also share a remarkable 3dimensional tertiary structure that is more conserved than expected from the only ca. 25% sequence similarity. Family III transferases are di mers (BbsEF is reported as a tetramer); FRC, homologue YfdW, CaiB, and MCR comprise two subunits that thread through one another like links in a chain (97, 197, 210, 235). The N-termi nus begins in the large domain, travels down

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41 Figure. 1-7. Cartoon representations of Family III CoA transferases. E. coli YfdW (pdb 1pt5), Mycobacterium tuberculosis -methylacyl-CoA racemase (MCR; pdb 1x74) (210), E. coli crotonobetainyl-CoAcarnitine CoA transferase (C aiB; pdb 1xa3) (235), and O. formigenes FRC (Protein Data Bank accession number 1p5h). Monomers in FRC are shown in black and white. CoA is shown in spheres bound in the active site, at the interface of the large and small domains, just above the catalytic Asp-169 residue, sticks through a linker domain to the small domain and then back up to te rminate in the large domain. The four structures were aligned and a structural sequence comparison was constructed (Figure 1-7). The large domains have relatively hi gh similarity, while more diversity is apparent in the small domains. Substrate specificity, rang ing from formyl-CoA to bulky steroids in MCR,

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42Table 1-1. Summary of Family II I CoA Transferases. Structural data are summar ized for the known Family III CoA transferases. AA refers to the number of amino acids in the monomer and %FRC is sequence identity with FRC. Name Description X-ray Structure Quaternary Structure Subunit MW (kDa) AA % FRC Organism/Pathway BaiF (266) cholyl-CoA hydrolase; proposed CoA transferase --47.5 426 27 anaerobic bile acid transformation BbsEF (149) succinyl-CoA: (R)-benzylsuccinate CoA transferase -22 44 and 45 410, 409 23, 28 first step of anaerobic toluene catabolism in Thauera aromatica CaiB (79) -butyrobetainyl-CoA: (R)-carnitine CoA transferase apo, CoA homodimer 405 24 anaerobic carnitine metabolism in E. coli and Proteus sp. FldA (137) (PLCT) (E)-cinnamoyl-CoA: (R)-phenyllactate CoA transferase -homodimer, heterotrimeric complex with FldABC 46 405 25 Stickland-fermentation in Clostridia ( sporigenes and difficile) FRC (124) formyl-CoA: oxalat e CoA transferase multiple ligands (see text) homodimer 47.2 428 100 oxalate metabolism in Oxalobacter formigenes HadA (137) ( R)-2-hydroxyisocaproyl-CoA: ( E)-2isocaprenoate CoA transferase homodimer 43 25 leucine fermentation in Clostridum difficile MCR (210) (Amacr) -methylacyl-CoA racemase multiple ligands (see text) homodimer 39 (89) 360 23 bile acid synthesis in Mycobacterium tuberculosis YfdW (97, 245) formyl-C oA transferase apo, AcCoA homodimer 48.3 415 60 E. coli

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43 appears to result from variation in the residues at the interface of the large and small domains, as well as from large differences in both the linke r and small domains. Another key difference is that the formyl-CoA transferases all share a tetraglycine loop th at is proposed to protect the active site and prevent hydrolysis of labile formyl-CoA (197). Research Objectives Three Fam ilies of CoA transferases, based on sequence similarity and kinetic mechanism, have been reported. Family I transferases use a ping-pong me chanism with an enzyme-CoA intermediate. Family II transferases form a tern ary complex that involves direct attack of the incoming receptor acid on to the donor acyl-CoA substrate. So far, the kinetic mechanism of Family III CoA transferases, of which FRC is th e representative and best characterized enzyme, is not completely understood. It is known that FRC forms a ternary complex with formyl-CoA binding first followed by oxalate in a sequential mech anism. X-ray crystal structures of FRC and OXC show variations in the conf ormation of peptide loops near th e active site. It is proposed that these motions play an important role in catalysis. Finally, the gene product of yfdW in E. coli has been expressed, purified, and crystallized. This enzy me is a putative formyl-CoA transferase based on sequence homology a nd structural similarity; however, E. coli has no reported ability to metabolize oxalate. Conf irming that YfdW is in fact a formyl-CoA transferase would be validation of structural genomics. The goals of this project were to 1. Determine if the mechanism of FRC includes an enzyme-substrate intermediate like that of Family I transferases, and if so, to determine the nature of the intermediate species; 2. Examine the nature of the tetraglycine loop in FRC and the C-terminal loop in OXC by site-directed mutagenesis and st eady steady state kinetics; and, 3. Test YfdW for formyl-C oA transferase activity.

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Figure. 1-8. Structure-based sequence alignment of Family III CoA transferase family members. This alignment, following page was generated by the superimposition of the crystal structures of FRC (pdb 1p5h), YfdW (pdb 1pt5), MCR (pdb 1x74) (210), and CaiB (pdb 1xa3) (235). -Helical and -strand secondary stru ctural elements are colored orange and blue respectively. Specific residue s discussed in the text are highlighted. Asterisks indicate re sidues identical in all four transferases. Taken from Toyota 2008 (245).

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45

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46 CHAPTER 2 CATALYTIC MECHANISM OF FORMYL-COA TRANSFERASE1 Introduction CoA-transf erases catalyze reversible transfer reactions of coenzyme A carriers from CoAthioesters to free acids. Most members of the enzyme class are grouped into the well characterized Family I and II Co A-transferases, but recently a third class of enzymes was identified, differing in sequence and three-dimens ional structure to the other CoA-transferases (102). Members of this third class are mostly from bacteria, but puta tive genes have been identified in Archaea and Eukarya as well. Fa mily III enzymes are known to be involved in Stickland fermentation and the metabolism of oxalate, carnitine, toluene, and bile acid. The first Family III CoA-transferase identified wa s formyl-CoA transferase (FRC) from Oxalobacter formigenes (102). The first Family III CoA-transferase identi fied was formyl-CoA transferase from Oxalobacter formigenes (102). Formyl-Coenzyme A transfer ase is the first of two enzymes involved in oxalate degradation in the gut-dwelling bacterium O. formigenes (1). Formyl-CoA transferase catalyzes the transfer of a CoA moiety between form yl-CoA and oxalate and thereby activates oxalate in the form of oxalyl-CoA ( 9, 191). Oxalyl-CoA is then decarboxylated by the second enzyme on the pathway, oxalyl-CoA decarboxylase, which regenerates formyl-CoA (10, 15). Oxalate catabolism has a central role in O. formigenes where oxalate serves as vital source of energy as well as carbon (1, 51). The crystal st ructure of formyl-CoA tr ansferase revealed an interesting new fold composed of two subunits li nked together in an in terlocked dimer like two rings of a chain (197) (Figure 2-3). This fold proved to be characteristic for the Family III 1 Reproduced in part with permission from Journal of Biological Chemistry, Vol. 283 (10), Berthold, C. L., Toyota, C. G., Richards, N. G., and Lindqvist, Y. Pages 6519-6529. Copyright 2008 Journal of Biological Chemistry

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47 family as the crystal structures of the formyl-CoA transferase ortholog in Escherichia coli coded by the yfdW gene (97) and the close homolog -butyrobetaine-CoA:carnitine CoA transferase (196, 235) were determined. The Family I Co A-transferases, includ ing mostly enzymes involved in fatty acid metabolism, have a well established mechanism described in Figure 2-1 ( Mechanism 1 ). The formation of covalent intermedia tes involving a glutamate residue of the enzyme results in a classical ping-pong mechan ism with exchanging substrate product glutamylacyl anhydrides and -glutamyl-CoA thioesters (217, 229). The -glutamylCoA thioester was Figure. 2-1. Summary of kine tic mechanisms for the three known CoA transferase Families. Mechanism 1 is the ping-pong scheme show n for GCT. The enzyme-CoA covalent intermediate is highlighted. Mechanism 2 is the transferase re action with acetyl-CoA and citrate in citrate lyase. Mechanism 3 is the 2004 proposed kinetic mechanism for Family III CoA transferases from (124). Putative tetrahedral intermediates have been omitted to save space.

PAGE 48

48 first identified in a Family I transferase in 1968 by electrophoresis and chromatography studies with isotope-labeled borohydride (229) and was recently trapped in a crystallographic study, giving the first structural proof of its existe nce (196). The smaller group of Family II CoAtransferases catalyzes a partial reaction in the citrate and citramalate lyase complexes. These reactions do not include covalent enzyme intermediates, and the transfer of a dephospho-CoA, which is covalently bound to an acyl carrier prot ein (ACP) in the enzyme complex, is carried out through a ternary complex where a mixed anhydride is formed between the two acids during the transition state (Figure 2-1, Mechanism 3 ) (33, 63). During initial studies of the Family III CoA-tr ansferases, steady state kinetics showed that the reaction is not consistent wi th a ping-pong mechanism as in the Family I CoA-transferases. The mechanism was instead interpreted to pr oceed through a ternary complex, where both formyl-CoA and oxalate need to be bound to the enzyme before catalysis (60, 124, 149). The crystal structure of an aspartyl-oxalyl mixed a nhydride led to the suggestion that the reaction was initiated in the ternary complex with both subs trates by the formation of an aspartyl-formyl anhydride and CoA-S-. The CoA-Swas then kept bound in the activ e site as a spectator while oxalate replaced formate, before attacking th e aspartyl-oxalyl anhydride yielding oxalyl-CoA (124). A freeze-trapped crystal stru cture reveals that the enzyme-aspartyl-CoA thioester intermediate is also formed during catalysis by formyl-CoA transferas e, a finding leading to reassessment of the catalytic mechanism of Family III CoA-transferases. The mechanistic investigation is complemented w ith the crystal structure of a trapped aspartylformyl anhydride similar to the previously charac terized aspartyloxalyl anhydride complex (124) and two mutant protein structures, where one contains the complex with -aspartyl-CoA and oxa late. Central to the catalyzed reaction is a glycine-rich loop that adopts two different c onformations controlling

PAGE 49

49 the accessibility of the ac tive site. Mutations in the loop seri ously affect the activity, proving its importance during catalysis. A modified mech anism in concordance with all information obtained is proposed, where catalysis includes form ation of both the aspartyl-formyl and -oxalyl anhydrides and the -aspartyl-CoA thioester and where the carboxylate product remains bound to the enzyme until release of the acceptor thioester. Results Wild-Type FRC Activity By first determ ining the inhibitory effects of free CoA (Table 2-5), a ubiquitous contaminant resulting from the hydrolysis of fo rmyl-CoA, the kinetic parameters for FRC and mutant variants were improved (see Figure 2-2). The values obtained for FRC by this method were similar to previously reported values (124). Fortuitiously, the inhi bition constant for CoA ( KiCoA) was 16.7 0.7 M and high enough that there wa s little effect on th e original kinetic analysis. Enzyme-Aspartyl-CoA Thioester Complexes Crystals, produced with a precipitant mixt ure of polyethylene glycol and magnesium chloride, were soaked with formyl-CoA for 1, 5, and 10 min respectively and with oxalyl-CoA for 2, 4, and 10 min, respectively. In spection of the crystal struct ures from different soaking times revealed that all formyl-CoA soaked crys tals contained the same intermediate and all oxalyl-CoA soaked crystals contained the same intermediate, with no difference over time. The best data set of each, formyl-CoA soaked for 2 min and oxalyl-CoA soaked for 5 min, were used for further analysis. Close inspection of the freeze-trapped formyl-CoA and oxalyl-CoA intermediates, show that the formylas well as the oxalyl moietie s are cleaved off by the enzyme, and a covalent bond is formed between the carboxyl group of Asp169 and the thiol-group of the CoA carrier.

PAGE 50

50 Figure. 2-2. Double-reciprocal plot for the inhibition of FRC by free CoA against varied [formyl-CoA] at constant saturating [oxa late] = 77 mM showing lines fitted to the data by linear-regression methods. CoA concentrations were 1.5 M ( ), 11.5 M ( ), and 24.3 M ( ). Ki(CoA) of 16 .7 0.7 M was determined from the replot of KMapp/Vapp vs. [CoA] ( insert ). The resulting intermediates from the formyl-CoA and oxalyl-CoA soaks are thus highly similar and the dimeric structures superimpose with an r.m.s. deviation of 0.3 over 851 C atoms. An intriguing feature is that in bot h complexes, the two subunits of the dimer adopt different active site conformations with the pant etheine arm of the CoA molecule bound in different orientations (Figures 2-4 and 2-6). Several residues show di fferent conformations in the two subunits of the dimer. Tyr-139 is centrally positioned in the acti ve site and moves with the side chain hydroxyl group shifted approximately 3 allowing the tw o different orientations of the pantetheine moiety. Lys-137 is positioned on the same side of the CoA molecule and shows a shift of 4.5

PAGE 51

51 Figure. 2-3. Two formyl-CoA transferase monom ers displayed separately and in the dimer. The figure displays the structure of the -aspartyl-CoA thioester derived from oxalylCoA. The C trace and CoA molecules (shown as stick models ) are coloured by Bfactor, with blue representing the lowest B-factor and red the highest. The arrow indicates the central hole in Monomer B. Modified from Berthold 2008 (18). at the side chain amino group. Residues Arg-38 and His-15 also adopt different side chain conformations in order to ad apt to the two CoA conformations. Finally, Gln-17 takes on two different rotamer conformations, with a posit ion behind Asp-169 in subunit A and above the thioester bond in subunit B. As was observed already for the apo-enzyme the side chain conformation of Trp-48 is flipped 90 between the two monomers and the glycine loop (258GGGGQ261) then assumes the open and closed conformations in subunits A and B, respectively (Figure 2-4). Neither formate nor oxalate was detected in the active site. Interestingly, subunit A contains density interpreted as one chloride ion bound behind the active site residue Asp-169, at a position occupied by residue Gln-17 in the ot her subunit, and subunit B has density interpreted as two chloride ions bound, one on each side of th e pantetheine arm of CoA, where one chloride ion is interacting with the closed glycine loop and the other with the main chain amides of Gln17 and Ala-18 (Figure 2-4).

PAGE 52

52 Figure. 2-4. Stereoview of the overlay of the two active site conformations of the -aspartylCoA thioester complex. The resting conformation A, shown in grey with one bound chloride ion in orange was observed in subunit A with an open glycine loop. The other activated conformation in cyan shows two bound chloride ions (green ) and subunit B with a closed glycine loop. Note the two rotamer conformations of Q17. Taken from Berthold 2008 (18). In both complexes Val-16 is positioned in the disallowed or generously allowed part of the Ramachandran plot in both monomers which wa s also observed in the complex of FRC with bound CoA reported earlier (197). In spection of the structures reve als that Val-16 has a strained conformation in order to f it the CoA moiety. In the -aspartyl-CoA thioester complex, Glu-140 is in the disfavored part of the Ramachandran plot in subunit B, which can be explained by the structure adopted by the adjacent residue Tyr-139, enforced by the different conformation of the CoA-moiety in that subunit. The B-factors show a clear difference in the region of the small domain comprising the two loops 230-247 and 282-347 between the two subunits (Figure 2-3).

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53Table 2-1. Data collection and refinement statistics. Values in parentheses represent the highest resolution shell. Data Collection -aspartyl-CoA thioester from formyl-CoA -aspartylCoA thioester from oxalylCoA Aspartylformyl anhydride Q17Aaspartyl-CoA thioester and oxalate G260A FRC Beamline ID14 eh1 ID14 eh1 I911-2 I911-2 ID23 eh2 Space group I4 I4 I4 I4 P43212 Unit cell a,b,c () 151.8, 151.8, 100.1 151.9, 151.9, 99.5 151.7, 151.7, 98.9 153.6, 153.6, 98.1 97.3, 97.3, 193.4 Resolution () 2.0 (2.11-2.0) 2.0 (2.11-2.0) 1.87 (1.971.87) 2.2 (2.32-2.2) 2.0 (2.11-2.0) Rsym 0.11 (0.39) 0.11 (0.30) 0.067 (0.36) 0.12 (0.57) 0.15 (0.50) Mn(I/ (I)) 8.7 (1.8) 10.1 (2.4) 15.9 (4.2) 9.3 (1.9) 7.2 (2.5) Completeness (%) 98.3 (94.3) 99.2 (97.2) 97.1 (81.1) 99.5 (100) 99.6 (99.9) Wilson B-factor 28 25 23 34 14 Refinement Resolution () 30-2.0 30.0-2.0 30-1.87 30-2.2 30-2.0 Reflections in working set 71459 74942 85088 54545 59799 Reflections in test set 3765 3910 4473 2894 3206 R-factor / R-free (%) 19.7 / 24.1 17.3 / 21.4 17.2 / 20.3 20.7 /24.8 16.9 / 21.5 Atoms modeled 7565 7565 7509 7197 7566 No. of amino acids / B-factor (2) 854 / 35.4* 854 / 29.7* 854 / 25.1* 854 / 33.0 854 / 12.5 Number of ligands / B-factor (2) 2 / 47.3** 2 / 42.4** 4 / 30.5** 3 / 41.3 0 / Number of waters / B-factor (2) 693 / 38.2 788 / 35.5 737 / 31.6 430 / 29.2 873 / 21.8 RMS Deviations from ideals Bonds () 0.007 0.008 0.008 0.008 0.009 Angles () 1.08 1.12 1.06 1.15 1.16 Ramachandran zone distribution (%) 92.1, 7.5, 0.1, 0.3 92.5, 7.2, 0.1, 0.1 91.8, 7.9, 0.3, 0 92.1, 7.6, 0.3, 0 92.0, 7.6, 0.3, 0.1 PDB Accession code 2vjl 2vjk 2vjm 2vjo 2vjn (includes amino acid part of residue 169) ** (includes CoA/formyl part of covalent complex at residue 169)

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54 Figure. 2-5. Mass spectrum of formyl-CoA transferase incubate d with formyl-CoA. The main peak of 47,927 Da corresponds with the covalent aspartyl-CoA thioester intermediate. No peak is observed at the molecular ma ss of the monomer (47,196 Da). Taken from Berthold 2008 (18). Figure. 2-6. Electron density maps of -aspartyl-CoA thioester and chloride ions. Annealed composite omit 2Fo-Fc electron density maps are contoured at 1 around the aspartyl-CoA thioester and chloride ions Subunits are labeled A and B. Figure courtesy of Catrine Berthold. Taken from Berthold 2008 (18).

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55 In subunit B this region is much more flexible, and in the -aspartyl-CoA thioester complex obtained from formyl-CoA, re sidues 286-316 have no interpreta ble electron density and are modeled with zero occupancy. Inspection of the crystal packing reveal s that the corresponding region of subunit A forms crystal contacts with the adjacent molecule while this region in subunit B is freely exposed to solvent. Inhibition of FRC by Chloride Ions and Glyoxalate The identification of chloride ions bound in the active sites was followed up by kinetic m easurements showing that chloride has an inhibitory effect on the transferase activity in FRC. Chloride is a weak competitive inhibitor against oxalate with Kic of 3 2 mM (Figure 2-7). Glycolate may act as an oxalate analogue and be a good tool for as certaining the oxalate binding Figure. 2-7. Double-reciprocal plot of competitive Clinhibition of FRC against varied [oxalate] (1.0 77.0 mM) at 21.4 M [formyl-CoA] and 10 M [CoA] with 8.8 nM enzyme. KCl concentrations were 5 mM ( ), 15 mM ( ), and 30 mM ( ). Kic = 3 2 mM was determined from the replot of KMapp/Vapp vs. [KCl] (insert ). Taken from Berthold 2008 (18).

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56 site in FRC. Thus, the effect of glycolate on FRC activity was determine d. Glycolate is also a competitive inhibitor against oxalate with Kic = 6 6 mM. Hydroxylamine and Sodium Borohydride Trapping Fa mily I CoA-transferases are inactivated by hydroxylamin e and sodium borohydride in the presence of donor CoA-thio esters (217, 229). In the Family I enzymes treatment with hydroxylamine gives formation of a hydroxa mate at the glutamate bound in the -glutamyl-CoA thioester while sodium borohydride reduces glut amyl-CoA to the corresponding alcohol. The effect of both these inhibitors on FRC preincubated with formyl-CoA at different concentrations were tested. Previous experiments on Family II I transferases have yiel ded ambiguous results. Table 2-2. Formyl-CoA dependence of inactiva tion of FRC (0.52 M) by hydroxylamine trapping under turnover conditions with saturating oxalate (77 mM) and varied concentration of formyl-CoA. [Formyl-CoA] (M) Residual Activ ity (%)Residual Activity (U/mg) 0.0 100 7 7.4 0.5 5.5 81 2 6.0 0.1 10.0 75 6 5.6 0.3 38.0 33 3 2.4 0.1 77.0 12 18 0.9 0.2 Table 2-3. Formyl-CoA dependence of inac tivation of FRC (0.26 M) by hydroxylamine and borohydride trapping in the absence of oxalate. [Formyl-CoA] (M) Hydroxylamine Residual Activity (%) Sodium Borohydride Residual Activity (%) 0.0 100 3 100 5 0.1 71 7 0.2 93 2 0.3 32 2 1.1 19 1 2.4 12 0.2 5.0 13 1.4 24.0 14.8 0.3 188.0 15 7 262.0 1 0.1

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57 The (E )-cinnamoyl-CoA:( R )-phenyllactate CoA transferase from Clostridium sporogenes was not inactivated by hydroxylamine and retain ed 50% activity when treated with NaBH4 (60). Activity of succinyl-CoA:( R )-benzylsuccinate CoA transferase from Thauera aromatica was also unaffected by hydroxylamine but could be reduced to 3.5% in the presence of benzylsuccinyl-CoA and 10 mM NaBH4 (149). The effect of both of these inhibitors on formylCoA transferase preincubated with formyl-CoA at different concentrations was examined. FRC incubated with oxalate and formyl-CoA was subsequently treated with hydroxylamine. As any activated acyl groups ar e expected to be trapped as hydroxylamine adducts, oxalyland formyl-acyl as well as acyl -thioester intermediates in the transferase reaction should be trapped. The enzyme showed a reduced activity after removing all small molecules (i.e. excess CoA and hydroxylamine) by gel filtration. Table 2-2 shows that the addition of 77 M formyl-CoA followed by hydr oxylamine reduces the activity about 88%. The same experiment performed without addition of oxalate also displaye d a trapping effect by hydroxylamine (Table 2-3). A clear dependence on formyl-CoA concentration was discovered for the inactivation of FRC by hydroxylamine. Trapping experiments with sodium borohydride to reduce possible thioester intermediates in FRC also led to reduced activity (Table 23). The resulting alcohol from the borohydride reduction has not been identified. Comparison with Previous FRC Complexes Previously determ ined structures of wild type FRC include the apoenzyme structure (pdb code: 1p5h) an inhibitory complex with CoA bound in the active site (p db code: 1p5r) and a structure where co-crystallization with oxalyl-Co A resulted in a crystal structure where CoA is bound in both subunits, but where one subunit also contains the aspartyl-oxalyl mixed anhydride (pdb code: 1t4c) Superimposition of thes e three existing structures with the -aspartyl-CoA

PAGE 58

58 thioester intermediate structures results in r.m.s. deviations of 0.4-0.5 over 854 C atoms for the dimer. The differences between the structures are mainly found in the flexible segments of the small domain in subunit B (residue 230-247 a nd 282-347) and among the active site residues that shift orientations in the two active sites. The orientation of the CoA moiety observed in subunit B of the -aspartyl-CoA thioester complex (Figur e 2-4 and 2-6B) has not been observed before and most probably represents a new state in the catalytic cycle. This conformation will be referred to as the "activated" conformation of CoA while the conformation in subunit A is described as the "resting" conformation. Aspartyl-Formyl Anhydride Complex A structure of FRC containing the aspartyl-form yl anhydride complex was obtained in the absence of chloride ions a nd oxalate upon flash-freezing a cr ystal 10 min after addition of formyl-CoA. At a resolution of 1.87 subunit A of the dimer was interpreted to contain the covalent -aspartyl-CoA thioes ter and subunit B the -aspartyl-formyl anhydride and free CoA (Figure 8B). The formyl group of the aspartyl -formyl anhydride was modeled at occupancy 0.6 to best fit the observed electr on density. In the subunit containi ng the trapped mixed anhydride, the active site is nicely shielded by the glycine loop which adopts the closed conformation. The other subunit has an open glycine loop and noi se in the electron density map indicates flexibility/disorder in the activ e site, especially in the regi on of the glycine loop and Tyr-139. The CoA moieties are found in the resting conf ormation in both subunits. Superposition of the aspartyl-formyl anhydride and aspartyl-oxalyl an hydride (pdb code: 1t4c) complex structures results in an r.m.s. deviation of 0.4 over 427 C atoms of the monomer (Figure 8A). The structures show very small changes in the enzyme core and the active sites are highly similar, while the flexible solvent exposed areas display some differences.

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59 Figure. 2-8. Steroview overlay of FRC aspart yl-formyl and aspartyl-oxalyl anhydride active sites. A the aspartyl-formyl ac tive site is shown in green and the aspartyl-oxalyl is shown in light blue. The glycine loop is in the closed conformation in both structures. The C trace of the enzyme is displayed. B Fo-Fc electron density map contoured at 3 calculated with the aspartyl-f ormyl anhydride and CoA molecule omitted from the structure. C, stereoview overlay of th e aspartyl-formyl anhydride active site ( green ) and the Q17A formyl-CoA transf erase mutant enzyme active site with the aspartyl-CoA thioester and oxalate bound to the open glycine loop ( pink ). Taken from Berthold 2008 (18).

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60 Figure. 2-9. Stereoview of oxalate and formate modeled into the FRC active site. Oxalate and formate are modeled into the anion binding si tes occupied by chloride ions in subunit B of the -aspartyl-CoA thioester complex. Po ckets are calculat ed with a probe radius of 1.4 and are displayed in a su rface representation. Both of the pockets have a connecting channel to the surf ace. The glycine loop in the closed conformation protects the th ioester from an attack from above. Hydrogen bonds are indicated by dashed yellow lines and the red dashed line shows where nucleophilic attack will take place. Taken from Berthold 2008 (18). Q17A FRC Mutant and Oxalate Binding The active site residue Gln-17, positioned in the close proxim ity of Asp-169, has two distinct rotamer conformations corresponding to the two different active site conformations in the -aspartyl-CoA thioester complexes (Figure 2-4). Replacement of this residue by an alanine results in severely impaired activity, with a 45-fold reduced kcat (Table 2-4). A crystal structure of the Q17A mutant was solved to 2.2 resolution from a crystal incubated with formyl-CoA and oxalate. The enzyme variant still could bind formyl -CoA and the reaction proceeded until formation of the -aspartyl-CoA thioester, which was observed in the resting conformation in both active sites of the dimer. Interestingly, the glycine loop displayed the open

PAGE 61

61 conformation in both subunits and an oxalate molecule was bound to the loop in subunit B (Figure 2-8C). Oxalate is hydrogen bonded with one of its carboxyl groups to the main chain nitrogens of Gly-260 and Gln-262 in the loop wh ile the other carboxyl gr oup points towards the active site and mainly interacts with water molecules. Th e distance between the closest oxygen of oxalate and C of Asp-169 is 5.3 in this conformation and the orient ation is not favorable for a nucleophilic attack by oxalate. Behind Asp-169 where the Gln-17 side chain normally is positioned when the glycine loop takes the clos ed conformation, a strong spherical electron density is present. A chloride ion, like in the -aspartyl-CoA thioester complex structures, could be refined into this position forming a str ong interaction with Ser170. The crystallization Figure. 2-10. Initial velocity pl ot of initial velocities of G261A variant against varied [oxalate] (0.063 77.0 mM) at 9.9 ( ), 39.2 ( ), and 78.4 M [formyl-CoA] with 45.3 nM enzyme. Data were fitted with the Mich aelis-Menten equation modified for substrate inhibition (Equation 2).

PAGE 62

62 conditions or protein buffer for th is complex contained no chloride ions and it is likely that the ion was bound during expression or pur ification of the mutant and remained due to the lack of a glutamine side chain occupying the site. G259A, G260A, and G261A FRC Loop Mutants Three FRC variants, where Gly-259, Gly-260, and Gly-261 were m utated into alanine residues, were prepared to investigate the im portance of the glycine loop. The mutations were expected to impact the loop movement, because the peptide geometries of the Gly-259 and Gly260 residues are positioned in the disallowed region of the Ramachandran plot for alanine. As Figure. 2-11. Substrate inhibition of the G261A variant by oxalat e against varied formyl-CoA. Initial velocity plot of initial velocities against varied [formyl-CoA] (9.9 mM 78.4 M) at 7.5 ( ), 30.5 ( ), and 77.0 mM [oxalate] with 45.3 nM enzyme. Data were fitted to the Lineweaver-Burk equation. Apparent inhibition constants for substrate inhibition by oxalate, Kic = 4 mM and Kiu = 73 mM, were determined from the replots of slopes and intercepts.

PAGE 63

63 expected the mutated enzyme is im paired. For the G260A mutant, the KM value for oxalate increases almost 5 times and kcat/ KM is 75 times reduced (Table 24). The 2.0 resolution crystal structure of the G260A mutant, sh owed clear strain in the loop, which could not adopt the same conformation as in the wild type enzyme in the closed form (Figure 2-12). The mutation containing loops were modeled in the most pr obable conformation based on the electron density in an omit map, although difference density around the loops shows them to be partly disordered. Figure. 2-12. Ramachandran pl ot showing loop glycine residues (258GGGG261). Plot generated in Swiss-PDB Viewer V3.7 from the apo-enzyme structure (1p5r). Chain A is in the closed conformation and chain B is in the open conformation. The black regions represent the generous ly allowed regions.

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64 Figure. 2-13. Stereoview of G260A tetraglycine loop. The open, blue and closed, cyan loops of FRC are shown as ribbons. The grey loop in the G260A varian t is unable to fully close. The specificity constant for G261A variant was reduced nearly 50 times relative to wildtype FRC, primarily due to an order of magnit ude increase in KM for formyl-CoA (26.6 M). In contrast to the other loop mutants, kcat/ KM for oxalate increases slightly for the G261A variant and oxalate was a mixed-type substrate inhibitor of G261A with an apparent Kic of 4 mM and Kiu of 73 mM (see Figures 2-10 and 2-11). Assuming that there is only one ox alate binding site (per monomer) in G261A, there are two plausible explanations for this behaviour: the role of the glycine loop is primarily that of protecting the active site or it is implicated in half-sites regulation of the second site. If the ability of the tetraglycine loop is disrupted, oxalate may be allowed to bind first and exclude formyl-CoA from the active site. If the G261A variant exhibits half-sites reactivity and the loop is critical to that reactivity, i.e. loop A closes down on the active

PAGE 65

65 site allowing loop B to open, then disruption may allow oxalate to again bind out of turn in the second site. The competitive and uncompetitive components of CoA inhibition, 2 M and 41 M, respectively, are the lowest seen for any FRC variant and these results also support the critical nature of the loop. Table 2-4. Summary of kinetic consta nts for wild-type FRC and mutants Table 2-5. Summary of the inhibi tion constants and patterns for wild-type FRC and mutants. (M) FRC Q17A G258A G259A G260A G261A CoASH competitive mixed-type mixed-type mixed-type mixed-type Kic 16.7 0.7 16.0 0.6 6.0 1.0 55 19 2 1 Kiu -100 14 460 129 290 5 41 1 Hydroxylamine Trapping of G261A Variant The proposed m echanism for formyl-CoA de pendent hydroxylamine inactivation requires that the nucleophilic hydroxylamine attacks the carbonyl of Asp-169 in the putative formylaspartyl anhydride. Chemically, it makes better sense for the attack to occur at the formyl group. An explanation is that the enzyme active s ite protects the formyl carbonyl from attack; interference with te traglycine loop may allow addition to the less hindered formyl group. If this is the case, a reduction in effectiv e inactivation is expected. Thus the G261A variant was tested Table 2-6. Formyl-CoA dependent inactiv ation of G261A (0.27 M) by hydroxylamine [Formyl-CoA] (M) G261A Residual Activity (%) 0 100 8 77 30 10 260 30 14 kcat (s-1) KM(F-CoA) ( M) kcat/ KM(F-CoA) (s-1M-1) KM(oxalate) (mM) kcat/ KM(oxalate) (s-1M-1) FRC 5.3 0.1 2.0 0.3 2.7 0.4 x 1063.9 0.3 1.4 0.1 x 103 G259A 1.9 0.1 4.7 0.8 4.1 0.6 x 10512.1 0.5 160 7 G260A 0.23 0.02 18 3 1.3 0.2 x 10418.0 1.6 12 1 G261A 1.65 0.01 26.6 0.9 6.2 0.2 x 1040.47 0.08 3.5 0.2 x 103Q17A 0.12 0.1 3.3 0.5 3.6 0.6 x 10413.2 0.6 8.7 0.9

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66 for inactivation by hydroxylamine in the presence of formyl-CoA. At up to 260 M formylCoA, the G261A variant was only inactivated about 70%. Mass Spectrometry Analysis of Proteolysed FRC In order to confirm the form ation of enzyme-substrate anhyd ride intermediates during FRC turnover, an 18O-oxalate labeling experiment was designed where the Asp-169-containing peptide could be monitored for isotopic label by MS. However, the Asp-containing proteolytic peptide was not reliably detected. Cleavage by glutamyl endopeptidase (V8) was expected to generate a M+H 1041.48 m/z peptide (GPPTVSGAALGD169) and this peptide was detected once (see Figure 2-13), but these condi tions could not be reproduced. A proteolysis map for FRC was generated by digestion with tr ypsin or glutamyl endopeptidase with both denatured and folded FRC (Figure 2-14). MS data were an alysed by ESI-MS and peptides were Figure. 2-14. Spectrum of FRC digested with glutamyl endopeptidase and analysed by MALDITOF mass spectrometry. The As p-169-containing peptide (1041.48 m/z ) was detected, but not reproducibly.

PAGE 67

67 Figure. 2-15. Combined sequence coverage by mass spectrometric peptide analysis was 74%. Peptides identified from tr ypsin digest are shown in dark grey ; peptides from glutamyl endopeptidase are shown in light grey ; and, regions of ove rlap are shown in black Identification was accomplished by tandem MS with the Mascot MS/MS ions search (185). The catalytic Asp-169 is highlighted with a black circle identified by MASCOT search engine (185). The central helix proved to be remarkably resistant to proteolysis (or detection by MS). Of the undet ected residues (26%), the majority were from this region in the protein. Engineering Trypsin-Friendly FRC In an effort to facilitate detection of this residue, an arginine residue was engineered into FRC to afford a 28-residue peptide with m a ss 2908.46. The P159R mutant was designed after close analysis of a structural sequence alignment of FRC, YfdW, CaiB, and MCR (Figure 1-8).

PAGE 68

68 Pro-159 corresponds to Arg-146 in MCR and both residues are f ound in a small, solventaccessible loop connected to -helix-9. Clostripain, a cysteine protease from Clostridium hystolytica was chosen as an alterna tive to trypsin as it cleaves Arg-Pro peptide bonds (172). The peptide was expected to retain a positiv e charge upon ionization and should be a good candidate for MALDI-TOF MS. However, the protein was not overexpressed under the normal expression and purification conditions. Half-Sites versus Independent Active Sites Reactivity Half-sites reactivity is an extrem e limit of negative coope rativity (see Sey doux for a review (220))ligand-binding induces struct ural changes and alterations in subunit interactions lower enzyme affinity for the substrate in a second, otherwise equivalent, active site. Half-sites reactivity is common in nature, examples in clude glyceraldehye-3phosphate dehydrogenase (48), thymidylate synthase (121), and the pyruvate dehydrogenase complex, where a proton wire is proposed to mediate half-sites reactivity (86). Family I CoA transferases exhibit this form of cooperativity, e.g. SCOT (1 56) and acetyl-CoA transferase from E. coli (233), mass spectrometric analysis of NaBH4-treated protein, and use of the 1,N6-etheno-CoA, a fluorescent CoA analogue, demonstrated that in both cases, only one monomer was active at a time. Based on the asymmetry of the FRC structure with CoA bound, it has been sugge sted that the dimer might exhibit half-sites reactivity as well (124). A construc t in the Novagen pET-Duet vector containing two cloning sites was therefore prepared allowing co -expression of FRC with the D169S inactive mutant Constructs with a pol y-histidine tag on FRC or the D169S mutant allowed the purification of heterodimers and histidine-tagged homodimers. If dimer formation is statistical, a 1:2:1 ratio of hi stidine-homodimer: histidine-he terodimer: heterodimer can be expected. Table 2-7 shows the predicted specific activities for both independent and half-sites

PAGE 69

69 models and the experimental specific activities a ssayed with saturating formyl-CoA and oxalate. It appears that the activ e sites of FRC work independently of each other. Table 2-7. Predicted and experimental activities of half-sites constructs. S.A. (U/mg) % wild type Half-sites (%)Independent (%) WT-FRC 6.5 0.4 100 6 DuetHisWT/D169S 4.0 0.4 62 6 100 66 DuetHisD169S/WT 1.8 0.2 28 2 66 33 Discussion The reaction catalyzed by both Family I and III of CoA-transferases includes the formation of aspartyl(Family III) or gl utamyl(Family I) mixed anhydr ide intermediates with the oxyacids, as well as covalent thioester intermedia tes to the CoA moiety (Figures 2-1 and 2-15). A distinction between the two families is that the Family I enzymes catalyze a classical pingpong reaction while the kinetics of Family III en zymes differ; release of donor oxyacid is not observed prior to binding of the acceptor oxyacid. This leaves two possibilities, either the requirement of a ternary complex for catalysis, or the completion of the reaction before any product can be released. It can be settled from the kinetic tr apping experiments and crystal structures presented above that hydrolysis of both formyl-CoA and oxalyl-CoA as well as formation of the mixed anhydride can be accomplished in FRC in the absence of acceptor carboxylic acid. Thus, the reaction does not need the formation of a ternary complex to proceed and the most probable interpretation of the kineti c data is that the leaving oxyacid remains bound in the enzyme and is released together with the acceptor thioester. Based on all available data a new proposal for the FRC reaction mechanism is presented in Figure 2-15. The glycine loop (258GGGGQ261) plays a central role during catalysis in FRC, and together with Gln-17, it protects the different intermediates from hydrolys is. The X-ray data suggest that upon binding of formyl-CoA, the CoA carrier adopts the resting conformation observed in most structures

PAGE 70

70 including the mixed anhydride complexes. The glycine loop is presumed to close down upon formation of the aspartyl-formyl anhydride complex [ B ] in Figure 2-15. The Gln-17 side chain is positioned behind Asp-169. During the next catalytic step, CoASperforms a nucleophilic attack on the mixed anhydride resulting in the aspartyl-CoA thioester [ C ]. Now the glycine loop opens up and Gln-17 flips its side chain out abov e the thioester, protec ting it from hydrolysis. The released formate molecule binds to the ope n glycine loop at the site where oxalate was observed in the Q17A mutant structure (Figure 2-8C). As the loop closes, formate is pushed Figure. 2-16. The proposed reaction mechanism for formyl-CoA transferase. All complexes observed in crystal structures are highli ghted. Letters and numbers correspond to structures and steps seen in Figure 2-17. The mechanism corresponds to Mechanism 1a in Figure 1-6. Putative tetrahedral in termediates are not shown to save space. down in the active site simultaneously as the CoA moiety reorganizes [ C ] into the newly observed activated conformation and Gln-17 moves back above Asp-169. The thioester is at this stage protected from hydrolysis by the closed glycine loop and formate is bound in one of the anion sites identified in subunit B of the aspartyl-CoA thioester complex (Cl1 B in Figures 2-4

PAGE 71

71 and 2-6B). Binding of formate at this site can result in hydrogen bonds to both the pantetheine arm and the main chain amide of Gln-262 (Figur e 2-9). The activated CoA conformation creates a cavity below the aspartyl-CoA thioester with connecti on to the surface, where oxalate can enter and bind in the second anion si te identified in subunit B of the aspartyl-CoA thioester complex (Cl2 B in Figures 2-4 and 2-6B). Manual modeling of oxalate at this site results in strong hydrogen bonds to the amides of Gln-17 and Ala18 and minor shifts would place also His-15 and Asn-96 within hydrogen bonding distances, ensu ing bonds to all four oxygens of oxalate (Figure 2-9). With a favorable orientation, and a distance of approximately 3.7 to C of Asp169, oxalate is perfectly aligned for a nucleophilic attack at the aspartyl-CoA thioester [D ]. The second mixed anhydride, the aspa rtyl-oxalyl anhydride results, and CoASshifts back to its resting conformation. The final attack by CoASat the oxalyl moiety regenerates the aspartate together with oxalyl-CoA [ E ]. Finally, in the product leaving step, opening of the glycine loop allows release of the acceptor thioester togeth er with formate. Experimental Methods Site-Directed Mutagenesis and Protein Production The Q17A, G259A, G260A, and G261A variants were prepared by QuikChange sitedirected m utagenesis (Stratagene) with the F RC gene in the pET-9a vector (Novagen, San Diego, CA) with the following primers: 5 -Q17A 5-GCT TGA CTT TAC CCA CGT CGC GGC AGG TCC TGC CTG TAC ACA GAT GA T GGG, 3-Q17A 3-CCC ATC ATC TGT GTA CAG GCA GGA CCT GCC GCG ACG T GG GTA AAG TCA AGC, 5-G259A 5GGT GCG GGC GGC CAG CCA GGC TGG, 3-G2 59A 3GCC CGC ACC TGC GTT ACC ACC ACG TGG, 5-G260A 5GGC GCG GGC CA G CCA GGC TGG ATG CTG, 3-G260A 3GCC CGC

PAGE 72

72 Figure. 2-17. Models and crystal structures showing assumed important features in the active site between the catalytic steps in Figure 2-15. For clarity, the amino acid residues are only labeled in A. Glycine loop is shown as C trace. A model of formyl-CoA in the active site. B aspartyl-formyl anhydride formed after step 1; C the enzyme-CoA thioester; D the activated conformation of the enzy me-CoA thioester observed in subunit B of the crystal structure; E the second anhydride; and, F the apoenzyme with both products modeled in the active site. Taken from Berthold 2008 (18).

PAGE 73

73 GCC ACC TGC GTT ACC ACC ACG, 5-G2 61A 5-GGT GGC GG C GCG CAG CCA GGC TGG, and 3-G261A-3 GCC GCC GCC CGC TG C GTT ACC ACC. PCR primers were obtained from Integrated DNA Technologies, Inc. (Coralville IA). DNA sequencing was performed by the DNA Sequencing Core of the Interdisciplinary Center for Biotechnology Research at the University of Florida. Recombinant FRC and variants were produ ced and purified follo wing the procedure previously described In short, the plasmids were transformed into the E. coli strain BL21(DE3) (Novagen) where the genes were expressed. Puri fication was then carr ied out sequentially by four steps of chromatography; DEAE anion ex change, Blue-Sepharose fast flow affinity, Sephadex G-25 size-exclusion, and QHP anion ex change. The final purified enzymes were stored at -80 C in 25 mM sodium phosphate pH 6.2 with 300 mM NaCl, 1 mM DTT, and 10 % glycerol. Purity was verified by SDS-PAGE and protein concentrations were determined by the Bradford method (27) with Commassie Plus reagent (Pierce) based on a standard curve constructed with known amounts bovine serum albumin or the Edelhoch method (see below). Determination of Protein Concentration by the Edelhoch Method. Common spectrom etric methods for determini ng protein concentrati ons depend heavily on the chosen standard (209). Methods em ploying the molar absorption coefficient, are more accurate, but are usually based on concentrations determined by dry weight, nitrogen, or amino acid analysis. The Edelhoch method as reported by Gill and von Hippel (89) is based on the data of Edelhoch (70) for the absorbance at 280 nm of tryptophan, tyrosine, and disulfide bonds and is the best way for determining for a protein. Based on 116 measurements from 80 proteins (181), the 280 for a protein can be predicted using the following equation: 280 (M-1 cm-1) = (nTrp)(5,500) + (nTyr)(1,490) + (ncystine)(125)

PAGE 74

74 where nTrp is the number of tryptophans, nTyr is the number of tyrsosine residues, and ncystine is the number of disulfide bonds in the protein in question. This method yields values with standard percent deviation of 3.836 when compar ed to a literature se t of concentrations determined by dry weight method, amino acid anal ysis, Kjeldahl nitrogen determination, or the Edelhoch method for 80 proteins. While the general equation above does a good job of predicting 280, the Gill and von Hippel method is slightly more accurate, accounting for the slight change in absorption of buried amino acid residues, and involves measuring A280 in denaturing 6 M guanidinium hydrochloride. This 280 (6MGuHCl) can then be used to calculate the 280 (buffer), which can then be used to easily and non-destructively determine concentration of samples in storage buffer. The following equation uses values reported by Pace: 280 (M-1 cm-1) = (nTrp)(5,685) + (nTyr)(1,285) + (ncystine)(125) Aliquots of equal volumes of FRC in storag e buffer were lyphophili zed and subsequently resuspended in either 100 L of FRC storag e buffer (100 mM potassium phosphate, pH 6.5 with 300 mM NaCl) or storage buffer with 6 M GuHC l. The absorbances at 280 and 333 nm were collected. The 280 (6MGuHCl) was corrected for the effects of light scattering by subtracting 1.929 x A333 and used to calculate the c oncentration in 6 M GuHCl. Th is concentration was used to calculate the 280 (buffer) 58, 202 M-1 cm-1. Assay for Coenyzme A Esters Concentrations of for myl-, oxalyl-, and succi nyl-CoA were determined with the singlepoint HPLC assay developed by Jnsson (123, 124) CoA ester separation was achieved by C18 reversed-phase HPLC (Dynamax Microsorb 60-8 C18, 250 x 4.6 mm or Varian Pursuit XRs C18 150 x 4.6 mm) with a singlewavelen gth detector at 260 nm. Va rian Galaxie Chromatography Data System software version 1.9.3.2 was used for data analysis.

PAGE 75

75 HPLC gradient methods Separation m ethods were optimized for the above C18 columns. Analysis with the Dynamax Microsorb column was achieved with pr eviously described HPLC methods (123, 124). Methods for separation of CoA esters with the Varian Pursuit XRs C 18 column and varyied gradients of Buffer A and Buffer B are summar ized below. Buffer A was 50 mM sodium acetate, pH 4.7 and Buffer B was 50 mM sodium acetate with 90% CH3CN, pH 4.5. Table 2-8. Oxalyl-CoA HPLC method Time, min Buffer B, % Flow, mL/min 0.00 5 1.0 0.50 Wait (close) 6.00 9 6.10 95 9.00 95 9.10 5 11.00 5 Figure. 2-18. Representative chroma togram for separation of oxalyl-CoA (tR = 5.2 min).

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76 Table 2-9. Formyl-CoA HPLC method Time, min Buffer B, % Flow, mL/min 0.00 6 1.0 0.15 Wait (close) 10.00 11 10.10 95 14.00 95 14.10 6 16.00 6 Figure. 2-19. Representative ch romatogram for separation of CoA (tR = 9.10 min) and formylCoA (tR = 10.10 min). Note the 2-phosphorya lated iso-formyl-CoA at around 11.2 min. Table 2-10. Succinyl-CoA HPLC method Time, min Buffer B, % Flow, mL/min 0.00 6 1.0 0.50 Wait (close) 11.50 14 11.60 95 14.00 95 14.10 6 18.00 6

PAGE 77

77 Figure. 2-20. Representative chromat ogram for separation of succinyl-CoA (tR = 10.65 min) Assay for coenzyme A concentration Ellm ans reagent (5,5 -dithio-bis(2-nitrobenzoic acid; DTNB)) was used to accurately assess CoA stock concentrations for inhibition studies. Samples of CoA were allowed to react in 100 mM potassium phosphate, pH 6.7, with 1 mM DTNB in a total volume of 150 L for 15 minutes at room temperature. Absorbance readings were taken at A412 and an extinction coefficient of 14150 M-1cm-1 was used to determine CoA concentrations after adjustment to the absorbance of a blank cont aining DTNB, but no CoA. Enzyme Kinetic and Inhibition Studies Form yl-CoA transferase activity was assayed by monitoring the formation of oxalyl-CoA by an HPLC point assay developed by Jnss on (124). Formyl-CoA and oxalyl-CoA were prepared by previously described methods (124). Reaction mixtures containing 60 mM potassium phosphate, pH 6.7, approximately 80 ng of enzyme, and appropriate amounts of formyl-CoA and oxalate were prep ared in a total volume of 200 L. Reactions were started by the addition of formyl-CoA and quenched by the addition of 30% acetic acid. The formation of

PAGE 78

78 oxalyl-CoA was measured by separating the que nched reaction mixtures by reverse-phase chromatography, monitoring the absorbance at 260 nm, and integrating th e area under the peak. The effects of contaminating CoA were controlled by first determining the inhibitory effect of CoA against varied concentrations of formyl-CoA. Kinetic constants Vmax, KiCoA, and KM(formylCoA) were then used to fit initial velocity plots of varied oxalate concentra tion at constant formylCoA concentrations to determine the apparent KM(oxalate) and Kia. The inhibition of FRC by chloride ions was determined at different oxalate concentrations at saturating concentration of formyl-CoA (21.4 M) and 10 M CoA with KCl concentrations of 5, 15, and 30 mM. Hydroxylamine and Sodium Borohydride Trapping Experiments The experim ents were carried ou t in a reaction volume of 500 L, containing 6.2 g of recombinant wild-type FRC in 60 mM potassium phosphate buffer, pH 6.7, and 77 mM oxalate. The reaction was started by the addition of 173 M formyl-CoA and was allowed to run for 10 seconds before treatment with 15 mM hydroxylam ine at pH 7 for 30 seconds at 30 C. Small molecules were immediately removed from the reaction solution by size exclusion chromatography (5 mL G-25) after which the resi dual specific activity of FRC was assayed using the normal HPLC point assay. The trapping experi ment was repeated in a reaction mixture in absence of oxalate with FRC or G261A inc ubated for 30 seconds at 30 C with varied concentrations of formyl-CoA (0.14 to 260 M). As before, the reaction mixture was separated by gel filtration chromatography 30 seconds af ter the addition of hydroxylamine. Residual activity of the protein treated only with NH2OH was also assayed. Borohydride trapping experiments were carried out as above with the exception that the reaction was trapped with the addition of NaBH4 (1M NaBH4 in 1M NaOH) to a final

PAGE 79

79 concentration of 33 mM, immediat ely followed by addition of an equal volume of 1 M HCl. The reaction mixture was allowed to incubate at room temperature for 30 min prior to gel filtration. Crystallization and Freeze-Trapping Experiments Crystallization and analysis of FRC variants was carried out by Dr. Catrine L. Berthold at the Karolinska Institutet, Stoc kholm Sweden. FRC was crysta llized by the hanging-drop vapor diffusion method using conditions previously optimized for the wild type enzyme (197). 2 L of the protein solution containing 7.5 mg/mL FRC in 25 mM MES buffer pH 6.2 and 10% glycerol was mixed with 2 L precipitant solution and set up to equi librate against 1 mL well solution at 293 K. A precipitant solution of 21-25 % PE G 4000, 0.1 M HEPES buffer pH 7.2-7.5 and 0.5 M MgCl2 resulted in approximately 0.1 x 0.1 x 0.2 mm singl e crystals that grow to full size within 48 h. The freeze-trapping experiments were performe d by transferring the crystals to a drop containing a modified well so lution (30% PEG 4000, 0.5 M MgCl2, 0.1 M HEPES buffer pH 7.2) mixed in a 1:1 ratio with 20 mM formyl-C oA or oxalyl-CoA in 50 mM sodium acetate buffer pH 5.0. The crystals were flash frozen in liquid nitrogen after th e desired reaction times. The crystals, diffracting to 2.0 resolution, belong to space group I4 with an asymmetric unit containing two 47 kDa FRC monomers, comprising the biological dimer. Crystals where the aspartyl-formyl anhydrid e complex was trapped were obtained by a new crystallization condition de void of chloride ions. An opt imized well solution of 1.35 M sodium citrate and 0.1 M HEPES buffer, pH 7.2-7.5, was used when setting up the crystallization experiments using the same protein solution and mixing conditions as above. The crystals belong to the same space group and were isomorphous with the previous ones.

PAGE 80

80 In order to form the anhydride complex 2-3 L of a formyl-CoA solution was slowly added to the crystals in the dr op and crystals were then transferred to an ethylene glycol cryo solution (1 M sodium citrate, 75 mM HEPES bu ffer, pH 7.2 and 25 % ethylene glycol) after approximately 10 minutes. The formyl-CoA solution was prepared by mixing equal volumes of 20 mM formyl-CoA in 50 mM sodium acetate buffer, pH 5.0, and well solution. Crystals of the G260A and Q17A mutants of FRC were obtained using the same conditions as for the aspartyl-formyl anhydride complex. Crysta ls of the G260A mutant were directly frozen in liquid nitrogen after transfer through silicon oil while crystals of Q17A were used for complex formation. For the ternary complex the drops containing the Q17A mutant crystals were supplemented with formyl-CoA as for the wi ld type aspartyl-formyl anhydride complex, followed by the addition of 1 L 40 mM potassium oxalate mixed into the well solution. For this complex, the ethylene glycol cryo-solution wa s supplemented with 40 mM potassium oxalate. Crystals of the Q17A mutant belong to the space group I4 wi th unit cell dimensions a = b = 153.6 and c = 98.1 while the G260A mutant crystallized in space group P43212 with cell dimensions of a = b = 97.3 and c = 193.4 Data collection, Structure Determination, and Refinement Data were collected at beam lines ID14 eh1 and ID23 eh2 at the European Synchrotron Research Facility, Grenoble, France and at beamline I911-2 at MAX-lab, Lund, Sweden. Data collection and refinement statistics are summarized in Table 2-1. All images were integrated with MOSFLM (146) and further processed using SC ALA (11). Phases from the originally determined apoenzyme (pdb accession code: 1p5h) ( 197) were used to solve the structures by molecular replacement using MOLREP (248). Re finement by the maximum likelihood method was carried out in REFMAC5 (174) interspersed with manual model building in WinCoot (157)

PAGE 81

81 where water molecules were assigned and checke d. The quality of the final structures were validated using PROCHECK (144)and WinCoot (157) and annealed omit maps calculated in CNS (29) were used to confirm the conformations in the active sites. All images of protein molecules were generated using PYMOL (59). Synthesis of [18O4]-Oxalate Normalized H2 18O (95% enrichment) was obtained from Cambridge Isotopes, Inc. (Andover, MA). The 18O-enriched oxalic acid was prepared by dissolving approximately 6.5 mg of (COOH)2 2H2O in 500 L of H2 18O in a literature procedure (6). The sample was sealed in an ampule and lyophilized after storage at room temperature for 5 weeks. The 18O content of the oxalic acid (79%) was determined by LC-MS (Ma ss Spectrometry Laboratory, University of Florida). Oxalate was resuspende d in water and the pH brought to 7 with the addition of solid KOH. Oxalate concentration was determined by oxalate decarboxylase enzymatic assay (143). Isotope (18O)-Labelling Experiment 18O-labelling of FRC Asp-169 was attempted by incubating 12.4 g of wild-type recombinant FRC with 236 M formyl-CoA and 19.8 mM 79% enriched [18O4]-oxalate for 1 and 2 minutes at 30 C. The unquenched reaction mixt ures were immediately buffer exchanged into 300 mM sodium chloride in 25 mM sodium phosphate, pH 6.2 on Amicon Microcon concentration devices to a final volume of about 50 L. Half was subsequently reincubated with 236 M formyl-CoA and 77 mM [16O4]-oxalate for 2 minutes. Peptide Generation by Proteolysis The trapped sam ples (theoretically 12 g of protein for hydroxylamine experiments or 6.2 g of FRC each for the 18O-labelling experiments) were buffer exchanged into about 50 L each of 50 mM NH4HCO3, pH 8.5 or 50 mM sodium phosphate, pH 7.5 with Amicon Microcon centrifugal filter devices (Millip ore). Concentrators were prepared by storing in 4% Tween 20

PAGE 82

82 overnight at 4 C, rinsed in deionized water, and membranes were washed by centrifuging twice with 500 L of water to reduce non-specific protein-me mbrane interactions. The samples were then heated to 60 C for 5 minutes and subsequen tly digested with 4% (w/w) trypsin or glutamyl endopeptidase for 16 hours at 37 C. Peptide Generation by Proteolysis (with GuHCl) The sam ple (12.4 g of FRC) was buffer exchanged into 100 L of 50 mM sodium phosphate, pH 6.7 with 6 M guanidinium HCl and h eated for 5 minutes at 60 C. The solution was cooled to room temperature and then dilu ted to 1 M GuHCl with 50 mM sodium phosphate, pH 7.5. V8 protease (Glu-C) was ad ded (25:1) and the sample was incubated 18 hours at 37 C. Concentrators (Amicon Microcon centrifugal filter devices, Milli pore) were prepared by storing in 4% Tween 20 overnight at 4 C, rinsed in deionized water, and wa shed by centrifuging twice with 500 L of water to reduce non-specific protein-membrane interactions. Mass Spectrometric Analysis Protein d igests were submitted for analysis by HPLC/(+)ESI-MS on a ThermoFinnigan (San Jose, CA) LCQ with electrospray ionizatio n (Mass Spectrometry Laboratory, University of Florida). MS and MS/MS data were compared against a database generated from FRC with hydroxylamine and single and double 18O labels allowed on acidic amino acid residues as variable modifications with the MA SCOT search engine (185). Mass spectrometric analysis of whole FRC was carried out by Dr. Gunvor Alvelius at the Karolinska Institutet, Stockholm, Sweden. A samp le of formyl-CoA transferase incubated with formyl-CoA in the absence of oxalate was prep ared according to an experiment by Lloyd and Shoolingin-Jordan (156). A 125 L reacti on mixture containing 0.153 mM formyl-CoA transferase in 25 mM MES buffer, pH 6.2, with 10% glycerol and 0.596 mM formyl-CoA was incubated for 1 min at room temperature. The r eaction mixture was then immediately desalted at

PAGE 83

83 277 K into 1 mM HCl using a prepacked NAP-5 column (Amersham Biosciences). The protein elution of 1 ml was mixed with an equal volum e of 98% acetonitrile and 2% formic acid. Data were immediately acquired in positive mode on a QTOF ULTIMA API instrument (Waters Corp., Milford, MA) equipped with the standard Z-spray source with a capillary voltage of 1.5 kV. The instrument was calibrated between 300 and 1400 m/z with myoglobin prior to the run. The sample was introduced with a metal-coated borosilicate glass cap illary needle (Proxeon Biosystems A/S, Odense, Denmark). Data we re collected over a mass range between 300 and 2500 m/z and with a scan time of 1 s for about 5 min. The spectra were combined and deconvoluted to zero charged ions with MaxEnt 1 in the Masslynx software (Waters Corp., Milford, MA). Half-Sites (pET-Duet) Constructs W ild-type FRC and D169S mutant sequences were cloned from pET-9a constructs (197). Forward primers included a clamp region and restriction site terminating in the start codon for the gene. The reverse primers comprised a clam p region, restriction site and an in-frame stop codon: 5-FRC BamH1 5-AGG AGA TAT AGG ATC CG A TGA CTA AAC CAT TAG ATG GAA TTA ATG TGC, 3-FRC HindIII (stop) 5ACA GGT AGT TTG AAG CTT AGA CTT, 5Nde1 5-AGG AGA TAT ACA TAT GAC TAA ACC ATT AGA TGG AAT TAA TGT GC 3-D169S Xho1(stop) 5-ACA GGT AGT TTG ACT C GA GAG ACT T. The amplified products were subjected to restriction enzyme digest with BamH1 and HindIII for the D169S PCR product and Nde1 and Xho1 restriction enzymes for wt-FRC. Th e resulting D169S and FRC fragments were isolated and ligated one at a time into the pETDuet-1 multiple cloning sites 1 (with Nterminal His6 tag) and 2 (no fusion tag) to generate the DuetHisDW and DuetHisWD constructs. JM109 competent cells (Stratagene) were transformed with the re sulting plasmids, screened for correct insert size, and confirmed by DNA sequencing.

PAGE 84

84 CHAPTER 3 FORMYL-COA TRANSFERASE (YFDW) FROM ESCHERICHIA COLI2 Introduction W ith the completion of genome se quences for several strains of Escherichia coli (23, 101, 186, 256), attention has turned to the annotati on of proteins encoded by specific genes of unknown function (244). Deletion st udies have shown that the yfdXWUVE operon (Figure 3-3), in which the yfdX gene is under the control of the E vgAS regulatory system (169), encodes proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions (168). Although the molecular mechanisms underlyi ng this phenotypic behaviour remain to be elucidated, the proteins encoded by the yfdW and yfdU genes in this operon (YfdW and YfdU, respectively) are homologous to the formyl-CoA transferase (FRC) (2, 29, 45) and the oxalylCoA decarboxylase (OXC) (10, 15) present in the obligate anaerobe Oxalobacter formigenes (237). FRC and OXC are essent ial for the survival of Oxalobacter in that they mediate the conversion of oxalate into formate and CO2 in a coupled catalytic cycle (Figure 3-1). In combination with an oxalate:formate antiporter (OxlT) ( 15), this cycle is thought to maintain Figure. 3-1. Coupled enzymes of oxalate catabolism in O. formigenes 2 Reproduced in part with permission from Journal of Bacteriology, Vol. 190 (12), Toyota, C. G., Berthold, C. L., Gruez, A., Jonsson, S., Lindqvist, Y., Cambillau, C., and Richards, N. G. Pages 2556-2564. Copyright 2008 Journal of Bacteriology

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85 the electrochemical and pH gradients need ed for ATP synthesis (3, 108, 142). It has therefore been proposed that (i) YfdW catalyzes the conversion of oxalate into oxalyl-CoA using formyl-CoA as a donor, and (ii) the YfdU protein mediates ox alyl-CoA decarboxylation (97). High-resolution X-ray crystallography supports the likely functional similarity of FRC and YfdW in that the two proteins adopt the same unusual interlocked, cat alytically active dimer (Figure.3-2) (92, 97, 197) despite having only 61% sequence identity (Figure 1-8). On the other hand, the ability of Escherichia coli to metabolize oxalate to formate and CO2 does not seem to have been reported, and the relevance of such an activity to survival unde r conditions of low pH remains to be established. Figure. 3-2. Superimposition of apo-YfdW (c yan) and apo-FRC (white) dimer structures. Coordinates were obtained from the Pr otein Data Bank files 1pt7 and 1p5h, respectively, and the figure was made using PyMOL (59).

PAGE 86

86 Results Kinetic Characterization of YfdW The initial e xperiments examined whether Escherichia coli YfdW could cataly ze the synthesis of oxalyl-CoA from formyl-CoA and oxalate, as infe rred on the basis of stru ctural genomics (92, 97). Incubating formyl-CoA and oxalate with YfdW in phosphate buffer, pH 6.7, did indeed result in the appearance of oxa lyl-CoA, and the amount of this product could be quantified by direct HPLC measurement (Fi gure 2-17). YfdW differed from FRC in that stronger quenching conditions were required. As s een in Figure 3-4, when attempts were made to stop the YfdW catalyzed reaction with 10% HAc, YfdW continue d to form oxalyl-CoA even when incubated on ice. Increasing the concentration of HAc to 20%, however, clearly abolished YfdW activity, but did not increase oxalyl-CoA hydrolysis. In addition, more stringent wash conditions for syringes, the HPLC injection port, and column we re necessary to preven t carryover activity. Figure. 3-4. Quench conditions for YfdW. Reactions were quenched and mixtures were inbubated with either FRC or YfdW. Oxal yl-CoA concentration was monitored over time. YfdW with 10% HAc incubated at 0 C( ), YfdW with 20% HAc at 32 C ( ), and FRC with 10% HAc at 32 C ( ).

PAGE 87

87 Figure. 3-3. Graphical representation of putative formyl-CoA transferase ( orange ) genes in various organisms for which total genome sequencing data are available. Putative oxalyl-CoA decarboxylase genes are shown in blue Arrows repres ent the direction of transcription. Yellow circles in E. coli and S. flexneri annotate identified EvgAS regulatory binding motifs in the yfdXYWUVE operon. Gene and gap lengths are given (bp).

PAGE 88

88 Thus the wash portion of the HPLC method grad ient was increased to 98% Buffer B with 2% Buffer A and run for 3 minutes. The analysis of in itial rate data using st andard fitting methods (47) gave a value of 510 30 M for the apparent KM of oxalate, almost an order of magnitude less than the cognate parameter determined for th is substrate in the FRC-catalyzed reaction (3.9 0.3 mM) (197). Variation of oxalate at different fixed concentrations of formyl-CoA gave intersecting lines in the Lineweav er-Burk plot (Figures 3-6 and 37), suggesting an ordered bi-bi sequential kinetic mechanism as reported previously for FRC (124) and other Family III CoA Figure. 3-5. Double-reciprocal plot for the inhibition of YfdW by free CoA against varied [formyl-CoA] at constant satu rating [oxalate] = 2.5 mM. Li nes are fitted to the data by linear-regression methods. CoA concentrations were 59 M ( ), 111 M ( ), 165 M ( ), and 273 M ( ) Kic and Kiu, 218 21 and 213 16 M, respectively, were determined from the replots of Kmapp/Vapp and 1/ Vapp vs. [CoA] (inserts). KM(F-CoA) of 351 4 M was determined by fitting th e initial velocity pl ots with the mixedtype inhibition equation w ith appropriate [CoA].

PAGE 89

89 transferases (60, 77, 84, 102, 137, 149). The finding th at oxalate concentrations higher than 2.5 mM inhibit the activity of YfdW (Figure 3-14) is again in sharp contrast to the kinetic behaviour of FRC, which is not inhibite d by oxalate at concentrations in excess of 230 mM (124). The evaluation of steady-state kinetic parameters fo r formyl-CoA in the Yf dWcatalyzed reaction was, however, complicated by the presence of free CoA in this substrate as a result of the procedures used to remove a 2'-phosphorylated isomer of this compound, which exhibited a slightly longer retention time than formyl-CoA on reverse-pha se HPLC (RP-HPLC) (Figure. 218). Contamination of the commercially availabl e CoA used in the synthesis of formyl-CoA Figure. 3-6. Double reciprocal plot of initial ve locities of YfdW with varied [oxalate] (0.125 2.5 mM) at 9.9 ( ), 29.6 ( ), and 49.3 M ( ) [formyl-CoA].

PAGE 90

90 Figure. 3-7. Double reciprocal plot of initial ve locities of YfdW with varied [F-CoA] (9.9 49.3 M) at 0.125 ( ), 0.375 ( ), 0.750 ( ), and 2.50 mM ( ) [oxalate]. (124) has been reported previously in studies of enzymes for which malonyl-CoA (170) and hydroxybutyryl-CoA (36) are substrates. The extent to which free CoA inhibited YfdW activity was assessed using standard kinetic methods, and inspection of the double-reciprocal plot showed a mixed-type inhibition ag ainst formyl-CoA (Figure. 3-5). After fitting to the appropriate kinetic equation, values of Kic and Kiu values of 220 21 M and 210 16 M, respectively, were obtained for inhibition by free CoA, which then permitted the apparent KM of formyl-CoA to be estimated as 352 4 M. The turnover number, kcat, under these conditions could then be determined as 130 17 s-1, which is considerably greater than that of FRC for which the cognate value is 5.3 0.1 (Table 3-3). Give n the presence of a poly-histid ine tag at the N-terminus of YfdW, a similarly tagged varian t of FRC was prepared and its steady-state kinetic parameters measured using the HPLC-based end-point assay (Figure 3-8). These experiments showed that

PAGE 91

91 Figure. 3-8: Double reciprocal plot of initial velocities of HisFRC with varied [oxalate] (2.5 75 mM) at 10.1 ( ), 30.3 ( ), and 80.8 M ( ) [formyl-CoA]. the observed difference in kcat values for YfdW and FRC (3-3) cannot be attributed to this structural modification. The crystallographic observation of a YfdW /acetyl-CoA/oxalate ternary complex (97) suggested that YfdW might be inhibited by acetyl-CoA, and therefore the steady state kinetic behavior of the enzyme in the presence of this compound was assayed (Figure 3-9). As in earlier experiments, the concentration of free Co A was maintained at a fixed value (52 M) as formylCoA was varied. Given that acetylCoA binds to the CoA site in the YfdW crystal structure (97), it was assumed that acetyl-CoA and CoA were mutu ally exclusive inhibitors at a given active site. This permitted the separation of their contri butions to the overall rate equation (78), and acetyl-CoA proved to be an uncompetitiv e inhibitor of formyl-CoA, with a Kiu value of

PAGE 92

92 Figure. 3-9. Double-reciprocal plot for the i nhibition of YfdW by acetyl -CoA against varied [formyl-CoA] at constant saturating [oxalate] = 2.5 mM and constant [CoA] = 51.6 M. Ac-CoA concentrations were 0 M ( ), 53.81 M ( ), 217.8 M ( ). Kiu, 94 2 M, was determined from the replot of 1/ Vapp vs. [CoA] ( insert ). There was no effect on KMapp/Vapp within experimental error. Lines were modeled with the Michaelis-Menten equation modified for uncompetitive inhibition. Figure. 3-10. Double-reciprocal plot for the inhibition of FRC by acetyl-CoA against varied [formyl-CoA] at constant saturating [oxalate] = 77 mM and constant [CoA] = 1.5 M with lines fitted to the data by linear-regression methods. Acetyl-CoA concentrations were 0 M ( ), 12.1 M ( ), and 36.4 M ( ). Ki(Ac-CoA) of 56 6 M was determined from the replot of Kmapp/Vapp vs. [acetyl-CoA] ( insert).

PAGE 93

93 94 2 M. In contrast, acetyl-CoA is a competitive inhibitor of FRC with respect to formylCoA, exhibiting a Kic value of 56 6 M at a fixed CoA concentration of 1.5 M (Figure 3-10). Size-Exclusion Chromatography Measurements. Size exclusion chrom atography of YfdW resu lted in a MW value of 85.5 kDa (Figure 311). Thetheoretical mass of the dimer is 96.6 kDa (monomer 48.3 kDa), suggesting that the active conformation is dimeric, sim ilar to that of FRC and consistent with crystal structure data. The slightly low MW may be a resu lt of a tight overall qua ternary structure and is also similar to the results seen for FRC (124). Figure. 3-11. Size-exclusion chromatography data used to estimate the molecular mass of catalytically active YfdW. Retention coefficients (KD) for the molecular weight standards, used to calibrate the column, are shown by filled circles ( ) and the open circle ( ) represents the experimentally determined KD of recombinant YfdW.

PAGE 94

94 Alternate Substrate Studies A variety o f CoA donors and acceptors were incubated with the recombinant, tagged YfdW to elucidate the substrate specificity of the enzyme (Tables 3-1 and 3-2). YfdW showed high levels of substrate specifici ty, being unable to catalyze CoA transfer from formyl-CoA to acetate, maleate or glutarate. Given that ma lonyl-CoA and succinyl-CoA are known metabolic intermediates in Escherichia coli however, whether either of these diacids could function as substrates was tested. In the case of malonate, Yf dW exhibited very low specific activity (0.01%) Table 3-1. FRC and YfdW substrate specificity for alternate CoA acceptors All activities are reported based on the rate of CoA transfer from formyl-CoA to oxalate for each enzyme (n.d. not determined). Formyl-CoA Oxalyl-CoA FRC YfdW FRC YfdW Formate --13 48 Acetate 0 0 0 0 Oxalate 100 100 --Succinate 909 4 n.d. n.d. Glutarate 273 0 n.d. n.d. Maleate 36 0.2 n.d. n.d. Table 3-2. FRC and YfdW substrate specificity for alternate CoA donors with either formate or oxalate as the acceptor. All act ivities are reported based on the rate of CoA transfer from formyl-CoA to oxalate for each enzyme. Formate Oxalate FRC YfdW FRC YfdW Formyl-CoA --100 100 Acetyl-CoA 0 0 0 0 Oxalyl-CoA 13 48 0 0 Succinyl-CoA 727 32 82 0.2 Malonyl-CoA 0 0 0 0 Methylmalonyl-CoA 0 0 0 0 Propionyl-CoA 0 0 0 0 using formyl-CoA as the donor. Control experiment s were also performed to ensure that any malonyl-CoA formed did not undergo exte nsive uncatalyzed d ecarboxylation under the conditions. Acetyl-CoA formation was also below the detection limits of the HPLC-based assay

PAGE 95

95 when the enzyme was incubated with formyl-CoA and malonate. In contrast, when succinate was used as an acceptor, succinyl-CoA was formed, albeit with a low specifi c activity (4%) relative to that observed for oxalate with formyl-CoA. A complete determination of the steady-state kinetic parameters for YfdW-catalyzed conversio n of succinate to succinyl-CoA was therefore performed to evaluate the substr ate specificity of the enzyme (Figure 3-12). These studies gave 80 40 mM for the apparent KM of succinate, and a turnover number of only 5.3 0.4 s-1 when formyl-CoA was employed as a donor. In contrast to observations on YfdW, succinate was an excellent substrate for FRC, the specificity cons tant being two orders of magnitude greater for Figure. 3-12. Double reciprocal plot of initial ve locities of YfdW with varied [succinate] (50 125 mM) at 19.7 ( ), 39.8 ( ), and 59.3 M ( ) [formyl-CoA]. KM(succinate) = 80 40 mM and Kia = 30 19 M. this substrate when compared with that of oxala te using formyl-CoA as a donor (Table 3-4). In a similar manner, it was observed that FRC coul d employ succinyl-CoA as an alternate CoA donor for the synthesis of formyland oxalyl-CoA (Table 3-6). The pattern of the lines in the double

PAGE 96

96 reciprocal plot approached The specific activity of FRC with malonate and formyl-CoA FRC as substrates was also substantially lower (0.1%) th an that observed when oxalate was present as the CoA acceptor. No products were detected in the HPLC-based assay when malonyl-CoA was used as a substrate with either formate or oxalate. Kinetic and Structural Characterizati on of the W48F and W48Q FRC Variants The extent to which active site res idues must be modified in order to change the substrate specificity of enzymes remains an interesting problem in enzyme evolution (90, 180, 241), and Figure. 3-13. Double reciprocal plot of initial velocities of FRC with varied [succinate] (0.05 5.0 mM) at 13.6 ( ), 31.4 ( ), and 67.2 M ( ) [formyl-CoA]. KM(succinate) = 0.32 0.03 mM and Kia = 0.5 0.4 M. its resolution has important implications for efforts to redesign bi ological catalysts for biotechnological applications (42, 133). The active sites of FRC and YfdW, however, are composed of conserved residues, making it difficult to understand the observed differences in (i)

PAGE 97

97 substrate specificity, and (ii) the ability of oxalate to exhibit substrate inhibition only in the case of YfdW. Structural studies on FRC had, however, revealed the importance of a tetraglycine segment in stabilizing a putative reaction intermediate (124), and conformational changes in this FRC and the Trp-48 FRC mutants. loop appeared correlated with the orie ntation of the Trp-48 side chain in FRC (197). Super-imposition of the cr ystal structures for the two CoA transferases Table 3-3. Steady-state parameters for the formyl-CoA/oxalate transferase activ ities of YfdW, Enzyme Formyl-CoA Oxalate kcat (s-1) KM(app) ( M) kcat/ KM(app) (mM-1s-1) KM(app) (mM) kcat/ KM(app) (mM-1s-1) His-YfdW 130 17 352 4 370 0.51 0.03 255 WT FRC 5.3 0.1 2.0 0.3 2650 3.9 0.3 1.36 His-FRC 5.5 0.4 4.7 1.6 1200 1.2 0.3 4.58 W48F FRC 17.1 0.2 0.7 0.4 24430 1.5 0.3 11.4 W48Q FRC 5.8 0.3 2.7 0.9 2148 0.43 0.03 13.5 Table 3-4. Summary of the inhibi tion constants and patterns fo r His-YfdW, FRC, His-FRC, and and variants. (M) FRC His-FRC His-YfdW W48F W48Q CoASH competitive competitive mixed-type mixed-type competitive Kic 16.7 0.7 9 7 218 21 11 5 55 19 Kiu --213 16 35 6 290 5 Table 3-5. Steady-state parameters for the formyl-CoA/succinate transferase activities of YfdW, FRC and the Trp-48 FRC mutants. Enzyme Formyl-CoA Succinate kcat (s-1) KM(app) ( M) kcat/ KM(app) (mM-1s-1) KM(app) (mM) kcat/ KM(app) (mM-1s-1) His-YfdW 5.3 0.4 180 14 29.4 80 40 0.07 WT FRC 149 13 16 2 9312 0.32 0.03 465 W48F FRC 42 6 12 6 3500 0.015 0.005 2800 W48Q FRC 17.9 0.5 6.7 0.9 2672 0.07 0.01 256 showed that this tryptophan residue was replaced by glutamine in YfdW (Figure 3-16). Moreover, for YfdW, an oxalate molecule was seen to bind to a closed conformation of this tetraglycine loop (corresponding to residues 246GGGGQ250 in YfdW) although the observed glutamine side chain rotamer was the same as seen for Trp-48 in FRC when the cognate loop segment was in its open conformation (46). Th us, it was investigated whether site-specific mutagenesis of Trp-48 in FRC mi ght yield variant enzymes exhibi ting modified kinetic behavior

PAGE 98

98 that was similar to that determined for YfdW. Two variants were prepared in which Trp-48 was replaced by phenylalanine (W48F) and glutamine (W48Q), and characterized under steady-state conditions. Relatively little change in the specificity constants ( kcat/ KM) of the two FRC variant enzymes for formyl-CoA and oxalate was evident when compared with the wild type enzyme (Table 3-3). Perhaps more importantly, the W4 8Q FRC variant exhibite d substrate inhibition with oxalate as observed for YfdW having a Ki value of 74 mM (Figure 3-15). In contrast, the W48F FRC variant was not inhib ited by oxalate at concentrations up to 154 mM, suggesting that hydrogen bonding to the Gln-48 side chain is an essential element for the interaction of this substrate with the site, as suggested by the YfdW/acetyl-CoA/oxalate crystal structure (97). So as to understand the structural effects of changing th e tryptophan residue in mo re detail, the crystal structures of the two FRC variants were obtained. Neither the W48Q nor the W48F FRC variant displayed any major structural changes when compar ed to wild type FRC, with the rmsd of the C atoms being 0.2-0.3 2 and 0.6-0.7 2 relative to subunit A a nd subunit B of apo-FRC, respectively. In both variant enzymes, the tetraglycine l oop (corresponding to residues 258GGGGQ262 in FRC) was seen to adopt a closed conformation (197). In wild type FRC, a 90o reorientation of the Trp-48 side chain seems to be important in controlli ng the tetraglycine loop conformation. This flipping of the indole mo iety, however, is accompanied by repositioning of Met-44 when the loop adopts its open conformation. For YfdW, in which a glutamine residue (Gln-48) replaces tryptophan however, oxalate can bind to the tetraglycine loop in the closed conformation, even though Gln-48 adopts the rotamer conformation corresponding to that of Trp-48 in FRC when the cognate loop is ope n. Comparison of the YfdW and W48Q FRC variant structures showed that Gln-48 in W48Q, in the absence of oxalate does not take the side chain rotamer conformation seen for the cognate residue in YfdW, presumably because of the

PAGE 99

99 Figure. 3-14. Initial velocities measured for Yf dW as function of oxala te concentration at 73.3 M formyl-CoA. The line is computed from a fit to the Michaelis-Menten equation modified for substrate inhibi tion (Eqn. 2). The apparent Ki for oxalate inhibition is 23 mM. Taken from Toyota 2008 (245). Figure. 3-15. Initial velocities measured for the W48Q FRC mutant as function of oxalate concentration at 70.3 M formyl-CoA. The line is computed from a fit to the Michaelis-Menten equation modified for s ubstrate inhibition (Eqn. 2). The apparent Ki for oxalate inhibition is 74 mM. Taken from Toyota 2008 (245).

PAGE 100

100 Table 3-6. Data collection and refinement statistics for the W48F and W48Q FRC mutants. Values given in parentheses represent those of the highest resolution shell. proximal methionine residue (Met-44) (Figure 318). For the W48Q FRC variant to bind oxalate in the site with the tetraglycine loop in a closed conf ormation, Gln-48 and Met-44 would both have to change rotamer conformation. In YfdW, th e methionine position is occupied by a smaller valine residue. Formyl-CoA Hydrolysis in the Presence a nd Absence of F RC, D169S, and YfdW In contrast to FRC and the D169A variant of FRC, YfdW does not appear to mediate the hydrolysis of formyl-CoA when pseudo-first orde r rate constants for the hydrolysis of formylCoA were determined. The half-lives for form yl-CoA hydrolysis in the presence of FRC, D169A, and YfdW were 51, 58, a nd 198 minutes, respectively. Formyl-CoA hydrolysis has been reported with a half-life of 150 minutes at pH 6.7 and 30C (124). Data Collection W48Q FRC W48F FRC Beamline ID14eh1 (ESRF) ID23eh2 (ESRF) Space Group C2 I4 Unit cell () 214.2, 98.9, 152.5 152.7, 152.7, 99.45 ( ) 90, 135.3, 90 90, 90, 90 Molecules in asymmetric unit 4 2 Resolution () 1.8 (1.9-1.8)a 1.95 (2.06-1.95) Rsym (%) 6.3 (40.8) 13.0 (53.2) Mean (I/ (I)) 12.4 (2.3) 8.8 (2.7) Completeness (%) 92.0 (68.7) (97.9) (99.5) Wilson B-factor 22.6 17.8 Refinement W48Q FRC W48F FRC Resolution range 30-1.8 30-1.95 R factor/ Rfree (%) 18.2 / 21.1 16.7 / 20.0 Atoms modeled 14725 7608 Number of residues 1708 854 Number of waters 1309 936 Mean B-factor model (2) 26.1 20.1 RMS deviation, bonds () 0.008 0.009 RMS deviation, angles ( ) 1.08 1.11 Ramachandran zone distribution (% ) 92.0 / 7.5 / 0.5 / 0 91.7 / 7.9 / 0.1 / 0.3 PDB deposition ID 2vjq 2vjp

PAGE 101

101 Table 3-7. Formyl-CoA hydrolysis in the presen ce of FRC, YfdW, and D 169A variant of FRC. Conditions t min Formyl-CoA 153 Formyl-CoA + FRC (328 nM) 51 Formyl-CoA + YfdW (28 nM) 198 Formyl-CoA + D169A (254 nM) 58 Figure. 3-16. Initial velocity plot of His-YfdU activity with varied oxalyl-CoA in the absence of added ADP ( ) and in the presence of 150 M ( ) and 1500 M ADP ( ). Expression, Purification, and Enzyme Activity of OXC Homologue HisYfdU The yfdU gene in E. coli is the hom ologue of oxc in Oxalobacter formigenes and the theoretical partner of YfdW. In an attemp t to understand the physiological role of the YfdW/YfdU enzyme pair in E. coli a construct containing the yfdU gene product cloned from genomic DNA was generated. The protein wa s expressed, purified by nickel affinity chromatography, and assayed by HPLC. The His-tagged YfdU fusion has a KM of 180 M and kcat of 15 s-1, and a turnover number of 8.6 x 104 M-1s-1. The corresponding values for wild-type

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102 OXC are 23 M, 88 s-1, and 3.8 x 106 M-1s-1. Unlike the Oxalobacter enzyme, YfdU does not appear to be activated by ADP (see insert in Figure 3-16.) Discussion These experim ents clearly demonstrate that YfdW is a formyl-CoA:oxalate CoA transferase, as anticipated on the basis of its seque nce and structural similarity to Oxalobacter formigenes FRC (197). Although this may seem an obvious fi nding, recent studies have shown that assigning enzyme function on the basis of sequence simila rity can often lead to mis-annotation in metabolic databases (203). Moreover, the location of the gene encoding a CoA transferase in an operon that confers resistance to acidic environments seems, at fi rst sight, unexpected. A further interesting outcome of these bioc hemical studies concer ns the high level of substrate specificity that is exhibited by YfdW. Thus, despite consider able efforts to identify other CoA acceptors and donors, only formyl-CoA and oxalate (and equivalently, oxalyl-CoA and formate) seem to be substrates for the enzyme. YfdW can th erefore mediate oxalate catabolism in Escherichia coli without affecting cellular succinyl-CoA levels. This observation stands in sharp contrast to the kinetic behavior of the Oxalobacter enzyme, for which succinate is a better CoA acceptor than oxalate when formyl-CoA is employed as a donor (Table 3-5). In light of the importance of oxalate as an energy source in Oxalobacter formigenes (3), the ability of FRC to synthesize succinyl-CoA is unexpected because this enzyme -catalyzed reaction removes a molecule of formyl-CoA thereby breaking the catalytic cycle (Figure 3-1). On the other hand, this side activity of FRC may be one mechanism by which Oxalobacter can use oxalate in the biosynthesis of other carbon-containing compounds given that succinyl-CoA is a key component of lysine biosynthesis and other biosynthetic pathways (94, 95). Th e presence of succinate in the cytoplasm of Oxalobacter is suggested by studies that have shown the presence of succinate

PAGE 103

103 Figure. 3-17. Comparison of the active-site residues in YfdW ( cyan ) and FRC ( white ). Conserved residues are indicated by a one-let ter code for amino acids. For positions where amino acids differ, the first letter refe rs to the residue present in YfdW. Taken from Toyota 2008 (245). dehydrogenase, fumarase, and malate dehydrogena se, which can be employed to interconvert oxaloacetate and succinate in the latter part of the ci tric acid cycle (51, 52). YfdW is inhibited by a variety of components, including acetyl-CoA, free CoA, and oxalate. On the basis of previous work on Oxalobacter formigenes (124), it was anticipated that CoA derivatives would compete with formyl-CoA for the free enzyme. Acetyl-CoA and free CoA are uncompetitive and mixed-type inhibitors however, with respect to both formyl-CoA and oxalate. Hence it seems that these compound s can both bind to YfdW/substrate complexes that are formed during catalytic turnover. The simp lest explanation for such kinetic behavior is

PAGE 104

104 that the two active sites in th e YfdW dimer can communicate so that only a single active site can catalyze the reaction at a given time (half -sites reactivity) (141). As a result, if CoAderivatives bind to a free CoA site in a YfdW/sub strate complex (or catalytic intermediate), then the enzyme undergoes a conformational change that precludes the formation of critical intermediates (18, 124) or product release at the other site. A mo re interesting observation was that YfdW is inhibited by elevated levels of oxa late, a kinetic behavior th at is not seen for the Oxalobacter formyl-CoA transferase. This inhibiti on was hypothesized to arise from oxalate binding at a second non-productive site defined (in part) by th e Gln-48 side chain in YfdW. Such binding is precluded by the presence of a tryptophan residue in FRC, and replacement of Trp-48 by glutamine to give the W48Q FRC va riant yields an enzyme for which oxalate inhibition is observed. Hence, it seems that replacing the indole si de chain by that of glutamine opens a hole in the FRC active site into which oxalate can bind in a non-productive conformation. This mutation also results in altere d conformational preferences of a tetraglycine loop that is known to be important for cat alytic function (18, 123, 124, 197), implying that altered active site dynamical moti ons may play a role in modulating kinetic properties (24, 72). The high Ki determined for oxalate in YfdW inhibition seems to preclude any physiological importance for this behavior. With the identifi cation of YfdW as a formyl-CoA:oxalate CoA transferase, questions are raised concerning the extent and importance of oxalate-related metabolism in Escherichia coli especially because this work de monstrates that YfdU is a ThDPdependent oxalyl-CoA decarboxylase. Although Escherichia coli has been implicated in the biomineralization processes leading to formati on of calcium oxalate crystals (41), recent measurements suggest that Escherichia coli does not degrade oxalate in media containing this compound at 5 mM concentration (247). The experi ments, however, did not systematically vary

PAGE 105

105 Figure. 3.18. Active-site struct ure in the W48Q FRC variant. A superimposition of apo-FRC with the tetraglycine loop in its open ( white ) and closed ( green ) conformations and the W48Q FRC variant ( pink ). B superimposition of apo-YfdW ( cyan) with the open conformation of the tetraglycine loop the YfdWacetyl-CoAoxalate ternary complex with the tetraglycine l oop in its closed conformation ( blue ), and the W48Q FRC variant with the tetraglycine loop in its closed conformation ( pink ). In both panels, the catalytic residue, Asp-169, and si de chains important in controlling the conformational properties of the tetraglycine l oop are displayed as stick models Met44 in the W48Q FRC variant is modeled in two conformations, and the carbonyl group of acetyl-CoA also adopts two confor mations in the stru cture of the YfdW acetyl-CoAoxalate ternary complex (97). Taken from Toyota 2008 (245). the incubation conditions and so it is po ssible that conditions exist under which Escherichia coli can metabolize exogenous oxalate. On this point, it should be noted that the YhjX gene product has been annotated as a possible formate:oxalate antiporter based on 25% sequence identity to Oxalobacter formigenes OxlT, which has been extensively characterized (108, 255). In a recent transcriptomic profiling study, YhjX has been id entified as a transporter upregulated by rapid cellular acidification (pH 5.5) in Escherichia coli (129). Work is therefor e needed to establish if Escherichia coli can mediate oxalate degradation, especia lly when in low pH environments. It is therefore interesting that the coupled action of YfdW and YfdU results in the consumption of a

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106 proton, as employed in the AR2 and AR3 mechanis ms of acid resistance mediated by the PLPdependent enzymes, glutamate decarboxylase and arginine decarboxylase (87). It appears that Escherichia coli has the required cellular machinery fo r either oxalate dependent AR and/or oxalate metabolism analogous to that in Oxalobacter formigenes YfdU, in contrast to OXC, does not appear to be activated by ADP; thus, YfdU imay not be involved in metabolism. However the question of whether oxalate catabolism can take place in Escherichia coli upon upregulation of the yfdXWUVE operon and YhjX expression under conditions of low pH remains. Experimental Methods Materials Unless otherwise stated, all ch emicals and r eagents were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scie ntific (Pittsburgh, PA), and were of the highest available purity. Recombinant, wild type FRC was expressed and purified following literature procedures (197). Protein concentrations were determined using a modified Bradford assay (Pierce, Rockford, IL) (27) for which standard curves were construc ted with bovine serum albumin as previously reported (124)), or the Edelhoch method (89). P CR primers were obtained from Integrated DNA Technologies, Inc. (Coralville, IA), a nd DNA sequencing was performed by the DNA Sequencing Core of the Interdisci plinary Center for Biotechnology Re search at the University of Florida. Formyl-CoA and oxalyl-CoA were prepared as described elsewhere (124). Expression and Purification of His-Tagged YfdW The subcloning and expression of the yfdW gene have been descri b ed in detail elsewhere (240, 251). Briefly, the yfdW gene was PCR amplified from the genomic DNA of Escherichia coli K12 and subcloned into the pDest17 vect or using Gateway t echnology (Invitrogen, Carlsbad, CA). Protein produc tion was carried out in the T uner(DE3)pLysS strain of Escherichia coli, and the His-tagged YfdW protein purified by me tal-affinity chromatography

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107 and subsequent gel filtration on a Superdex 200 column eluting with 5 mM HEPES buffer containing 150 mM NaCl, pH 7.5. Expression and Purifica tion of His-Tagged FRC The gene encoding Oxalobacter formigenes FRC (225) was cloned into the pET-28b vector (Novagen, San Diego, CA) so as to introduce a His-tag with 10-am ino acid linker at the N-terminus of the recombinant protei n. Primers used were as follows: 5Nde1 5-AGG AGA TAT ACA TAT GAC TAA ACC ATT AGA TGG AAT TAA TGT GC and 3BamH1 (stop) 5AAG TCT GGA TCC TCA AAC TAC CTG T BL21(DE 3) competent cells were transformed with the resulting construct and protein expressi on was induced by the addition of IPTG at an OD600 of 0.6. After harvesting and pe lleting by centrifugation at 5000 g for 15 min, the cells were re-suspended in lysis buffer (50 mM potassium phosphate, pH 7.2, containing 300 mM NaCl, 10 mM imidazole and 1 mM -mercaptoethanol) and sonicated. Cell debris was removed by centrifugation at 10,000 g for 15 min, and the supernatant was loaded on to an 0.5 mL NiNTA column (Novagen) equilibr ated with lysis buffer at 4 oC. The column was washed with lysis buffer containing 50 mM imidazole, and Hi s-FRC was eluted (5 x 0.5 mL) with elution buffer (50 mM potassium phosphate, pH 7.2, cont aining 300 mM NaCl and 250 mM imidazole). Size-exclusion chromatography on a Sephadex G-25 column (30 mL) equilibrated with storage buffer (25 mM sodium phosphate, pH 6.7, 300 mM NaCl, and 1 mM DTT) removed the imidazole, and the purified pr otein was stored at -80 C in 10% glycerol. Expression and Purification of FRC Variants The expression and purification of wild-type FRC and the W 4 8F and W48Q FRC variants lacking the N-terminal histidine fusion tag were performed by following procedures described in the literature (124, 197).

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108 Size-Exclusion Chromatography Measurements. A BIOSEP SEC-S2000 colum n (300 x 7.8 mm with 75 x 7.8-mm guard column) was calibrated using lysozyme (14.4 kDa), carbonic anhydrase (29.0 kDa), per oxidase (44.0 kDa), bovine serum albumin (66.0 kDa), alcohol dehy drogenase (150 kDa), amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kD a) in 100 mM potassium phosphate buffer, pH 7, with 100 mM KCl at a flow rate of 1 mL/min. A 75 L aliquot of 53.8 g/ L YfdW in 100 mM potassium phosphate, pH 6.7 was injected to give a single peak with retention time corresponding to a molecular mass of 85 kDa. KD is calculated as (Velution Vvoid)/(Vcolumn Vvoid). Confirmation of Quench Conditions Kinetic assays of CoA transferase activity in FR C were quenched in 10% acetic acid (124). Conditions for stopping the transfer ase reaction with YfdW were examined. Reactions were run as below: 90 L of reaction mixture were quenched in 10 L of either 10% or 20% HAc, followed by incubation at either 0 oC or 32 oC. Aliquots were removed at times of up to 100 minutes and oxalyl-CoA concentration was ascertained by measurement of absorbance at A260 ( vida infra ). Steady-State Kinetic Assays All kinetic m easurements were performed us ing an HPLC-based assay, as reported in previous studies on FRC (124, 197). For measuremen ts of YfdW-catalyzed CoA transfer, assay mixtures consisted of YfdW (54 ng) and the carboxylic acid acceptor in 100 mM potassium phosphate, pH 6.7 (total volume 100 L). The concentration of free CoA in all samples was normalized to that present as a contaminant in the assay mixtures containing the largest amount of formyl-CoA. After inc ubating this solution at 30 oC, reaction was initia ted by the addition of formyl-CoA. An aliquot (90 L) was taken after 60 seconds, a nd quenched by addition to 20%

PAGE 109

109 aq. HAc (10 L). The amount of the appropriate thio ester product was then quantitated by injection of these samples onto a C18 analytical column (Dynamax Microsorb 60 C18, 250 x 4.6 mm reverse-phase analytical column equ ilibrated with 86% Buffer A (25 mM NaOAc, pH 4.5) and 14% Buffer B (Bu ffer A containing 20% CH3CN)) at a flow rate of 1 mL/min. Immediately after injection the pr oportion of Buffer B was increas ed to 6% over 210 s, then to 100% for 90 s before the wash using 98% Buffer A and 2% Buffer B. Co A-containing species were observed by monitoring absorbance at 260 nm, and their concentrations were determined by integrating the peak areas and comparison with those for known amounts of authentic material. These measurements were calibrated us ing independent determinations of formyl-CoA concentration using (i) a hydroxylamine-based colo rimetric assay (227) and (ii) the oxalate concentration in hydrolyzed a nd nonhydrolyzed samples of oxal yl-CoA as measured with a standard detection kit (Sigma). No formation of CoA ester products wa s observed in control experiments when the enzyme or either substrate was omitted from the mixture. In kinetic assays of FRC, His-tagged FRC and the two FRC variant enzymes, a similar HPLC-based procedure was followed except that assays contained either FRC (41 ng), Histagged FRC (46 ng), W48F (41 ng) or W48Q (43 ng). Reactions were quenched in 10% aq. HAc (10 L) and aliquots were eluted initially wi th 96% Buffer A (25 mM NaOAc, pH 4.5) and 4% Buffer B (Buffer A containing 40% CH3CN) at a flow rate of 1 mL /min. The amount of buffer B was then increased to 11% over 210 s and then to 100% Buffer B for 90 s, after which it was returned to 4%. Determination of Steady-State Kinetic Constants Kinetic cons tants were obtain ed by curve-fitting to the fo llowing equations for sequential bi-bi kinetics (Eqn. 1), substrate inhibition (E qn. 2), competitive inhibition (Eqn. 3) and mixedtype inhibition (Eqn. 4) (47)):

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110 ][ 1][ ][ 1 ][maxA K B A K K BVMA ia MB (Eqn. 1) S i MK S SK SV2 max][ ][ ][ (Eqn. 2) ][ ][ ][maxS K I K SVc i M (Eqn. 3) iu ic MK I S K I K SV ][ ][ ][ 1 ][max (Eqn. 4) In these equations, Kia, KMA and KMB represent the dissociation of the first substrate to bind to the enzyme (formyl-CoA) and the KM values for formyl-CoA and oxalate, respectively, KiS is the substrate inhibition constant of oxalate against varied formyl-CoA concentration, and Kic and Kiu are the inhibition constants for competitive and uncompetitive mechanisms, respectively. Patterns of intersecting lines in double-reciprocal plots (supporting information) were used to ascertain the mode of inhibiti on, and hence the correct equatio ns for use in evaluating the inhibition constants (47, 216) Kic and Kiu, and the formyl-CoA KM were determined by fitting initial velocity plots of CoA inhibition direc tly with the Michaelis-Menten equation for mixedtype (Eqn. 3) or competitive inhibition (Eqn. 4). KM values for oxalate and succinate and Kia could then be determined by fitting initial velocity plots using an ordered bi-bi equation when the second substrate (oxalate or succinate) was va ried (Eqn. 1). In the cas e of YfdW, attaining saturating formyl-CoA concentrations proved to be impractical. In this case, apparent KM and Vmax values for either oxalate or succinate were ob tained by fitting to the initial velocity plots at

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111 fixed, varied formyl-CoA concentrations and va rious concentrations of the appropriate acid acceptor. Linear fits to the replots of ( KM/ Vmax)app and (1/ Vmax)app against [formyl-CoA] were then used to estimate KM and Kia (216). All curve fitting was performed with KaleidaGraph 3.5 (Synergy Software, Reading, PA). Determination of the Specific Activity of FRC and YfdW w ith Alternate Substrates The specific activities of FRC with alternate substrates were determined by incubating 8.8 nM enzyme (83 ng FRC in 200 L) in 60 mM pot assium phosphate, pH 6.7, 125 mM in the CoA acceptor, and 80 M in the CoA donor at 30 C. In the case of YfdW, 11.2 nM (108 ng YfdW in 200 L) was used with 75 mM acceptor and 350 M CoA donor. Specific activities for YfdW with succinate or oxalate were determined from initial velocity experiments. Substrates were regarded as having no activity when no products were detected in reactions that were run for 60 min. Crystallization and Structure Determinat ion of the W48F and W48Q FRC Variants Crystallization and analysis of FRC variants was carried out by Dr. Catrine L. Berthold at the Karolinska Institutet, Stockholm Sweden. Crystalliza tion of the FRC mutants was performed by the vapor diffusion method in 24-well plates where hanging drops of 2 L protein solution and 2 L well solution were set up to equilibrate against 1 mL well solution at 293 K. A protein solution containing 7.5 mg/mL of the desi red mutant in 50 mM MES buffer, pH 6.2 with additional 10% glycerol was used when screenin g for optimal conditions for crystallization. The W48Q variant of FRC was crysta llized against a we ll solution of 1.35 M sodium citrate and 0.1 M HEPES buffer, pH 7.2-7.4, resulting in crys tals of the tetragona l space group I4. These crystals were protected in a cr yosolution of three parts well so lution mixed with one part 100% ethylene glycol before being flash-frozen in liquid nitrogen. For the W48F FRC mutant a well

PAGE 112

112 solution of 1.9 M malic acid, pH 7.0, gave crysta ls belonging to the monoclinic space group C2. The crystallization drops containi ng the W48F mutant were covere d in silicon oil, through which the crystals were dragged before being flash-frozen. X-ray data were collected in a nitrogen stream at the beamlines ID14 eh1 and ID23 eh2 at the European Synchrotron Research Facility, Grenoble. All crystallographic data were pr ocessed with MOSFLM (147) followed by SCALA of the CCP4 program suit (11). The structure of the apoenzyme (pdb code: 1p5h) (197) was used to retrieve the phases by molecular replacement using the program MOLREP (248). Refinement was carried out with REFMAC (174) and manual model building was performed in COOT (76) where water molecules were assigned and the stru ctures were validated. The stereochemistry of the structures was checked with PROCHECK (144). Formyl-CoA Hydrolysis in the Presence a nd Absence of F RC, D169S, and YfdW Pseudo-first order rate constants for the hydr olysis of formyl-CoA at pH 6.7 and 30C were determined by standard fitt ing procedures. Half-lives fo r hydrolysis in the presence of FRC, D169A, and YfdW were 51, 58, and 198 mi nutes, respectively. Formyl-CoA hydrolysis has been reported with a half-life of 150 minutes at pH 6.7 and 30C (124). Cloning, Expression, and Purifica tion of HisYfdU and HisYfdW The E. coli genes yfd W and yfdU were cloned from genomic DNA isolated from BL21(DE3) by nested PCR. DNA was purified by phenol-chlorofor m extraction (208). The first PCR primers were 5CGC CTG GCC GGT GTT GGC GTA ATG G and 3-5CCC TGT TTG CCC GAG TAA TAG ATA CAA ATA GAG CCG C. Nested primers were designed to include upstream Nde1 and downstream HindIII restriction endonuclease sites: HisYfdW 5A GGT ATT CAT ATG TCA ACT CCA CTT C AA GGA ATT AAA GTT CTC GAT TTC, His YfdW 3-5GGG AGC AAG CTT CCC CCG TTA ATA TCA GAT GGC G, HisYfdU 5CGA GGT TAT TAC ATA TGT CAG ATC AAC TTC AAA TGA CAG ATG G and HisYfdU

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113 3-5-CTC ACC ATC GCA TAA TGA GTT AAG CTT AGG AGA CGA TGT CAG. The second PCR products were digested with Nde1 and HindIII and inserted into gel-purified pET28b (Novagen) linearized with the same restrict ion enzymes. PCR primers were obtained from Integrated DNA Technologies, Inc. (Coralville IA). Constructs were confirmed by DNA sequencing by the DNA Sequencing Core of the Interdisciplinary Cent er for Biotechnology Research at the University of Florida. A single BL21(DE3) cell transformed with eith er the HisYfdU or Hi sYfdW construct was used to inoculate a culture of LB with 50 g/m L kanamycin. The culture was allowed to grow all day at 37 C and shaken at 215 rpm. At 4 pm, 1 mL of the culture was used to inoculate 500 mL of ZYM 5052 autoinducing media (239). The culture, shaken overni ght at 37 C and 215 rpm, was harvested by centrifuga tion at 5000 xg for 10 minutes (OD600 of ~6). The His-tagged protein was purified as described previously in this chapter. Activity of HisYfdU The activ ity of HisYfdU was assayed as previo usly described (15). Prior to analysis, HisYfdU was incubated with 60 M ThDP on ice fo r at least 30 minutes. In a total volume of 100 L, 3.4 nM HisYfdU was combined with 60 M ThDP and 6 mM MgCl2, in 60 mM potassium phosphate, pH 6.7. The reaction was started with the additi on of the appropriate amount of oxalyl-CoA. The reaction was que nched with 30% HAc and the formyl-CoA produced was measured by single point HPLC assay.

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114 CHAPTER 4 OXALYL-COA DECARBOXYLASE3 Introduction Oxalyl-co enzyme A (CoA) decarboxylase (OXC) is one of two enzymes in the oxalate degradation pathway in the gastrointestinal bacterium Oxalobacter formigenes (10). OXC is a typical thiamine diphosphate (ThDP)-dependent nonoxidative decarboxylas e, converting oxalylCoA to formyl-CoA and CO2. In the catalytic cycle (Figure 4-1) that is almost certainly common to all ThDP-dependent enzymes in this family ( 67), turnover is initiated by activation of ThDP through deprotonation of C2 in the th iazolium ring to give the ylide [ 1 ]. This is facilitated by a conserved glutamate, which donates a hydrogen bond to N1 of ThDP and stabilizes the 1,4imino-pyrimidine tautomer enabling the 4NH to abstract a proton from C2 (134, 151). Nucleophilic attack by the cofactor ylide on the -carbonyl of the substrate [ 2 ] and protonation of the oxygen atom of the carbonyl then gives ri se to a covalent substrate-ThDP adduct [ 3 ]. The positively charged thiazolium ring then facilitates decarboxylation to form an carbanion/enamine [ 4 ] complex that is protonated at the -carbon [ 5 ] before C-C bond cleavage takes place to yield the product [ 6 ] and the ylide, completing the catalytic cycle. Previously, it was reported that the first crystal structure of the OXC holoenzyme, a homotetramer with each of the 60 kDa subunits containe d one tightly bound ThDP, Mg2+, and ADP (15). The presence of ADP is required for maximal decarboxylase acti vity, presumably because it stabilizes the functional conformation of the enzy me. On the basis of this struct ure, a catalytic mechanism for the formation of formyl-CoA was proposed. In addition, it was specula ted that the 4-amino group of the pyrimidine ring of ThDP might be i nvolved in stabilizing the developing negative 3 Reproduced in part with permission from Structure, Vol.15, Berthold, C. L., Toyota, C. G., Moussatche, P., Wood, M., Leeper, F., Richards, N. G., and Lindqvist, Y. Pages 853-861. Copyright 2007 Cell Press.

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115 charge on the oxygen bound to the -carbon atom as the substrat e reacts with the ylide intermediate and that a water molecule, anchor ed by hydrogen bonds to the side chains of Tyr120, Glu-121, and the main chain carbonyl oxyge n atom of Ile-34, protonates the carbanion/enamine intermediate formed after de carboxylation. Here, the structures of OXC in complex with its substrate oxalyl-CoA [ 2 ], with its product formyl-CoA [ 6 ], and with a trapped covalent reaction intermediate [ 5 ] are presented (numbering from Figure 4-1). In the substrate complex [ 2 ], a ThDP analogue, 3-deazathiamine diphos phate (dzThDP), was used in place of ThDP to prevent turnover. In order to further substantiate these findings two additional X-ray crystal structures are presented: a reference structure containing only dzThDP as well as a structure of active OXC in complex with CoA. Co mbining the structural data with kinetic data from several active-site varian ts has allowed profound insight in to the catalytic mechanism of OXC. Results Structure of OXC with dzThDP The thiam ine-analogue dzThDP is an extrem ely efficient inhibitor of ThDP-dependent enzymes (145, 167). As a substrate analogue, dzThDP is almost identical to ThDP, but with the nitrogen atom of the thiazolium ring exchanged for a carbon atom; the lack of positive charge prevents formation of the activated ylide and no attack on the substrate can take place. The charge state of dzThDP, however, mimics the ylid e with an overall neut ral thiazolium ring and has been shown to bind more tightly than ThDP to several of the enzymes utilizing the cofactor (145). To be able to draw conclusions from the structure of a nonreac tive substrate complex containing dzThDP, it was desirable to study struct ural changes that resulted from exchanging ThDP with dzThDP.

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116 Figure. 4-1. Scheme of OXC mechanism for ThDP-dependent oxalyl-CoA decarboxylation. 1 ThDP-ylide; 2 oxalyl-CoA; 3 pre-decarboxylation intermediate; 4 -carbanion/ enamine intermediate; 5 formyl-CoA-ThDP covalent complex; and 6 formyl-CoA. Crystal structures of OXC with oxa lyl-CoA bound, OXC with formyl-CoA, and covalent intermediate are highlighted.

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117 Figure. 4-2. OXC tetramer and active site. A the OXC tetramer repres ented with one of the catalytic dimers in a surface mode. ADP dzThDP, and oxalyl-CoA are represented as balls-and-sticks The C-terminal region that undergoes organization upon substrate binding is shown in red B the substrate binding site with side chains interacting with oxalyl-CoA shown as sticks The C-terminal residues after Arg-555 have been omitted for clarity. The main chain of two residues in the newly organized C-terminus is shown coloured in red Taken from Berthold 2007 (17). The structure of OXC with bound dzThDP, refined to 2.2 resolution (Table 4-1), is virtually identical to the holoenzyme structure so lved previously (rmsd, 0.24 for 546 C atoms) (15). Structure of the Oxalyl-CoA Complex and the CoA Complex The substrate binding site in OXC was iden tified by crystallizing OXC inhibited with dzThDP and then soaking oxalyl-CoA into the crystals. The structure was refined to 2.0 resolution. T he substrate is bound with the CoA carri er in the cleft betwee n the regulatory (R)and pyrophosphate (PP)-domain of one subunit, and, with a length of approximately 30 it reaches all the way into the active site where the oxalyl group is well positioned for attack by the cofactor (Figure 4-2). No significant reorganiza tion of the active site takes place upon substrate binding and superposition of the hol ostructure to the substrate complex results in an rmsd of 0.286 for 546 C atoms. The only significant structural changes upon substrate binding are

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118 seen at the C-terminus of OX C. The C-terminal residues 553, which, in the structure of the holoenzyme, are flexible and w ithout interpretable density (15) organize upon substrate binding and fold down over the active site (Figure 4-2A). Of the 1,070 2 total accessible surface of the substrate, 920 2 are buried upon binding to the enzyme; the C-terminal residues contribute approximately 200 2. The side chain of Arg-555 forms inti mate contact with the substrate by a hydrogen bond network bridging the diphosphate and the 3-phosphate of the ribose in the CoA moiety (Figure 4-2B). The main chain of re sidues 263 form a loop on the other side of the ribose ring and keep it in place by three direct hydrogen bonds and one linked by a water molecule. The diphosphate is positioned between the three arginine residues, 266, 408, and 555. Most of the interactions between the substrate and protein are formed between the ribose and diphosphate part of the substrate. Ther e are only a few hydrogen bonds linked by water molecules to the rest of CoA. The oxalyl gr oup of the substrate is precisely positioned by hydrogen bonds to all three substrate oxygen atoms. One of the carboxylate oxygen atoms is held by Tyr-483 and Ser-553 and the other by the ma in chain amino group of Ile-34 (Figures 4-3 A Figure. 4-3. Three snapshots of OXC intermediates. A close-up of oxalyl-CoA binding [ 2 ]. B structure of the postdecarboxylation intermediate [ 5 ]. C structure of the product complex [ 6 ]. For all images, the C-terminus after residue Arg-555 has been omitted for clarity. Residues from di fferent subunits are coloured differently and red spheres represent water molecules. Taken from Berthold 2007 (17).

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119 and 4-4 A). Tyr483 is in a strained conformation in the disallowed part of the Ramachandran plot and Ile-34 is followed by the conserve d Pro-35, the amide of which adopts a cis conformation (98). Consiste ntly, in all determined structures of OXC, a water molecule (W1) is observed, bound between the side chains of Tyr-120 and Glu-121 and the carbonyl oxygen atom of Ile-34. The substrate C -carbonyl oxygen atom makes hydroge n bonds to Tyr-120 and W1, bridging to Glu-121 and the cis-Pr o-35 loop. The substrate is thus perfectly positioned for attack by the activated cofactor with a distance of approximately 3 between the substrate C atom and C2 of the ThDP thiazolium ring. The structure of the CoA complex containing ThDP, solved to 2.2 resolution, is virtually identical (rmsd, 0.181 for 559 C atoms) to the substrate complex; the structured C-terminus also folds over CoA. Structure of a Trapped Covalent Intermediate A transien tly accumulated covalent intermediate [ 5 ], formed after attack of the C2 of the ThDP thiazolium ring on the substrate C atom and after decarbox ylation, was trapped in Figure. 4-4. Annealed composite omit maps calculated for the structures shown around the active site: in A the oxalyl-CoA complex [ 2 ]; B the postdecarboxylation intermediate complex [ 5 ]; and C the product complex [ 6 ]. The contour level is 1 Residue labels can be seen in Fi gure 4-3. Taken from Berthold 2007 (17).

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120Table 4-1. Data collection and refinement statisti cs for OXC structures Ligand Complex Substrate [ 2 ] Intermediate [ 5 ] Product [ 6 ] CoA Cofactor dzThDP ThDP ThDP dzThDP ThDP Data collection statistics Resolution () 152-2.06 (2.17-2.06) 76.0-1.82 (1.92-1.82) 51.92-2.15 (2.27-2.15) 63.89-2.20 (2.32-2.20) 63.89-2.20 (2.32-2.20) Cell axis a = b, c () 127.0, 151.8 126.2, 151.9 127.6, 152.0 127.7, 152.4 127.7, 152.1 Rmerge 0.104 (0.503) 0.090 (0.480) 0.100 (0.246) 0.137 (0.430) 0.008 (0.172) Mean ((I)/ (I)) 11.8 (2.5) 10.2 (2.0) 9.8 (2.8) 14.5 (2.4) 10.1 (3.8) Completeness (%) 96.2 (74.2) 99.2 (99.9) 98.8 (96.9) 97.8 (87.8) 99.2 (99.4) Wilson B-factor (2) 25.4 18.7 26.7 35.0 27.7 Refinements Statistics Resolution () 30.0-2.06 30.0-1.82 25.0-2.15 30.0-2.2 30.0-2.2 Reflections work/test set 76,781/3,994 112,438/5,831 69,803/3,658 64,803/3,413 65,382/3,439 Number of residues 1,118 1,118 1,115 1,094 1,118 Number of waters 908 1185 615 618 666 Rfact/Rfree (%) 17.4/21.2 15.0/17.6 18.9/23.1 17.7/21.5 19.9/23.8 Rmsd from ideal Bonds ()/Angles () 0.009/1.34 0.008/1.43 0.009/1.30 0.08/1.26 0.008/1.18 Ramachandran residues in region (%) Most favoured 88.9 89.7 89.5 89.8 89.4 Additional allowed 10.7 9.9 10.1 9.7 10.1 Generously allowed 0.2 0.1 0.2 0.2 0.2 Disallowed 0.2 0.2 0.2 0.2 0.2 Occupancy of ligands 1.0/1.0 0.8/1.0 0.6/0.8 -1.0/1.0 PDB deposition ID 2ji6 2ji7 2ji8 2ji9 2jib

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121 Figure. 4-5. Stereoview of postdecarboxylation intermediate complex data refined with both the enamine form [ 4 ] and protonated intermediate [ 5 ]. The nonplanar protonated intermediate [ 5 ] is shown with grey carbons and the enamine carbanion form [ 4 ] is shown with carbons in cyan. Both are viewed along the thiazolium plane. The annealed composite omit map is contoured at 1 and shows a better fit for the protonated intermediate. Take n from Berthold 2007 (17). crystallographic freeze-trapping experiments and re fined to 1.8 resolution (Figures 4-3B and 4-4B). No structural changes are observed when compared to the holoenzyme (rmsd, 0.216 for 546 C atoms) except the ordering of the C termi nus. The OXC intermediate was best modeled into the electron dens ity with a nonplanar C conformation (Figure 4-5), and not as the stabilized enamine previously seen in pos tdecarboxylation intermediate co mplexes of transketolase, the dehydrogenase of the Thermus thermophilus HB8 branched chain -ketoacid dehydrogenase complex (178), and pyruvate oxidase (POX) (262). The C is best modeled as lying slightly out of the thiazolium plane, although th is observation is at the limit of the error at this resolution. Whether the density in the calculated omit map (F igures 4-4B and 4-5) corresponds to a single homogenous intermediate or a mixture of intermedia tes at different states can not be certain, but modeling other intermediate conformations with varying occupa ncy does not improve the model and results in increased amounts of negative el ectron density in the difference map. In the

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122 intermediate structure, the C -OH and 4-NH of ThDP form a close contact, with a hydrogen bond distance of 2.55 The C -OH also makes a hydrogen bond to Tyr-120 (Figure 4-3B). At the approximate positions of the substrate carboxyl oxygen atoms in the oxalyl-CoA complex, two water molecules (W2 and W3 in Figure 4-3B ) are bound. W3 also interacts with Ser-553 and Tyr-483. W2 makes a hydrogen bond to the main chain nitrogen of Ile-34 and is ideally positioned (2.7 ) to have donated a proton to the C atom, and further, proton transfer to the carbanion is likely to have occurr ed. Thus, the structure repres ents the covalently bound product ( vida infra ). Structure of the Formyl-CoA Complex The product com plex [6] was formed by so aking crystals of ThDP-bound OXC in a solution of formyl-CoA (Figures 4-3C and 44C). No structural changes in the protein framework are observed in OXC complexed with formyl-CoA when compared with the Figure. 4-6. Initial velocity plot of S553A OXC variant. Oxalyl-CoA concentration was varied form 10 500 M and enzyme concentration was 9 nM. Inset progress curve for the formation of formyl-CoA over time.

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123 holoenzyme with the exception of the orderi ng of the C-terminus (rmsd, 0.237 for 546 C atoms). In the product complex structure, there are some rearrangements compared with the substrate complex around the CoA sulfur atom (Figure 4-6). The carbonyl group of formyl-CoA forms a hydrogen bond via a water molecule (W2) to the main chain ni trogen of Ile-34. Kinetic Validation of Active Site Residues Deduced from the OXC Crystal Structures Several site-directed OXC m utants and a vari ant containing a truncated C-terminal region were expressed and kinetically characterized by standard methods (Table 4-2) (15). Catalytic activity was abolished in the C-terminal truncat ion mutant and the OXC variant in which Glu-56 was replaced by alanine. This glutamate residue is strictly conserved in the POX family, and is needed for ThDP activation by promoting formation of the 1,4-iminopyrimidine tautomer of the cofactor, which then facilita tes deprotonation at C2. Mutation of this glutamate persistently results in severely reduced activity in all ThDP -dependent enzymes studied with the exception of glyoxylate carboligase which has a valine in place of the otherwise conserved glutamate (130). As expected, size exclusion expe riments showed that all the pr epared variants eluted as tetramers, but interestingly that the E56A mutant eluted with a retention time corresponding to that of a dimer. This disruption to the quatern ary structure of OXC might be a consequence of impaired cofactor binding due to the loss of the important hydrogen bond between Glu-56 and the N1 of ThDP. While truncation of the C-terminus, disorder ed in the holoenzyme, abolished activity, mutation of Arg-555 gave a significant rise in KM without affecting kcat. Replacement of Tyr120, Glu-121, Tyr-483, or Ser-553 with alanine resulted in significan tly reduced activity of the enzyme (Table 4-2) without greatly affecting the KM, showing that all four are important for

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124 Table 4-2. Summary of ki netic data for OXC, OXC variants, and HisYfdU. Enzyme KM, M kcat, s-1 %WT kcat/ KM, s-1M-1 OXC (15) 23 3.5 88 4 100 3.8 x 106 E56A --0 -Y120F 43 9 7.2 0.6 8.2 1.7 x105 Y120A 60 14 0.26 0.03 0.3 4.1 x 103 E121Q 18 4 3.3 0.3 3.8 1.8 x 105 E121A 41 8 0.1 0.01 0.1 2.4 x 103 Y483F 40 11 1.7 0.2 1.9 4.1 x 104 Y483A 24 7 1.4 0.1 1.6 5.6 x 104 S553A 21 5 13 1.5 15 6.2 x 105 R555A 66 8 85 4 96 1.3 x 106 553-565 --0.001 -HisYfdU 180 39 15 1 -8.6 x 104 efficient catalysis. However, the fact that act ivity is not abolished in any of these mutants suggests that none of them participate di rectly in the proton transfer reactions. Discussion C-Terminal Organization Upon Substrate Binding The structure of OXC in com plex with CoA rev eals that binding of th e carrier CoA is what induces the structural organizati on of the C-terminal 13 residues. The presence of the dzThDP analogue, with no net charge and thus an excelle nt mimic of the ylide state of ThDP, does not have this effect in the absence of CoA, sugge sting that deprotonation of ThDP alone is not inducing this conformational change. Formation of the ylide was previously suggested to trigger a loop to close down over the pyruvat e dehydrogenase E1 subunit from Escherichia coli (PDHE1) active site (145). The C-terminal segment cl oses down over the substrate and thus provides much of the binding energy, which explains w hy none of the single mutations have drastic effects on the KM. There are still sufficient interactions such that th e tight binding of the CoA carrier remains. On the other hand, the OXC muta nt with a truncated C-terminus can no longer bind the substrate and is inactive. It has previously been suggested for both acetohydroxyacid synthase (AHAS) and Zymomonas mobilis pyruvate decarboxylase (zPD C) that the C-terminus

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125 might play an important role in the catalytic cy cle by moving aside to let the substrate access the active site and then closing down during catalysis (40, 136). These structural data provide evidence for this hypothesis by showing that substrate binding in OXC clearly induces Cterminal folding. Substrate Alignment fo r Ylide Attack The oxalyl-CoA binding site is organized to perfectly position the substrate for nucleophilic attack by the ThDP-ylid e. For the attack to occur, the negative charge developing on the C -carbonyl oxygen of the oxalyl-m oiety must be stabilized. The cofactor 4-NH2 was suggested for this task (15), a nd here the structural data show this to be probable. Jordan et al (160) have also concluded th at the predecarboxylation intermed iate exists in its 1,4iminopyrimidine form in agreement with th e postulated mechanism in which the 4-NH2 is responsible for proton donation. The carboxyl group of the substrate is perpendicular to the positively charged thiazolium ring, which promotes decarboxylation by permitting overlap of the sand p*-orbitals (68, 246). Tyr-120, Tyr-483, and Ser-553 participate in positioning the oxalyl group in the active site. A lthough mutating these residues only has a minor effect on KM, the specificity constant is severely reduced, demonstrating their impor tance in aligning the substrate favorably for cofactor attack. Postdecarboxylation Intermediate A postdecarboxylation interm ediate complex [ 5 ] could be observed in crystals soaked with oxalyl-CoA at 4 C for 8 min; shorter soaks showed no or low occupancy complexes, and longer soak times resulted in a heterogeneous composition of complexes in the crystal where some of the substrate molecules had turned over to formyl-CoA. From this, it can be concluded that the decarboxylation proceeds rapidly, in agreement with existing proposals for other ThDPdecarboxylases (126). A water molecule, W 2, is at 2.7 distance from the C atom of the

PAGE 126

126 intermediate, ideally positioned for transfer of a proton to a C -carbanion intermediate, and provides an explanation for the obs ervation that no single mutation of active site residues (except Glu-56) abolishes catalysis. Replacement of resi dues Tyr-120 or Glu-121 with alanine has an effect on kcat due to the importance in firmly positioning the water molecule, W1. W1 also makes a hydrogen bond to the Ile-34 carbonyl oxygen with the adjacent Pro-35 in the cis-conformation, an interaction that is cruc ial for positioning the Ile-34 NH group (Figure 4-3). The Ile-34 NH group is involved in binding substrate, and mo st importantly, the water molecule W2, involved in transfer to the -carbanion intermediate. Tyr-483 and Ser-553 also participate in positioning W2 via W3, and there is also a significant reduc tion in the turnover numbe r when these residues are replaced by site-directed mutagenesis. The short distance between water molecules W2 and W3 (2.5 ) suggests that W2 might have some hydroxide ion charac ter, which would be consistent with a proton having been transferred to the -carbanion. Thus it appears that the covalently bound product is present, indicating that in the catalytic cycle, product release from the cofactor is the slowest step of the reaction cata lyzed by OXC. There is also a close contact between C -OH and 4-NH2, 2.55 which confirms particip ation of the 4-amino group of ThDP in proton transfers to and from the s ubstrate carbonyl oxygen and stabilization of the intermediate. In OXC, the enamine/ -carbanion before protonati on may be nonplanar and thus has more -carbanion character than would a planar en amine. A planar enamine would not have the proton donor, W2, optimally positioned for proton transfer, and the C carbon would be less basic. The difference from other enzymes in which a planar enamine state has been observed (82, 178, 262) may be explained by the fact that the latter oxidative enzymes require a second acceptor substrate. To allow the intermediate to proceed via the energetically more stable enamine might then be a way for these enzymes to protect the intermedia te from protonation of

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127 C during binding of the second substrate. For OXC, on the other hand, relaxation into the planar enamine structure would only impede the subsequent C protonation step in the proposed mechanism. The complete removal of the st rain and relaxation would then occur only upon product dissociation, and would fu rther drive this process. Until 2006, a planar enamine-like structure was consistently seen in all postdecarboxylation intermediate structures (82, 178, 262). A crystallographic study was then published in which the decarboxylase subunit of the human -ketoacid dehydrogenase complex (BCKDC-E1b), in complex with several substrate analog intermediates, consistently showed a nonplanar (and therefore more -carbanion-like) structure (161 ). Common for the BCKDC-E1b analog complexes and the OXC natural intermed iate structure is the fact that the C is best modeled as lying slightly out of the thiazolium plane. This out -of-plane distortion, as discussed by (161), might force the intermediate into its most active state. When the orbitals are not Figure. 4-7. Stereoview of the aligned OXC structures. The substrate ( green ), intermediate ( pink ), and product complexes ( blue ) are overlayed. Water molecules of three structures are shown in the same colours, but in a lighter shade. The C-terminus after residue Arg-555 has been omitted for cl arity. Taken from Berthold 2007 (17).

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128 completely aligned, the negative C charge cannot be as eff ectively delocalized into the thiazolium ring, the basicity of C is increased, and the subsequent step of the reaction when C is protonated is enhanced. This is likely to be true in this case as well, because it allows the proton donor (W2) to come close to C (2.7 ). The close C -OH-to-4-NH2 contact and out-ofplane distortion was observed in the predecarboxylation intermed iates of PDH-E1 (5) and POX (262). The authors of these studies claimed that the strain was a contributing force for the decarboxylation step and that it would be rela xed upon enamine formation. The strained out-ofplane distortion might persist also in the postd ecarboxylation intermediate of OXC, and could therefore act as a contributi ng force throughout the reaction. Formyl-CoA Release The reason f or the relative stabili ty of the covalent product inte rmediate might be the close contact to the 4-amino group of ThDP. The strained confor mation, with the C out of plane with the thiazolium ring and the positive charge on the thiazolium ring, might be the factors leading to cleavage of the C2-C bond and product releas e from the cofactor. Conclusions on Catalysis in Simple Decarboxylating ThDP Enzymes Common to OXC and many other ThDP-depende nt enzym es catalyzing a decarboxylation, mutations of active site residues, other than the conserved glutamate needed for activation of the cofactor, hardly ever lead to abolished activity (Table 4-2) (88, 125) showing that only the cofactor itself is essential for catalysis. In agreement with the postulated mechanism for OXC in Figure 4-1 and the structure of the intermediate complex presen ted here, it has been shown by CD data on several ThDP enzymes (126, 176), and directly observed by NMR (134), that the 4N atom of the pyrimidine moiety performs mo st of the acid-base reactions involving the substrate C -carbonyl oxygen atom in the reaction sequence by c onversion between the 4amino and the 1,4-imino form of the cofactor OXC and other related simple decarboxylating

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129 ThDP enzymes achieve a significant fraction of their catalytic power from setting up the central -carbanion intermediate for proton transfer to C (269). In the case of OXC, the proton is derived from a bound water molecule in the active site. The cis-Pro loop containing Ile-34 has a central role in the OXC active site by positioni ng the substrate, the product, and the water molecule, W2, donating the proton to the -carbanion intermediate. The conformation of the loop is maintained by a hydrogen bond network i nvolving the invariant water molecule, W1, in the active site. There have been several stud ies reporting on communicat ion between the active sites through a proton-conducting channel resul ting in alternating s ite reactivity (66, 86, 125, 127). In OXC, there is no proton conduction ch annel and no evidence for alternating site reactivity in the structures of OXC. Although th e electron density of lig ands is often better defined in one subunit than in the other due to different occupancies, in no case are different species visible in the active sites. Transketolase has also been reported to lack alternating site reactivity (82). Experimental Methods Protein Expression, Mutagene sis, and Purification OXC was produced reco mbinantly in E. coli as described previously (15, 16). Sitespecific OXC variants were produced by the QuikChange site-directed mutagenesis method (Stratagene) or the overlap extension method (109), and were expressed and purified according to protocols used for the wild-t ype enzyme (15, 16). Briefly, this procedure includes affinity chromatography on a Blue-sepharose fast flow affinity column followed by desalting on Sephadex G-25 size-exclusion column, and fu rther purification by QHP anion-exchange chromatography. The C-terminal truncation variant lost the capacity to bind to the affinity column, but could be retained and purified to homogeneity by ion-exchange chromatography. The OXC apoenzyme was prepared by dialysis overnight against 50 mM Tris (2-amino-2-

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130 (hydroxymethyl)propane-1,3-diol) buffer, pH 8.5, containing 1 mM dithiothreitol and 1 mM EDTA, followed by buffer exchange into 50 mM MES (4-morpholine-ethanesulphonic acid) buffer, pH 6.5. No activity was observed for th e OXC apoenzyme, or for apoenzyme incubated with dzThDP and Mg2+, although full activity was regained when ThDP and Mg2+ were added to the apoenzyme. Samples of oxalyl-CoA and fo rmyl-CoA were prepared and purified as described previously (124). Enzyme Assay The OXC va riant enzymes were assayed by th e previously described high-performance liquid chromatography point assay mon itoring the formation of formyl-CoA (15) Typically, the enzyme was diluted with 25 mM sodium phospha te buffer, pH 6.5, containing 300 mM NaCl, to a final concentration of about 0.7 mg/ml, and initi al velocities were reco rded as a function of oxalyl-CoA concentration (10 mM). Crystallization and Complex Formation Crystallizations were perfor med Dr. Catrin e Berthold by the hanging drop vapor-diffusion method under conditions very similar to those desc ribed previously for the holoenzyme structure (16). Well diffracting crystals were produced wi th a precipitating solution containing 0.5 M CaCl2, 0.1 M BisTris propane (2-[bis(2-hydroxy ethyl)amino]-2-(hydroxymethyl) propane-1,3diol), pH 6.5, and 26% polyethyle ne glycol 550 monomet hyl ether. In contra st to the holoenzyme crystals (16), no twinning was detected among the complexes. The dzThDP-inhibited (5 mM) OXC crystals were produced at 20C, while active OXC containing ThDP was crystallized at 4 C through streak seeding after 2 hr equilibration with the well solution. The lower temperature facilitated the freeze-trapping experiments by re ducing the reaction rate. The CoA complex was produced by cocrystallization of wild-type OXC with 1 mM Co A. The soaking experiments were performed by transferring the crystals to a new drop c ontaining 2 ml of 0.2 M BisTris

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131 propane, pH 6.5, and 52% polyethylene glycol 5 50 monomethyl ether mixed with 2 ml of 50 mM sodium acetate, pH 5.0, containing either 20 mM oxalyl-CoA or formyl-CoA. Crystals soaked in this mixture remained unaffected more than 12 hr without reduced diffraction quality. The crystals were flash frozen in liquid nitrog en after desired soaking times, and the soaking solution was sufficient as cryoprotectant. Soaking tim es for the substrate, covalent intermediate, and product complexes were 5 min, 8 min, and 12 min, respectively. Crystals inhibited by dzThDP were used to obtain the substrate comp lex by soaking rather than cocrystallization due to the instability of oxalyl-CoA. Data Collection and Structure Determination Data were collected at beam line I711 at M AX-lab in Lund, Sweden, and at ID14 eh1, eh3, and eh4 at the European Synchrotron Research Facility in Grenoble, France and analysed by Catrine Berthold. A summary of all data sets can be found in Table 4-1. All data were processed with Mosflm (146) and then scaled and further pr ocessed with programs in the CCP4 suite (11). Due to slight shifts in length of cell axes, mo lecular replacement with the previously solved holostructure as a search model (PDB code: 2c31) was used for phasing. The Rfree set was imported from the holoenzyme structure for a ll data sets. REFMAC5 (175) was used for refinement and model building as well as wa ter assignment were performed in COOT (76). Atomic displacement parameters were refined in REFMAC by the TLS (translation, libration, screw) method, with each of the two monomers in the asymmetric unit treated as a single TLS group. The soaked ligands were not included until the e nd of the refinements. Libraries were created with the Dundee PR ODRG2 server (http://davap c1.bioch.dundee.ac.uk/programs/ prodrg/). Refinement of the cova lent intermediate was performed in parallel with restrains for a planar and tetrahedra l conformation around C The geometric restrains were then loosened toward the end of refinement. Annealed omit maps calculated in CNS (29) were used to confirm

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132 the conformations at the active site (Figure 4-4), and the geometry of the refined structures were checked with PROCHECK (144). All Figures of protein molecules were produced with PyMol (59).

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133 CHAPTER 5 SUMMARY Kinetic Mechanism of Family III CoA Transferases Fam ily III CoA transferases differ from Family I transferases both in sequence similarity and in kinetic mechanism. Hydroxylamine a nd borohydride trapping experiments, as well as more direct evidence from MS and crystallographi c data, have demonstrated that, despite their divergent kinetic mechanisms, both Families mediat e the transferase reaction through a covalent enzyme CoA thioester intermediate. The form yl-CoA transferases, a subgroup of Family III CoA transferases, rely on a conserved flexible loop comprising four glycine residues to protect labile reaction intermediates and contribute to su bstrate specificity. Analysis of two formyl-CoA transferase homologs from E. coli and O. formigenes demonstrates that modification of the residues near the glycine loop, but outside of the active site, confers 100-fold increase for oxalate affinity and replacement of these residues with al anine severely impairs the ability of the enzyme to catalyze the formation of oxalyl-CoA from formyl-CoA FRC and OXC from E. coli This work has clearly demonstrat ed that YfdW and YfdU from E. coli are a formyl-CoA transferase and an oxalyl-CoA decarboxylase, re spectively; these enzymes are functionally homologous to FRC and OXC from O. formigenes The submillimolar KM values for formylCoA and oxalyl-CoA are an order of magnitude larger than for the O. formigenes enzymes, but are similar to each other as is expected with a complementary enzyme pair. The turnover number for oxalate with YfdW is 100 times higher than for the O. formigenes enzyme. Thus, despite the critical nature of the FRC reaction, YfdW is more st ringent for using formyl-CoA and oxalate than is FRC. In an X -ray structure (1pt8), oxalate is s een bound outside of the active site

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134 Figure.5-1. Pair-wise sequence alignment 10 around the active sites of FRC and YfdW. Residues that differ are highlighted in grey. in YfdW. This binding pocket, blocked by Trp-48 in FRC, has been identified as the basis for substrate inhibition of YfdW by oxalate. Due to th e exceptional similarity in active site residues (Figure 5-1) differences in catalyt ic specificity are likely the result of altered motion seen in the mobile tetraglycine loops in the two enzymes. The physiological role of YfdW and YfdU in E. coli remains unclear. It appears that YfdW was derived from FRC (Figure 5-2; bootstra p values in Appendix C) Sequence similarity remains high and, rather than losing specificity fo r oxalate, YfdW has evolve d tighter specificity. Both the yfdXWUVE operon and yhjX gene are induced by low pH and, in contrast to OXC, YfdU is not stimulated by ADP. Thus it seems that the yfdW and yfdU genes continue to be physiologically relevant in E. coli and evidence suggests a role in acid resistance rather than metabolism. Future Work Folding If the com plete tertiary structure is cons idered, knots in proteins are not uncommon. Disulfide bridges and links through metal centers can lead to knotted or closed loops (150). There are examples of proteins with true knots in their peptide backbone structure: trefoil knots in members of the / -knot superfamily of me thyltransferases (MTases) including TrmH from Auifex aeolicus (189), AviRb from Streptomyces viridochromogenes (173), TrmH from Thermus thermophilus (179), and YibK from Haemophilus influenzae (165), and in the chromophore-

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135 Figure. 5-2. Phylogram of FRC, putative form yl-CoA transferases, and known Family III CoA transferases from pair-wise sequenc e alignment of polypeptide sequences.

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136 binding domain of Deinococcus radiodurnas phytochrome (254); and figu re-of-eight knots in the crystal structure of the plant protein acetohydroxy acid isomeroreductase (243). FRC is similar to the disulfide-linked exampl es above with the exception that its two monomers are non-covalently linked through each other. A preliminary melting curve monitored by CD and fluorescence shows that the dimer denatu res at 57 C (Figure 53) in a process that appears to be irreversible (data not shown). Proteolytic MS data (Figur e 2-14) suggest that the central dimer interface which includes -helix-9 from both monomers is exceptionally resistant to enzymatic digestion. An FRC heterodimer, comprising an active monomer and an inactive monomer where Asp-169 has been replaced with alanine, was successfully overexpressed in E. coli The mechanism by which E. coli and O. formigenes (and all other organisms that employ this interlocked dimer scaffold) form the active dimer in vivo is an interesting problem and deserves careful study. Monitoring of the fluorescence of native tryptophan re sidues is a method that can be used to examine FRC folding(204). Trp-109 is found on -helix-6 in the large dom ain of FRC. It is located 10 and 24 from surface residues Ser-389 and Glu-394, respectively, both found on the linker region near the C-term inus of FRC. These residues are candidates for mutation to cysteine residues for labelling with IAEDANS; they should provide excellent reporters on unfolding FRC. Another good experiment will use ANS, or 8-anilino-1-naphthalenesulfonic acid (see Figure 5-4), an envir onment-sensitive fluorophore which is virtually nonfluorescent in water, but can be used as a probe for hydrophobi cityin nonpolar environments it emits a blue fluorescence with quantum yield ~0.70 (238). Thus, as FRC is treated with increasing concentrations of chaotropic agents, increased binding of ANS with exposed hydrophobic

PAGE 137

137 regions can be monitored fluorescently to charact erize folding intermediate populations as seen in experiments on -lactamase (96) and the trp repressor (166). 020406080100 -20 -18 -16 -14 -12 -10 CD290nm (millidegrees)Temperature (oC) Figure. 5-3. Melting curve for FRC monitored by CD at 290 nm. Due to the irreversible nature of FRC dimeri zation, it seems likely that chaperones play an important in the expression of the act ive enzyme. If this is true, both E. coli and O. formigenes, must both express this chaperone. A simple i mmuniprecipitation experiment in conjuction with proteolytic MS will likely identify this theore tical protein. As both FRC and YfdW can be expressed with polyhistidine fusion tags, commercial ly available anti-His antibodies can be used to coprecipitate the transferases with any proteins associated in their synthesis.

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138 Dynamics Clearly the tetraglycine loop (Gly-258-261) in F RC and the flexible C-terminus of OXC are important for efficient catalys is. Despite the vast amount of crystallographic data on the FRC structure, there are no data that confirm the catal ytic competence of the glycine loop movement. Protein folding studies monitor global changes in protein structure usi ng spectroscopic methods like UV/vis absorption, fluorescence detection, and ci rcular dichroism. Protein dynamics seek to report on conformational changes on catalytic time scales which ar e on the order of microto milliseconds (81). NMR methods have been used successfully to follow fluctuations near protein active sites (135). However, NMR analysis of proteins are extremely rare above 30 KDa (260), and thus complete assignment of the F RC monomer (47.2 kDa) backbone is not likely. Site-directed spin labelling (SDSL) experiments are also able to monitor structural changes in the millisecond timescale (116) and are therefore appr opriate for dynamics studies. FRET has been used to monitor the catalysis-li nked reduction of the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH)(257). Both SDSL and FRET methods require la belling the protein by chemically bonding fluorophores or spin labelling reagents to specif ic amino acids (253). Lysine groups can be derivatized with succinimidyl active ester, isothiocyanate, or sulfonyl chloride activated fluorophores. Sulfonyl chlorides and isocyanates are reactive species that can be used to label hydroxyl groups. Labelling of cyst eine residues is a common appr oach because they are reactive at physiological pH (6.5 8.0). One criterion for a ta rget protein is that th ere are no cysteines, or other target groups, accessible to labelling reagents. Table 5-1 summarizes the 6 cysteine residues per monomer in recombinan t wild-type FRC. Cysteine ti tration experiments can be run to prove that some residues are no t reactive towards labelli ng reagent. One met hod is to label the

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139 Table 5-1: Cysteine residues in wild type FRC. The MS column indicates whether the cysteine residue-containing peptides have been identified in MS expreriments. Conservation Location Interacts MS Environment Mutant Cys-22 Conserved in true FRCs: A, G, T Helix 6 Helix 9 V8DE Large domain; buried, hydrophobic A Cys65 R, S(YfdW), H or C Loop ( 57) possible H-bond with His379 ( 11) no Large domain; partially buried S Cys145 C, A, S, or G Helix 7 Sheet 7 no Large domain; buried, hydrophobic A Cys269 Highly conserved: F or Y Sheet 7 Near Helix 16 no Small domain; near surface, but buried, hydrophobic A Cys293 Conserved in true FRCs: A, S, R, M, L, or V Helix 12 Helix 13 V8DE Small domain, buried, hydrophobic A Cys347 Conserved: A, V, or Y Sheet 9 Helix 16 Trypsin Small domain, buried, hydrophobic A protein, proteolyse, and analyse by mass spectrometric methods. Table 5-1 also summarizes the total sequence coverage of ma ss spectrometric analysis of digests of FRC to date. Tryptophan has been used as a FRET donor in conjunction with ANS or IAEDANS acceptors (R0 = 22 )(219). IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1sulfonic acid, covalently attached to engineer ed cysteine residues has been used for FRET studies (93). FRC has 8 native tryptophan residues: Trp-48, 109, 156, 265, 272, 289, 301, and Figure. 5-4. IAEDANS and ANS.

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140 339. Trp-48 is located in a flex ible loop near the ac tive site. The native fluorescence of Trp-48 may act as a reporter on gross conformational changes in the enzyme or AEDANS-labelled cysteine mutants could be used to monitor ch anges in the environment of Trp-48. Trp-48 is found on a loop connecting helices 2 and 3 and is located near both the tetraglycine loop and catalytic Asp-169. Trp-48 is found packed against the glycine loop residues when in the closed position (124), and if these environments diffe r enough to cause discer nable changes in the FRET donor characteristics of Trp-48, it may be useful as a reporter on conformational changes linked to catalysis.

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141 APPENDIX A PRIMERS USED FOR MUTAGENESIS AND CLONING Primers for pET-28b constructs 5-Nde1 FRC 5-AGG AGA TAT ACA TAT GAC TAA ACC ATT AGA TGG AAT TAA TGT GC 3-FRC BamH1(stop) 5-AAG TCT GGA TCC TCA AAC TAC CTG T 5-Nco1 FRC 5-AAG GAG CCA TGG AGA TGA CTA AAC CGT TAG ATG 3-FRC Xho1(stop) 5-CTG ACC TCG AGA ACT ACC TGC TTG C 5'-BamH1FRC 5-AGG AGA TAT AGG ATC CGA TGA CTA AAC CAT TAG ATG GAA TTA ATG TGC 3-FRC Xho1 5-CCC AGA AAG TCT GAC CTC GAG AAC TAC CTG TTT TGC ATG C Primers for Duet constructs 5-FRC BamH1 5-AGG AGA TAT AGG ATC CGA TGA CTA AAC CAT TAG ATG GAA TTA ATG TGC 3-FRC HindIII(stop) 5ACA GGT AGT TTG AAG CTT AGA CTT 3-FRC Xho1(stop) 5ACA GGT AGT TTG ACT CGA GAG ACT T QUICKCHANGE primers for FRC variants 5-Q17I 5-GTC ATT GCA GGT CCT GCC TGT ACA CAG-3 3-Q17I 5-TGC AAT GAC GTG GGT AAA GTC AAG CAC-3 5-Q17A 5-GCT TGA CTT TAC CCA CGT CGC GGC AGG TCC TGC CTG TAC ACA GAT GAT GGG-3 3-Q17A 5-CCC ATC ATC TGT GTA CAG GCA GGA CCT GCC GCG ACG TGG GTA AAG TCA AGC-3 5-W48F 5-GAT ATG ACT CGT GGA TTC CTG CAG GAC AAA CC-3 3-W48F 5-GGT TTG TCC TGC AGG AAT CCA CGA GTC ATA TC-3 5-W48Q 5-GAT ATG ACT CGT GGA CAG CTG CAG GAC AAA CC-3 3-W48Q 5-GGT TTG TCC TGC AGC TGT CCA CGA GTC ATA TC-3 5-P159R 5-CCG GTT TCT GGG ATG GTC GTC CAA CCG TTT CCG GC-3 3-P159R 5-GCC GGA AAC GGT TGG ACG ACC ATC CCA GAA ACC GG-3 5-G258A 5-GGT GGT AAC GCA GCG GGC GGC GGC C-3 3-G258A 5-GGC CGC CGC CCG CTG CGT TAC CAC C-3 5-G259A 5-GGT GCG GGC GGC CAG CCA GGC TGG-3 3-G259A 5-GCC CGC ACC TGC GTT ACC ACC ACG TGG-3 5-G260A 5-GGC GCG GGC CAG CCA GGC TGG ATG CTG-3 3-G260A 5-GCC CGC GCC ACC TGC GTT ACC ACC ACG-3 5-G261A 5 GGT GGC GGC GCG CAG CCA GGC TGG 3-G261A 5 GCC GCC GCC CGC TGC GTT ACC ACC Nest primers for YfdW and YfdU 5-YfdVUW 5-CGC CTG GCC GGT GTT GGC GTA ATG G-3 3-YfdVUW 5-CCC TGT TTG CCC GAG TAA TAG AT A CAA ATA GAG CCG C 5-YfdW 5-AGG TAT TCA TAT GTC AAC TCC ACT TCA AGG AAT TAA AGT TCT CGA TTT C-3 3-YfdW 5-GGG AGC AAG CTT CCC CCG TTA ATA TCA GAT GGC G -3 5-YfdU 5-CGA GGT TAT TAC ATA TGT CAG ATC AAC TTC AAA TGA CAG ATG G-3 3-YfdU 5-CTC ACC ATC GCA TAA TGA GTT AAG CTT AGG AGA CGA TGT CAG-3 Nested primers for YhjX 5-YhjX nest 5-GCC GTT TTT CCC CAG GCA TAA AGT GC-3 3-YhjX nest 5-GCC CAG TAG CTC GCG GC-3 5-YhjX 5-GCA GGA ATA CTC ATA TGA CAC CTT CAA ATT ATC AGC GTA CCC GC-3 3-YhjX 5-CCA GTA GCT CGA AGC TTA GCA TTA AAG GGA GCC-3

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142 APPENDIX B SUMMARY OF KINETIC CONSTANTS Table B-1. S ummary of all kinetic cons tants for wild-type FRC and variants Table B-2. Summary of a ll inhibition constants and patterns for wild-type FRC and variants. (M) WT-FRC Q17A G258A G259A G260A G261A CoASH competitive mixed-type mixed-type mixed-type mixed-type Kic 16.7 0.7 16.0 0.6 6.0 1.0 55 19 2 1 Kiu -100 14 460 129 290 5 41 1 (M) WT-FRC His-FRC His-YfdW W48F W48Q CoASH competitive competitive mixed-type mixed-type competitive Kic 16.7 0.7 9 7 218 21 11 5 55 19 Kiu --213 16 35 6 290 5 kcat (s-1) KM(F-CoA) ( M) kcat/ KM(F-CoA) (s-1mM-1) KM(oxalate) (mM) kcat/ KM(oxalate) (s-1mM-1) Kia ( M) WT-FRC 5.3 0.1 2.0 0.3 2700 3.9 0.3 1.4 16 2 G259A 1.9 0.1 4.7 0.8 410 12.1 0.5 0.16 0.9 0.6 G260A 0.23 0.02 18 3 13 18.0 1.6 0.012 3 3 G261A 1.65 0.01 26.6 0.9 62 0.47 0.08 3.5 3 2 Q17A 0.12 0.1 3.3 0.5 36 13.2 0.6 0.009 79 3 His-YfdW 130 17 352 4 370 0.51 0.03 255 18 0.1 His-FRC 5.5 0.4 4.7 1.6 1200 1.2 0.3 4.58 22 6 W48F FRC 17.1 0.2 0.7 0.4 24430 1.5 0.3 11.4 10 7 W48Q FRC 5.8 0.3 2.7 0.9 2148 0.43 0.03 13.5 4 1

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143 Table B-3. Steady-state parameters for the formyl -CoA/succinate transfer ase activities of YfdW, FRC and the Trp-48 FRC mutants. Table B-4. Steady-state parameters for OXC Enzyme KM, M kcat, s-1 %WT kcat/ KM, s-1M-1 OXC (15) 23 3.5 88 4 100 3.8 x 106 E56A --0 -Y120F 43 9 7.2 0.6 8.2 1.7 x105 Y120A 60 14 0.26 0.03 0.3 4.1 x 103 E121Q 18 4 3.3 0.3 3.8 1.8 x 105 E121A 41 8 0.1 0.01 0.1 2.4 x 103 Y483F 40 11 1.7 0.2 1.9 4.1 x 104 Y483A 24 7 1.4 0.1 1.6 5.6 x 104 S553A 21 5 13 1.5 15 6.2 x 105 R555A 66 8 85 4 96 1.3 x 106 553-565 --0.001 -HisYfdU 180 39 15 1 -8.6 x 104 Enzyme Formyl-CoA Succinate kcat (s-1) KM(app) ( M) kcat/ KM(app) (mM-1s-1) KM(app) (mM) kcat/ KM(app) (mM-1s-1) Kia ( M) His-YfdW 5.3 0.4 180 14 29.4 80 40 0.07 30 19 WT FRC 149 13 16 2 9312 0.32 0.03 465 0.5 0.4 W48F FRC 42 6 12 6 3500 0.015 0.005 2800 12 8 W48Q FRC 17.9 0.5 6.7 0.9 2672 0.07 0.01 256 9 4

PAGE 144

144 APPENDIX C DENDOGRAMS Figure.C-1. Phylogram of Family III CoA transferases from pair-wise sequence alignment of nucleotide sequences.

PAGE 145

145 Figure.C-2. Phylogram of Family III CoA transferases from pair-wise sequence alignment of amino acid sequences.

PAGE 146

146 Figure.C-3. Phylogram of ThDP-dependent decarboxylases from pair-wise sequence alignment of nucleotide sequences.

PAGE 147

147 Figure.C-4 Phylogram of ThDP-dependent decarboxylases from pair-wise sequence alignment of amino acid sequences.

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169 BIOGRAPHICAL SKETCH Cory Glenn Toyota was born in 1970 in the Key C ity of the Kootenays, a sm all town at the foot of the Rockie Mountains called Cra nbrook. He was studying Ja panese language and illustration (Manga) in Osaka in the early 1990s, wh en he met Lori Lamb, his wife-to-be. After a wedding ceremony in the Mississippi June heat, Cory whisked his new bride back to Canada, where the young couple lived in a double-wide tr ailer for three years in Grand Forks, British Columbia; Cory sold furniture and Lori worked as a baker, travel agent, and cashier at the local supermarket. The next couple years saw them re turn to Cranbrook to help his father Ron with the family furniture and applian ce businessTaks Home Furnishers. When his father closed the business, Cory took the opportunity to go back to college. The small family (now with a Springer Spaniel named Gumbo) travelled to Loris hometown Jackson, MS. Upon graduation from Mississippi College with his Bachelor of Science degree in biochemistry, they pulled up stakes and moved to Gainesville, FL to attend graduate school in chemistry where, under the supervision of Nigel G. J. Richards, Cory bega n to study the metabolic enzymes of a bacterium called Oxalobacter formigenes Despite a lengthy and monument al struggle with an ancient HPLC, Corys time in Gainesvill e brought success in the form of papers published, awards, and fellowships. He and his wife celebrated by having their firs t son, Arwood Takeo, in January 2008. The family (now with a second dog named Weaver) plan to move back to Mississippi where Cory will take a postdoctoral fellowship at the University of Mississippi Medical Center in Jackson working with Michael Hebert.