Citation
Molecular characterization of a bifunctional enzyme (dehydroquinate dehydratase/shikimate dehydrogenase) and the post-prephenate pathway enzymes needed for phenylalanine and tyrosine biosynthesis in Nicotiana SPP

Material Information

Title:
Molecular characterization of a bifunctional enzyme (dehydroquinate dehydratase/shikimate dehydrogenase) and the post-prephenate pathway enzymes needed for phenylalanine and tyrosine biosynthesis in Nicotiana SPP
Creator:
Bonner, Carol Ann
Publication Date:
Language:
English
Physical Description:
xiii, 157 leaves : ill., photos ; 29 cm.

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Antibodies ( jstor )
Chromatography ( jstor )
Complementary DNA ( jstor )
Dehydrogenases ( jstor )
Enzymes ( jstor )
Gels ( jstor )
Plasmids ( jstor )
Purification ( jstor )
Sequencing ( jstor )
Botanical chemistry ( lcsh )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Microbiology and Cell Science thesis Ph.D
Nicotiana -- Chemotaxonomy ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 149-156).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Carol Ann Bonner.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. 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.
Resource Identifier:
030582268 ( ALEPH )
31920213 ( OCLC )
AKF8508 ( NOTIS )

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Full Text












MOLECULAR CHARACTERIZATION OF A BIFUNCTIONAL ENZYME
(DEHYDROQUINATE DEHYDRATASE/SHIKIMATE DEHYDROGENASE)
AND THE POST-PREPHENATE PATHWAY ENZYMES NEEDED FOR
PHENYLALANINE AND TYROSINE BIOSYNTHESIS IN NICOTIANA SPP.














BY

CAROL ANN BONNER


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


1994















ACKNOWLEDGEMENTS

Many thanks to Roy A. Jensen for the inspiration and

guidance throughout my studies for this dissertation. I

thank, also, my committee members, Dr. Richard P. Boyce from

the Department of Biochemistry and Molecular Biology; and

from the Department of Microbiology and Cell Science, Dr.

Lonnie O. Ingram, Dr. Robert R. Schmidt and Dr. Keelnatham

T. Shanmugam for their support and encouragement in

completion of this dissertation. Special thanks to all the

members of our lab who have been a great source of help to

me during these studies. Firstly, to Randy Fischer and

Premila Rao, my most grateful thanks for their help and

encouragement in so many ways from the very beginning of my

studies. Thanks to Astride Rodrigues, Jackie Miller,

Michelle Miller for all the work in the tissue culture lab.

Most special thanks to Tianhui Xia, Genshi Zhao, Prem

Subramaniam, Wei Gu and Jain Song for all the discussion and

work we have done together. I would also like to thank the

other members of the microbiology and cell science

department who have helped me with their knowledge and

advice, especially Philip Miller, for all the information

and insight that he gave me with respect to the molecular

biology portion of my dissertation.

ii
















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS........................................ ii

TABLE OF CONTENTS....................................... iii

LIST OF FIGURES......................................... v

LIST OF TABLES........................................ .. viii

ABBREVIATIONS ........................................... ix

ABSTRACT ................................................. xi

CHAPTERS

I LITERATURE REVIEW AND RATIONALE ............... 1

Aromatic Amino Acid Biosynthesis in Plants.. 1
Rational for Focus upon DQT and SDH.......... 8
Shikimate biosynthesis and quinate
catabolism...................... ........... 9
Multiple Species of Shikimate Dehydrogenase. 12
The Post-Prephenate Pathway in Plants....... 16

II MATERIALS AND METHODS.......................... 19

Organisms and Extract Preparation............. 19
Enzyme Assays................................. 23
Separation of Pathway Enzymes by
Chromatography.............................. 27
Purification of Pathway Proteins............. 29
Polyacrylamide Gel Electrophoresis........... 33
Kinetic Values............................... 34
Inhibition studies........................... 35
Antibodies .............. .................... 35
cDNA cloning and sequencing.................. 37
Biochemicals................................. 43


III PHYSIOLOGICAL PROFILE OF AROMATIC PATHWAY
ENZYMES IN PLANTS............................. 45

Results..................................... 45
Discussion.................................. 59
iii










IV PURIFICATION OF PROTEINS FROM THE AROMATIC
AMINO ACID PATHWAY........................... 62

Results .................................... 62
Discussion.................................. 53

V PROPERTIES OF THE S-PROTEINS................... 76

Results .................................... 76
Discussion.................................. .104

VI SPECIFIC ANTIBODY TO THE BIFUNCTIONAL
S-PROTEIN AND TO TWO POST-PREPHENATE
PROTEINS... ................................. 108

Results..................................... 108
Discussion.................................. .116

VII CLONING cDNAS ENCODING THE BIFUNCTIONAL
S-PROTEIN, ADH AND PAT....................... 118

Results..................................... 118
Discussion.................................. .132

SUMMARY.... ........... .................................. 148

REFERENCES.... .......................................... 149

BIOGRAPHICAL SKETCH ..................................... 157
















LIST OF FIGURES


Figure Pane

1-1 Aromatic amino acid biosynthetic pathway.......... 3

1-2 Multiple compartmentation of pathways in plant
cells ............................................. 7

1-3 Quinate catabolism and aromatic amino acid
biosynthesis...................................... 11

3-1 DEAE-cellulose chromatography of aromatic pathway
enzymes from N. silvestris....................... 48

3-2 DEAE-Cellulose chromatography of aromatic pathway
enzymes from C. sorokiniana...................... 51

3-3 Temperature optimum for activity of prephenate
aminotransferase in Chlorella sorokiniana........ 52

3-4 Growth curve of N. silvestris in suspension
cultures......................................... 56

3-5 Arogenate dehydratase activities followed
throughout a growth curve of N. silvestris....... 58

4-1 Elution profiles of SP-I and SP-II from Celite
545 column chromatography........................ 64

4-2 Elution profiles of SP-I and SP-II from DEAE-
cellulose chromatography......................... 67

4-3 Elution profiles of S-proteins from an NADP'
specific affinity column.......................... 70

4-4 Celite 545 column chromatography overlap of
arogenate dehydrogenase and SP-II................ 72

4-5 Hydroxylapatite column chromatography separation
of arogenate dehydrogenase and SP-II............. 73

5-1 Elution profile of SDH activities from a Celite
545 column run with a shallow gradient............ 78

5-2 Molecular weight determinations by SDS PAGE and
by silver stain gels of the two S-proteins....... 81
v









5-3 Gel filtration chromatography of S-proteins...... 83

5-4 Optimal PAGE activity staining for SDH in crude
extract .......................................... 86

5-5 PAGE activity staining of purified S1- and S2-
proteins......................................... 88

5-6 S-proteins pH optima of optima for activity...... 90

5-7 Temperature optima for S-proteins................ 93

5-8 Thermal inactivation of S-proteins............... 95

5-9 Substrate protection against thermal
inactivation of S-proteins....................... 97

5-10 Saturation curves and double reciprocal plots
for SP-I ......................................... 100

5-11 Saturation curves and double reciprocal plots
for SP-II ........................................ 102

6-1 Precipitation of S-proteins with SP-I specific
antibody on Ouchterlony plates .................. 110

6-2 Effect of SP-I specific antibody on SP-I and
SP-II activities ................................ 113

6-3 Comparison of Western blot and activity stained
gels............................................ 115

7-1 Agarose gels of cDNA encoding aromatic pathway
proteins......................................... 121

7-2 Preliminary alignment of translated sequences of
clone SP3 with established DQT sequences......... 124

7-3 Sequencing strategy for cDNA encoding the
bifunctional protein ............................. 126

7-4 Agarose gels of SP3 cDNA fragments during
stages of subcloning.............................. 128

7-5 Nucleotide and deduced amino acid sequence of
cDNA coding for the bifunctional S-protein....... 131

7-6 Multiple amino acid alignment of the AroD
domain of AroD*E with its homologues.............. 135

7-7 Dendrogram of dehydroquinase homologues........... 137









7-8 Multiple amino acid alignment of the shikimate
dehydrogenase domain of AroD*E and its
homologues.... ................................... 141

7-9 Dendrogram for shikimate dehydrogenase
homologues...................................... 143


vii















LIST OF TABLES


Table Pare

1-1 Molecular mass and Km values for DQT and SDH....... 15

3-1 Specific activities from N. silvestris and C.
sorokiniana aromatic pathway enzymes.............. 46

3-2 Prephenate aminotransferase amino acid donor
specificity in C. sorokiniana .................... 53

3-3 Prephenate aminotransferase activity after
thermal treatment in C. sorokiniana.............. 53

4-1 Purification of SP-I from N. silvestris
suspension cultured cells........................ 71

4-2 Purification of SP-II from N. silvestris
suspension cultured cells........................ 71

5-1 Kinetic parameters for SP-I and SP-II............. 103

5-2 Effect of PCMB on enzyme activity................ 104

6-1 S-protein antibody effects on N. silvestris and
E. coli extracts................................. 116

7-1 SDH/DQT activities in transformed aroD and aroE
mutant strains of E. coli ........................ 122

7-2 Pairwise comparisons of S-protein with its
homologues....................................... 144

7-3 NAD(P)H binding motif of enzymes................. 145

7-4 Transit peptides of aromatic pathway proteins
and other plant proteins......................... 143


viii
















ABBREVIATIONS


ADH
ADT
AGN
uKG
AMP
ANS
ANS-1
3ME
BSA
CHA
CM
CM-52
CM-I
CM-II
DAHP
DAHPS
DE52

DHQ
DHS
DQT
Ds-Co
Ds-Mn
DTT
E-cells

EDTA
EE-cells
EPPS

EPSP
E4P
HA
HPLC
HPP
ICBR
IPTG
KPO4
LB
LB-AMP
LES
MTT

NADP
NTPS


arogenate dehydrogenase
arogenate dehydratase
L-arogenate
a-ketoglutarate
ampicillin
anthranilate synthase
aneuploid N. silvestris suspension cell line
beta mercaptoethanol
bovine serum albumin
chorismate
chorismate mutase
carboxymethyl cellulose-52 cation exchange column
chloroplast chorismate mutase
cytosolic chorismate mutase
3-deoxy-D-arabino-heptulosonate 7-phosphate
DAHP synthase, enzyme 1 of aromatic pathway
(DEAE) diethylaminoethyl cellulose-52 anion
exchange column
dehydroquinate
dehydroshikimate
dehydroquinate dehydratase or dehydroquinase
cytosolic DAHP synthase of plants
chloroplast DAHP synthase of plants
dithiothreitol
cells of about two generations of exponential
growth
ethylenediaminetetraacetate
cells in continuous exponential growth
(N-[2-Hydroxyethyl]piperazine-N'-[3-
propanesulfonic acid];HPPS)
5-enolpyruvylshikimate-3-phosphate
erythrose 4-phosphate
hydroxylapetite column
high performance liquid chromatography
4-hydroxyphenylpyruvate
Interdisciplinary Center of Biotechnology Research
isopropylthio-o-D-galactoside
potassium phosphate buffer
Luria-Bertaini medium
LB plus ampicillin
lag, exponential and stationary phases of growth
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium-bromide; (thiazolyl blue)
nicotinamide adenine dinucleotide
nucleotide triphosphates
ix









OAA oxaloacetate
OPA orthopthalaldyhyde
PAGE polyacrylamide gel electrophoresis
PAT prephenate aminotransferase
PCA protocatechuic acid
PCMB p-chloromercuribenzoate
PDH prephenate dehydrogenase
PDT prephenate dehydratase
PEGo000 polyethylene glycol
PEP phosphoenolpyruvate
PLP pyridoxal 5'-phosphate
PMS N-methyldibenzopyrazine methylsulfate (phenazine
metasulfate)
PMSF phenylmethylsulfonyl fluoride
PPA prephenate
PPY phenylpyruvate
PCA protocatechuic acid
QA quinic acid
QDH quinate dehydrogenase
QDT quinate dehydratase
RE restriction enzymes
SA specific activity
S-protein shikimate-bifunctional DQT/SDH protein
SDH shikimate dehydrogenase
SDS sodium dodecyl sulfate
SP-I (S1-protein) bifunctional protein (DQT/SDH), major
peak from Celite 545 chromatography separation
SP-II (S2-protein) bifunctional protein (DQT/SDH), minor
peak from Celite 545 chromatography separation
SHK shikimate















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


MOLECULAR CHARACTERIZATION OF A BIFUNCTIONAL ENZYME
(DEHYDROQUINATE DEHYDRATASE/SHIKIMATE DEHYDROGENASE)
AND THE POST-PREPHENATE PATHWAY ENZYMES NEEDED FOR
PHENYLALANINE AND TYROSINE BIOSYNTHESIS IN NICOTIANA SPP.


By

Carol Ann Bonner

April 1994


Chairman: Roy A. Jensen
Major Department: Microbiology and Cell Science


Two bifunctional proteins containing catalytic domains

for dehydroquinate dehydratase (dehydroquinase) and

shikimate dehydrogenase were separated from extracts of

Nicotiana silvestris suspension cells by use of a decreasing

ammonium sulfate gradient on a Celite 545 column. The two

apparent isoenzymes, denoted as SP-I and SP-II, were

purified 1000- and 800-fold, respectively. Higher molecular

mass values were obtained for SP-II than for SP-I on the

criteria of sodium dodecyl sulfate (SDS) polyacrylamide gel

electrophoresis (PAGE) and gel filtration chromatography.

Each protein exhibited multiple bands on SDS-PAGE and on

native-PAGE monitored with a specific shikimate

xi









dehydrogenase activity stain. Temperature and pH optima for

catalysis were similar for each protein, as were the

temperatures of inactivation. However, Km values for

shikimate were 0.80 and 0.36 mM for SP-I and SP-II,

respectively. Antibody raised against SP-I cross-reacted

with SP-II, but not with Escherichia coli proteins

possessing the corresponding activities. Antibody screening

of a cDNA library of Nicotiana tabacum was used to isolate

clones expressing the S-protein. Functional complementation

of aroD and aroE E. coli auxotrophs transformed with

plasmids carrying cloned cDNA were successful. Activities

for each enzyme were 15-fold greater in the transformed

mutant strains than in the wild type strain of E. coli.

Analysis of the amino acid sequence deduced from the cloned

cDNA sequence revealed homology with the appropriate

functional domains of the pentafunctional Arol protein of

Saccharomyces cerevisiae and with the monofunctional AroD

and AroE proteins of E. coli. The post-prephenate pathway

enzymes were also studied in Nicotiana silvestris suspension

cells. Levels of arogenate dehydratase activity changed

throughout a growth cycle in suspension cells. Using

specific antibodies, several putative cDNA clones encoding

prephenate aminotransferase or arogenate dehydrogenase were

isolated for further study. The base of comparative

enzymology in higher plants for the aforementioned enzymes

was extended to the unicellular alga, Chlorella sorokiniana.

xii









Prephenate aminotransferase of Chlorella exhibited the

striking high-temperature activity optimum and specificity

for prephenate that is typical of higher plants.


xiii















CHAPTER I

LITERATURE REVIEW AND RATIONALE



Aromatic Amino Acid Biosynthesis in Plants

In higher plants, the three aromatic amino acids (L-

phenylalanine, L-tyrosine and L-tryptophan) are not only

required for protein synthesis and primary metabolite

synthesis (such as the hormone, indole acetic acid and the

structural component of woody plants, lignin), but also must

be available as starting substrates for a vast array of

secondary metabolites including alkaloids, coumarins,

isoflavones, and tannins (2, 78, 79). Intermediates of the

pathway are also precursors for synthesis of other essential

metabolites in plant cells. The vitamin-like derivatives,

folic acid and ubiquinone, are synthesized from chorismate,

the first branch point intermediate in the aromatic pathway.

Protocatechuic acid is derived from the common pathway

intermediate, dehydroshikimate (Fig. 1). It has been

estimated that up to 60% of plant carbon flows through the

aromatic amino acid pathway (40). Humans as well as other

higher forms of life depend heavily on plants to provide the

three essential aromatic amino acids, and knowledge of

aromatic biosynthesis in higher plants is fundamentally

























Fig. 1-1. Aromatic amino acid biosynthetic pathway.
The common portion of the biosynthetic pathway leading to
the branch point intermediate, chorismate, begins with
DAHP synthase [1] catalizing the condensation of
erythrose 4-phosphate and phosphoenolpyruvate and
consists of seven steps. These steps consist of
dehydroquinate synthase [2], dehydroquinate dehydratase
[3], shikimate dehydrogenase [4], shikimate kinase [5],
EPSP synthase [6], and chorismate synthase [7].
Chorismate, at the branchpoint of the pathway is utilized
in one direction to yield L-tryptophan (steps 8-12) and
in another direction by chorismate mutase [13] to produce
prephenate, another branch point intermediate. Organisms
in nature use different routings from prephenate. Higher
plant chloroplasts proceed through prephenate
aminotransferase [14] and arogenate dehydratase [15] to
form PHE, or through prephenate aminotransferase and
arogenate dehydrogenase [16] to form TYR. In other
organisms a second route proceeds through prephenate
dehydratase [19], then through aromatic (or PHE)
aminotransferase [20] to synthesize PHE, or through
prephenate dehydrogenase [17] and through aromatic (or
TYR) aminotransferase [18] to synthesize TYR.
























c-o
C-N
C No c 4C COO "


ON N NO.

ATP


CO" oo"N COON COON
& N"
1;1 f:C.coo @. Coom N OP.%cSi
N" ON On


0o
CMz-C-COON CCN -CH-COON


S g--t 0


/ /
191 (si


r-CHCOOM MOOC- CNZ-CN-COON


\ -\

\NAO*/NADP* 0 \NAD*ONADP*
1171 1161


CN,-C-COON CM-CCO0



0 OH


NC-Ow
NC-OW



COO"
engoozy


& r... ^oo"
N4











important. Figure 1 shows a composite schematic of the

biosynthetic steps of the common portion of the pathway and

of the three divergent amino acid branches.

It has been established that a complete aromatic

biosynthetic pathway is present in the chloroplasts of plant

cells (2, 41). The common portion of the pathway begins

with the condensation of erythrose 4-phosphate and

phosphoenol pyruvate to form 3-deoxy-D-arabino-heptulosonate

7-phosphate by DAHP synthase and continues with six more

enzymatic steps to form CHA, the major branchpoint

intermediate. From CHA, the pathway branches to form L-

tryptophan via anthranilate synthase (ANS, enzyme 8) and to

L-phenylalanine or L-tyrosine via chorismate mutase (CM,

enzyme 13). From prephenate, the product of the CM

reaction, different pathway variations have been shown in

nature to complete the synthesis of PHE and TYR (16). In

plant chloroplasts, the post-prephenate pathway begins with

the transamination of PPA to yield L-arogenate by prephenate

aminotransferase (PAT, enzyme 14). Finally, AGN is either

converted to PHE by arogenate dehydratase (ADT, enzyme 15)

or to TYR by arogenate dehydrogenase (ADH, enzyme 16). This

pathway is illustrated with dashed arrows in Fig. 1.

The basic hypothesis underlying my studies is that a

second pathway for aromatic amino acid biosynthesis exists

in the cytosol compartment of higher plants. The supportive

evidence is that DAHP synthase (the early-pathway enzyme)











(32), CM (the mid-pathway enzyme) (23) and ANS (the initial

TRP-branch enzyme) (15) are present as pairs of separately

compartmented isoenzymes (Fig. 1-2). It is also known that

starting substrates for aromatic biosynthesis (E4P and PEP)

are synthesized by duplicate pathways of carbohydrate

metabolism located in both the plastid and cytosolic

compartments (Fig. 1-2). Since most secondary metabolites

are synthesized in the cytosol of plant cells (39) (Fig. 1-

2), it seems likely that an aromatic pathway should be

available in the cytosol to produce aromatic amino acids not

only for protein synthesis but as starting substrates for

secondary metabolism as well.

Cytosolic isoenzymes of DAHP synthase and chorismate

mutase (Ds-Co and CM-II, respectively) are markedly

different from Ds-Mn and CM-I (the corresponding plastid-

localized isoenzymes) in both catalytic and regulatory

properties (32, 23). Such differences and indeed a lack of

homology would be consistent with the endosymbiotic

hypothesis of organelle evolution (55). If cytosolic

species of other pathway steps are present in plant cells,

it would not be surprising to find them to have equally

divergent properties that reflect their xenologous origin at

the time of endosymbiosis. Thus, the alternative post-

prephenate steps shown in Fig. 1 (solid arrows) might be

used in the cytosol. From this perspective it seems

unlikely that dehydroquinase and shikimate dehydrogenase















*H C-r- -H ,C 3-) 4
0 4 ,C 4 (rd

r-1 r a 3 f 3-
Sa sr o



S--,- > r "
o0 0 0 0'
r-l 0 C
(0 r0 ( 4
02 3.02
h o a, O


( U 0 o EU
, .O .r 4
"0 (o o

*-1.5 u U o; M
04 4 41 f :
nJJ0 0J








-o ( 0 0 r
--0 0 r- 0E



0 0
4-1 Hd~4 C=





04 r= 4.4 0


0 0 -


o.~ 00 0
-0 rH 0 q
S 0 rmd
- 5 oa ) "


no
0cd 0 0,
O- 4 4
a C (d 0 -4










W r= C: rq
0404- 0r^











(d u 91 4J U J3 M






















0~K SP
IIL

IL V-% IJ o




IL I i

0 IL




I 'IL











would co-exist as a bifunctional protein, as they do in

plastids. Alternative possibilities would be that they are

monofunctional species, or that they are domains of a large

pentafunctional protein like that present in fungi (21).

That the enzymes catalyzing the third and fourth steps

in the common pathway of plastids are bifunctional in higher

plants has been documented (14, 61). In E. coli, DQT and

SDH are each found as monofunctional proteins (18,19).

Yeast and fungi produce the arom protein (21), a

pentafunctional protein bearing functional domains

corresponding to steps 2 through 6 of aromatic amino acid

biosynthesis (Fig. 1-1). It is interesting that when the

arom pentafunctional protein of Neurospora crassa is

subjected to limited proteolysis, the DQT/SDH domains were

retained as an intact and functional fragment (77).



Rationale for Focus upon DOT and SDH

The rationale for choice of the DQT/SDH protein (or S-

protein) for study was as follows. (i) SDH is easily

assayed, has very high activity and the substrates are

readily available. (ii) The S-protein is in the middle of

the common portion of the pathway and provides a link from

the early-pathway isoenzyme pair of DAHP synthase to the

second known mid-pathway pair of isoenzymes of chorismate

mutase. (iii) Two separable shikimate dehydrogenases had

been demonstrated by Rothe et al. (67) in pea seedlings, and











my preliminary experiments had shown this result to be

repeatable in N. silvestris. (iv) Distinctly different

properties of the isoenzyme pairs for DAHP synthase,

chorismate mutase, and anthranilate synthase have been most

useful to demonstrate the separate spatial locations. Since

DQT and SDH were known to coexist as a plastid-localized

bifunctional protein, I thought it likely that the

corresponding cytosolic enzymes would be monofunctional. If

so, this would be a type of differential property that might

allow me to distinguish the two during cell fractionation

studies.



Shikimate biosynthesis and quinate catabolism

In monocots, such as Zea mays, there are two

dehydroquinases, one is bifunctional with SDH (specific for

NADPI) and the other is either bifunctional or stably

completed with a NAD' specific quinate dehydrogenase (36).

The pathway steps to quinic acid in plants have not yet been

rigorously established and may originate from E4P and PEP

via DHQ, or from unknown pathways. Quinate has been shown

to accumulate in plant vacuoles and to account for at least

10%, if not more, of the dry weight of certain plant tissues

(2, V. I. Ossipov, personal communications). The quinate

catabolic system from fungi (34,35) overlaps the

biosynthetic shikimate pathway with several analogous

dehydrogenase and dehydratase reactions (Fig. 1-3). An















u a) 4) 5 02t)' ro 0 a) :
-ri 4J 4- c 4 a0 0 -Hq 4J 0
.0 (d mU 0 Ea -H (d 10r :j
(d 0 :4-44 i1 24 4J 0 M
04 4) ..
O m 4 83
-1 0
a) U a( ) -- 0 i


() U
3: (L U 4-4

a)a~

a,) o
ON Q rHbl
4j c+ a) ma 0
rj d Q (d j Ia)

r~-I 4J t 4 r= Q 4 E
o r. "Ia) ) aE-14-
0-HH M4 H EO



ud UwrUa,02 a )
t4 0 M 4.) -C: 0
o-i i0 rl r A
04 M (d 04
-rl 0 P -ri 4 r=



004-) 0 rd 0oH








a)HO4 r
$44 M2 0 0
(d 4-) H 4-)k~










~ >02
4J r-i W a)
-H 4- rd' 0 0 -H 02
-H M -r 'd 02 0









4 .02 >1
. 2 0 -r -H 40
0 4j 4-) E r(j




r. a) r 4-E>
>4 4 0 d 5




41, U4 U J









-H H1- 1 0l -) a) 04I4-
4- Ua ~IJ ) 0 4-
r d d CJ2 -
04 ro o)S
.rq 41 >, ao 4 ) Q mb
::, 0 'o -ri 0 ril M -r-I Mr
CY> a 04 Eo (d$
j ) r= (d Q) 4

Q a) -rq 0 0 r. a

0 -rl I 0 a)
.H io rd o d 4)
04rd 0 (d

-r- 4 4 0 ) a) p P-r

5 M 0 :4 4 4 W
fd U rO 0 CO Q 4J> 4














O




A










..

6


/l


0
a-


/

I
ij=
0
$
A

Iu


0
o \O


0


0
O















t
t



M e
N A


0
OF


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0I


a
01b











inducible catabolic dehydroquinase takes DHQ that comes

directly from quinic acid (by a specific NAD' quinate

dehydrogenase) to dehydroshikimate which is converted to PCA

and on to carbon building blocks generated by the TCA cycle.

The dehydroquinase functional domain of the pentafunctional

arom protein of yeast and fungi channels DHQ formed from

DAHP to EPSP, thus bypassing PCA formation and avoiding

futile cycling (17, 21, 28). Both a NADPH specific QDH (V.

I. Ossipov, personal communication) and a quinate

dehydratase (V.I. Ossipov, personal communication) have

recently been shown. The NAD* specific QDH from E. nidulans

also will utilize SHK (34). These two systems offer some

interesting possibilities for overlapping metabolism in

plants. For example, it is conceivable that a QDT might

convert quinate to SHK for synthesis of aromatic amino acids

(Fig. 1-3) to be utilized as precursors for the synthesis of

lignin and other compounds.



Multiple Species of Shikimate Dehydrogenase

Shikimate dehydrogenase has been a popular enzyme

choice for a variety of isoenzyme studies and multiple

activities have been described on many occasions.

The bifunctional DQT/SDH protein (NADP' specific) was

examined in dicots (50). No evidence of a separate DQT or

SDH has been determined, thus far.









13

Cofactor specificity. Rothe et al. (67) separated two

SDH (NADP') activities from pea seedlings on a Celite 545

column with an ammonium sulfate gradient. Whether DQT

activity coeluted with one or both of the SDH activities was

not examined. Shikimate dehydrogenase was shown to be

located in the stroma of spinach chloroplasts and was

partially purified (30). Mousdale et al. (61) purified a

bifunctional DQT/SDH (NADP' specific) from pea seedling

chloroplasts and also showed by chromatofocusing that there

were two chloroplast isoenzymes and one unstable isoenzyme,

probably located in the cytosol. Two forms of nondissoc-

iable DQT/SDH (NADP') activities and one of QDH (NAD*) were

found in Phaseolus mungo seedlings by Minamikawa (58).

Koshiba (49) also found two forms of DQT/SDH (NADP')

activities from Phaseolus mungo seedlings. In 1988, Ogawa

and Tateoka (64) reported one SDH (NADP*) specific activity

and two SDH (NAD) specific activities (DQT apparently was

not assayed in these fractions) from Phaseolus mungo. This

is the first description of NAD* specific SDH activities and

is surprising since the same methodology for separation was

used by the three groups and each checked for specificity of

cofactors. A recent study of QDH in Phaseolus mungo by Kang

and Scheibe (44) found that the enzyme could use NADP' as

well as NAD*. When SHK was added to the reaction mix

containing quinic acid and NAD', some inhibition was found.

Apparently SHK was not tried as a substrate for QDH. If QDH











also functions as a SDH, competition for two products

(dehydroquinate and dehydroshikimate) would occur and this

could account for the inhibition seen with SHK added to the

reaction mix. This might also explain the earlier report of

the NAD* specific SDH activity by Ogawa and Tateoka (64), if

the enzyme was actually a nonspecific QDH. The qa gene

cluster of Neurospora crassa encodes a functional domain for

a QDH (NADH specific) that will also utilize SHK/NADH (34).

Properties. The range of molecular mass for DQT/SDH

(or SDH, where DQT was not studied) is from 44,000 to

73,000. It is not clear as to whether the lower Mr of

44,000 might represent a monofunctional SDH. SDH activity

is usually assayed in the reverse of the physiological

direction with the substrates, SHK and NADP', rather than

the forward direction with DHS and NADPH:



NADPH
Dehydroshikimate < > Shikimate
NADP*



Values of Km have been determined and vary from 0.060 to 1.3

mM for SHK, from 0.010 to 0.10 for NADP', and from 0.130 to

0.180 mM DHQ. Table 1-1 gives estimates of Mr and Km values

for DQT and SDH from several plant species, as well as Mr

from E. coli and from S. cerevisiae.

Inhibition studies with HgCl2, p-chloromercuri benzoate

and oxidized glutathione have suggested that SDH












Table 1-1. Molecular

Source Substrate

Sweet potato SHK
NADP
Mung bean 1 DHQ
SHK
NADP
Mung bean 2 DHQ
SHK
NADP


mass and

Km (mM)

1.300
0.100
0.130
0.220
0.025
0.180
0.910
0.025


15

Km values of DQT and SDH.

Mra Reference

-- 48

57,000 (GE) 49


57,000 (GE)


Tomato SHK 0.038 73,000 (GF) 52
Pea SHK 0.690 59,000 61
NADP 0.013
Corn 1 -- 55,000 (GE) 13
Corn 2 -- 49,000 (GE)
Spinach -- 67,000 (GF) 30
59,000 (GE)
Cucumber SHK 0.060 44,000 (GF) 53
NADP 0.010
Radish SHK 0.860 60,000 (GF) 71
NADP 0.090
Spinach SHK 0.480 44,000 (GF) 71
NADP 0.032 49,000 (GE)
E. coli -- 56,000 (AA) 28b
S. cerevisiae -- 68,000 (AA) 77

aMolecular mass estimated by gel filtration (GF), by PAGE
(GE) or calculated from the deduced amino acid sequence
(AA) .
bMolecular mass of monofunctional DQT and SDH were combined.




sulfhydryl groups act as functional groups in the catalysis

of the enzyme and that partial reversal of inhibition is

accomplished by thiols (48, 53). Aromatic compounds,

metals, and salts have been studied with respect to effect

on DQT and SDH activities. Protocatechuic acid was found to

be a competitive inhibitor of SDH activity in tomato (52,)

and Cl- inhibited DQT activity competitively in pea (61).

Ca2 stimulated SDH activity in Brassica rapa (71).









16

Molecular genetics. Plant genes encoding DQT or SDH

have not been sequenced, but the monocistronic aroD and aroE

genes have been cloned and sequenced in E. coli (1, 27).

Amino acid sequences are also available for discrete domains

encoding these activities in S. cerevisiae (28) and

Aspergillus nidulans (now known as Emericella nidulans) (17)

as part of the pentafunctional arom gene (aroM). The E. coli

amino acid sequences, aroD (DQT) and aroE (SDH) exhibit only

have about 25 and 21% identity, respectively, with the

corresponding functional domains of the yeast arom protein.

In higher plants, aromatic-pathway genes thus far

cloned and sequenced are limited to DAHP synthase (29),

shikimate kinase (70), EPSP synthase (73), and chorismate

synthase (69) of the common portion of the pathway.

Anthranilate synthase (63) and tryptophan synthase 0 subunit

(3) of the tryptophan branch of the pathway have been cloned

and sequenced.



The Post-Prephenate Pathway in Plants

The dual-pathway hypothesis of aromatic amino acid

biosynthesis specifies that an intact post-prephenate

pathway exists in the cytosol in addition to that already

demonstrated in the plastid compartment (41). If a separate

cytosolic pathway functions: (i) separate genes encoding

isoenzyme counterparts of the plastid-localized enzymes may

exist, (ii) separate genes encoding alternative enzymes











steps may exist, or (iii) the unprocessed preprotein

precursor of the plastid enzymes may function in the

cytosol.

Prephenate aminotransferase. PAT of higher plants was

first characterized in partially purified extracts,

separated from aromatic aminotransferases, and shown to be

unusually specific for its substrate, prephenate. Activity

was optimal at 700C for PAT, a temperature which inactivated

other interfering aminotransferases (5). PAT was purified

about 1000-fold (7), and localization studies showed the

enzyme to be located in the chloroplast (Bonner and Jensen,

unpublished data, 1985; 41, 76). A simple, inexpensive

spectrophotometric assay was developed for PAT by following

the increase in oxaloacetate when the L-aspartate/a-

ketoglutarate couple was used (6). In the same study, the

ASP/aKG couple was used with native PAGE activity stained

gels to relate the position of PAT with respect to other

aminotransferases and to show the disappearance of heat-

inactivated aminotransferases.

Arogenate dehydrogenase. Partially purified ADH was

studied in N. silvestris with respect to allosteric

regulation by TYR by Gaines et al. (31). ADH was partially

purified and regulatory properties were determined in

Sorghum bicolor (22). The enzyme was purified about 1000-

fold (8). PAT, ADH and SDH activities were followed

throughout a growth cycle in N. silvestris suspension cells











(12) and after wounding of potato (59).

Arogenate dehydratase. ADT activity was first

determined in N. silvestris cells by Jung et al. (43). The

enzyme was localized in spinach chloroplasts and allosteric

regulation (activation by TYR and inhibition by PHE) was

studied (43). Siehl and Conn (75) partially purified ADT

from Sorghum bicolor and studied some kinetic and regulatory

properties.

Rationale for focus upon the post-prephenate enzymes.

The rationale in choosing to study some aspects of the post-

prephenate enzymes for this dissertation is my long-term

objective to follow up earlier work with molecular-genetic

approaches. Studies of the individual enzymes may shed some

light as to the multiplicity of compartmentation. The

merits are as follows. A) Prephenate aminotransferase (i)

has been characterized and a well-developed HPLC assay

exists (5), (ii) has important biotechnological application

due to its unique aminotransferase properties and the value

of its product, L-arogenate, and (iii) can be purified by an

established procedure (7). B) Arogenate dehydrogenase (i)

has an established purification methodology (8) for

providing purified protein for antibody production that may

be used to probe cDNA libraries, and (ii) is easily assayed

fluorometrically. C) Arogenate dehydratase has an

established HPLC assay (43), but is by far the most

difficult of the three enzymes to assay and purify.















CHAPTER II

MATERIALS AND METHODS



Organisms and extract preparations

Eukaryotes

Nicotiana silvestris Speg. et Comes suspension cultured

cells originally isolated from haploid leaf material have

been maintained in our laboratory for about twelve years and

are well described physiologically and biochemically (10,

11, 12). Cell populations are subcultured in stationary

phase (7 day), diluted fivefold into fresh Murashige and

Skoog medium (62), and grown under specified conditions in

controlled growth chambers (10). For these experiments

(unless otherwise stated) seven-day old cells were harvested

by filtering through Miracloth (Calbiochem, LaJolla, CA),

rinsed with nanopure water, ground in liquid nitrogen in a

Waring blender and stored at -700C. until use.

C. sorokiniana cells cultured as described by Meridith

and Schmidt (57) were kindly provided by Dr. Robert Schmidt.

Bacterial strains and plasmids

E. coli strains used for transformation include DH5a,

AB1360 aroD (DQT-) and SK494 aroE (SDH-). Plasmids used for

subcloning were pUC18, pGEM5Zf(+) or pBluescript SK+.











Preparation of N. silvestris extract

Crude extracts for assay. Frozen, ground N. silvestris

cells were suspended (1:1) in 100 mM KPO4 buffer at pH 7.3

containing 20% glycerol, 1.0 mM phenylmethylsulfonyl

fluoride and 1.0 mM dithiothreitol, centrifuged at 35,000 x

g at 40C for 20 min. Extracts were treated to 80% of

saturation with ammonium sulfate, centrifuged as above,

followed by resuspension of pellets in EPPS-KOH buffer (pH

8.6) with all of the above components and dialyzed with four

changes of buffer until ammonia could not be detected by

HPLC. Extracts not treated with ammonium sulfate were

desalted using PD10 columns in Epps-KOH buffer as above or

dialyzed overnight with 4 changes of the Epps-KOH buffer.

Extracts for purification of ADH and PAT. Five-day N.

silvestris suspension cells were used in the purification of

ADH and PAT. Crude extracts were prepared as described

above and also included 0.1 mM pyridoxal 5' phosphate and

1.0 mM EDTA in order to stabilize PAT (5).

Extracts for S-protein purification. For purification

of the S-proteins, frozen, ground seven-day N. silvestris

suspension cells (300 g) were suspended into 300 ml of 100

mM KPO4 buffer, pH 7.8, containing 20% glycerol, 1.0 mM DTT,

1.0 mM benzamidine, 0.2 mM PMSF and 0.05 mM leupeptin. A

sample was immediately desalted using a PD10 column,











equilibrated with 50 mM EPPS-KOH buffer at pH 8.6,

containing the same components as above and labeled as crude

extract.

Preparation of cultured cells for assay of ADH levels

The isogenic suspension cell-culture line of N.

silvestris was grown in continuous light with gentle shaking

at 150 rev/min. The growth properties of a N. silvestris

suspension cell line (ANS-1) in lag, exponential, and

stationary phases of growth have been characterized

throughout a conventional growth cycle (referred to as E

cells) and for cells which have been maintained in

exponential growth for more than 10 generations, EE cells

(10). For routine subculture, 80 ml of suspension-culture

medium were inoculated with 20 ml of 7-day stationary-phase

cells in 500-ml Erlenmeyer flasks. EE cells (2 g wet

weight) were transferred to fresh medium on day 7 when cells

reached about 1 X 106 cells ml- Daily samples were

harvested using Miracloth for filtration, washed with

distilled water, ground and frozen in liquid nitrogen, and

stored at -700C until extract preparation. Daily sampling

was also done to determine a cell count by mixing 1 ml of a

50 ml culture with 12% chromium trioxide (helps to separate

cell clumps and visualize cells more easily). Samples were

heated to 700C for 2 to 5 min before counting in a Fuchs-

Rosenthal counting chamber at 20x magnification. The

remaining 49 ml of culture were filtered and cells were











dried on preweighed filters made from Miracloth in an oven

at 500C and were weighed daily until constant dry weight

values were obtained. From these two methods, growth curves

were determined.

Preparation of C. sorokiniana extracts

Frozen cell pellets of C. sorokiniana were suspended

into 50 mM PIPES buffer, pH 7.3, containing 1.0 mM DTT, 1.0

mM PMSF, 35 mM KC1, and were broken by 2 passes through a

French Pressure Cell at 20,000 psi. The slurry was

centrifuged at 18,000 x g, at 40C for 15 min, and the

supernatant was next centrifuged at 150,000 x g for 1 h at

40C. Extracts were either desalted and used for column

chromatography, or dialyzed with 50 mM EPPS-KOH buffer with

all protective components as listed above for use in enzyme

assays. (Pipes buffer plus KCl was used in place of EPPS

buffer for column chromatography in order to separate

isoenzymes of chorismate mutase for another study.)

Glycerol and PLP were added to column fractions to protect

several of the enzymes to be assayed (PAT and ADH).

Preparation of bacterial extracts

The mutant E. coli strains (aroD and aroE) carrying the

cDNA clones encoding functional proteins (DQT or SDH,

respectively) were grown in 200 ml of LB/Amp media, washed

and resuspended in KPO4 buffer containing 20% glycerol and 1

mM DTT at pH 7.6, and centrifuged at 150,000 x g, at 40C for











1 h. The supernatants were desalted by passage through

Sephadex G-25 columns before fluorometric assay.



Enzyme assays

Aminotransferases

Prephenate aminotransferase. Prephenate

aminotransferase was usually incubated with concentrations

of 20 mM L-glutamate and 0.8 mM prephenate (unless otherwise

stated), incubated at 370C for 10 min (or as otherwise

stated) and then assayed by HPLC as an orthopthalaldyhyde

derivative in a 60% methanol/40% of 20 mM KPO4 buffer

system. Peaks of PHE were quantified before and after

acidification of the product of the reaction, AGN, in 1N HC1

for 10 min at 370C.

Aminotransferase couples. Other aminotransferase

assays included an amino acid donor (ASP, TYR, LEU or ALA)

and keto acid (PPY) at concentrations as stated and were

assayed similarly by HPLC, before and after acidification

when applicable. Controls included samples without amino

acid donor or keto acid.

Arogenate dehydratase

Arogenate dehydratase was assayed according to Jung et

al. (43) by measurement of OPA derivatives following HPLC

where the peak of AGN formed were monitored before and after

acidification to PHE. Concentration of 0.5 mM AGN, 0.25 mM

TYR (activator), 50 mM EPPS-KOH buffer at pH 8.6 and enzyme











were included in the reaction sample of 100 Al that were

incubated at 320C for 30 min. Controls included samples

without AGN or enzyme.

Dehydroquinase

Dehydroquinase activity was assayed spectro-

photometrically at 234 nm at room temperature (240C) by

following the rate of increase of product, dehydroshikimate,

when dehydroquinate was provided as substrate. Unless

otherwise stated, a reaction sample included 0.5 mM DHQ,

enzyme and 50 mM EPPS-KOH buffer at pH 8.6. An extinction

coefficient of 12,000 was used to calculate activity (37).

DQT was also assayed fluorometrically via a coupled assay

with SDH. DHS formed from DHQ by the dehydratase reaction

was reduced with NADPH (cofactor for SDH) and the rate of

NADPH disappearance was followed. Reaction samples

contained 0.5 mM DHQ, 0.2 mM NADPH in 50 mM EPPS-KOH at pH

8.6 for column fractions or 50 mM Bis-tris propane buffer,

pH 7.5, for other studies as stated. Controls were carried

out for each enzyme assayed by adding only one substrate for

about three to five min before adding the second substrate.

Dehydrogenases

Dehydrogenase reactions were usually assayed

fluorometrically at room temperature (240C) with cofactor

NAD(P)* for 1 to 2 min before addition of substrate, and the

rate of formation of NAD(P)H was monitored (340 nm emission











and 460 nm excitation). Unless otherwise stated, a total

vol of 400 il contained substrates and 50 mM EPPS-KOH buffer

at pH 8.6. The rate is expressed as fluorometric units per

min (FU min-1). Activities were calculated as nmol min-' by

relating experimental values to a standard curve obtained

with authentic NADPH. Any background rate seen when

cofactor was present alone in reaction sample (only in some

crude extracts) was subtracted from the rate obtained from

reaction sample containing both substrates to give an

appropriate correction. A spectrophotometric assay was also

used occasionally by following the rate of formation of

NAD(P)H at A340o.

Arogenate dehydrogenase. Arogenate dehydrogenase

activity was determined when 0.5 mM AGN was added to the

reaction assay after 0.5 mM NADP' had been added or as

otherwise stated. When appropriate, NAD* was added as

cofactor to the reaction mix.

Prephenate dehydrogenase. Prephenate dehydrogenase

activity was assayed with 0.5 mM NADP* (or NAD*) and 1.0 mM

PPA.

Shikimate dehydrogenase. Shikimate dehydrogenase

activity was usually assayed in the reverse-of-physiological

direction with 1.0 mM NADP' and 4.0 mM SHK, unless otherwise

stated. When appropriate, NAD* was added to the reaction

sample. In the forward direction, SDH was assayed with











concentrations of 1.0 mM NADPH and 1.0 mM DHS or at

concentrations otherwise stated.

Quinate dehydrogenase. Quinate dehydrogenase was

assayed with 1.0 mM quinate and 1.0 mM NAD or NADP+ in 100

mM glycine buffer at pH 9.5.

Prephenate dehydratase

Prephenate dehydratase was assayed by incubation of PPA

and enzyme in 50 mM EPPS buffer (total vol of 100 Il) at

370C for 10 to 20 min before addition of 100 il IN HC1

followed by further incubation at 370C for 10 min. Controls

included PPA or enzyme with buffer only. Activity was

calculated from spectrophotometric readings at A20 nm and an

extinction coefficient of 17,500.

Protein estimations

Protein concentrations were estimated by the method of

Bradford (14) with BSA as a standard. A micro-assay was

used, as described, to estimate highly purified protein

concentrations.

Definition of activity and specific activity

Activity for all enzyme reactions in this study are

defined as nmol min-1 for the stated amount of protein added

into a standard assay volume. Specific activity is defined

as nmol min-' mg protein-.











Separation of pathway enzymes by chromatography

Column chromatography of N. silvestris extracts

A DEAE-cellulose column for separation of enzymes of

aromatic biosynthesis was prepared similarly as described

under purification. A 100 mg amount of protein was applied

to a bed volume of 102 ml of a DEAE column equilibrated in

50 mM KPO4 at pH 7.5. After washing the column with about 3

column volumes of buffer, a 600 ml gradient from 0 to 0.4 M

KC1 (KP04 buffer) was applied.

Column chromatography of C. sorokiniana extracts

A DEAE-cellulose column (20 ml) was prepared,

equilibrated in Pipes buffer (above), and about 29 mg of

protein from Chlorella were applied. The column was washed

with about 40 ml of the same buffer before a 35 mM to 500 mM

KC1 gradient was added. Finally, 1.0 M KCl/Pipes buffer was

added to remove residual protein from the column.

Appropriate fractions with DQT and SDH activities in the

gradient were pooled and dialyzed against 5 mM KPO4 buffer,

20% glycerol, and 1 mM DTT at pH 7.2. An HA column (10 ml)

was prepared with the 5 mM KPO4 buffer, pH 7.2. A 15-ml

sample from above was applied to the HA column and

activities for DQT and SDH eluted in the wash fractions.

Celite 545 (shallow-gradient application of Fig. 5-1)

A 200-g amount of ground, frozen 7-day suspension cells

was suspended in 200 ml of KPO4 buffer, 7.2 pH, containing

protectants and centrifuged as described above. A 3-ml









28
amount was removed for dialysis in Epps-KOH buffer and used

as a crude extract. Ammonium sulfate was added to give 75%

of saturation. From the slurry, 70 ml were removed and

prepared for use as a concentrated crude extract. To the

remaining slurry, 40 g of Celite 545 was added, stirred,

poured into a column (90 ml vol) and the eluate collected

into a cylinder. The column was washed with 65% (NH4),SO4 of

saturation in Epps-KOH buffer (about 180 ml) and 8-ml

volumes were collected. Buffer with (NH4)2SO4 at 55% of

saturation (120 ml) was added, followed by a shallow

gradient of 600 ml from 55% to 45% of (NH4)2SO4 saturation in

Epps-KOH buffer. A step gradient of 180 ml of buffer at 45%

of (NH4)2SO4 saturation was applied before the second

gradient of 45% to 0 of (NH4)2SO4 saturation (400 ml) was

added. A final wash of 120 ml of Epps-KOH buffer completed

the elution schedule.

Gel filtration

A Sephadex G-200 gel filtration column was used to

determine the native molecular weight of the S-proteins.

The column was equilibrated with 50 mM Epps buffer, pH 8.6

and DTT. Three ml of concentrated crude extract, and

concentrated SP-I or SP-II combined with 0.1 M sucrose were

applied individually to the same column (column vol of 471

ml, void vol of 119.4 ml). Protein standards of 5 mg each,











alcohol dehydrogenase (150,000 D), BSA (66,000 D) and

carbonic anhydrase (29,000 D) plus sucrose were applied to

the column.



Purification of pathway proteins

Purification of the S-proteins

Crude extract of 540 ml was brought to 60% of ammonium

sulfate saturation at 40C while stirring and was used for

the purification of the S-proteins.

Celite 545 chromatography. After stirring the 60% of

ammonium sulfate saturation slurry for 30 min, 70 g of

Celite 545 was added, with continued stirring for about 20

min. The slurry containing 605 mg protein was applied to a

column and the eluate collected into a large cylinder. The

column volume of 174 ml was washed with 60% of(NH4)2SO4

saturation-Epps-KOH buffer containing 20% glycerol and 1.0

mM DTT at pH 8.6 and was collected in a separate cylinder.

No activity was found in these eluates, although the protein

level was very high. A 500-ml gradient from 60 to 40% of

(NH4)2SO, saturation in the same buffer was applied and 8-ml

fractions were collected. Next a 40% of (NH4)2S04

saturation-Epps-KOH buffer of about 130 ml was applied. A

second 400 ml gradient of 40 to 0% of (NH4)2SO4 saturation-

Epps-KOH buffer was applied, followed by a wash of buffer

without (NH4)2SO4. Two peaks of SDH/DQT activities were

located, pooled individually, and dialyzed against the Epps









30
buffer as above. Samples of each were saved for assay, and

the remainder of each pool was used in the second

purification step (643 ml, 206 mg of SP-I protein and 348

ml, 97 mg of SP-II protein).

CM-cellulose chromatography. CM cation exchanger

columns were equilibrated with 50 mM Epps-KOH, 20% glycerol

and 1 mM DTT at pH 8.6. SP-I was applied to a bed volume of

123 ml and SP-II was applied to a bed volume of 80 ml. Both

SP-I and SP-II eluted in wash fractions and a small portion

was saved for assay. The remaining portions of the

preparations were used in the third step of purification.

DEAE-cellulose-52 chromatography. Two DEAE-52 anion

exchanger columns were prepared and equilibrated in the same

Epps-KOH buffer as the preceding step. Proteins were

applied, SP-I to bed volume of 107 ml and SP-II to a bed

volume of 72 ml, and washed with the same buffer. A 400-ml

(SP-I) or 300-ml (SP-II) gradient from 0 to 0.6 mM KC1 was

applied to the columns. Fractions of about 6.5 ml were

collected. Single peaks of activity, each eluting at about

0.3 mM KCL were individually pooled and dialyzed against 5

mM KPO4 buffer containing 20% glycerol and 1 mM DTT at pH

7.3. Samples were saved for assay, and the remainder of

each protein was used in the fourth step of purification.

Hvdroxvlapatite column chromatography. SP-I and SP-II

were applied to HA columns equilibrated in the KPO4 buffer

of the previous step (100 ml and 50 ml bed volumes,











respectively). Each protein eluted in the wash and was

applied to the fifth step of purification after samples were

removed for assay.

DEAE KPO4 chromatography. DEAE columns were

equilibrated in 50 mM KPO4 buffer at pH 7.3, before applying

the proteins from the previous step. Gradients up to 0.3 M

KC1 were applied to each column. Single peaks of each

protein were eluted, pooled and dialyzed into 50 mM Epps-KOH

containing 20% glycerol and 1 mM DTT at pH 8.6. Samples

were saved, and the remaining proteins were subjected to the

sixth step of purification.

2'5'ADP Sepharose-4B affinity chromatography. An

NADP'-specific 2'5'ADP Sepharose-4B affinity column was

prepared and equilibrated with the Epps-KOH buffer. The

SP-I sample was applied to the column and washed with

starting buffer before a 300-ml gradient from 0 to 0.18 M

NaC1 and 0 to 12 @8,25 AM NADP' was applied. The protein

eluted at about 4 iM NADP+ in the gradient. The SP-I

Activity peak was pooled and dialyzed into the Epps-KOH

buffer. A sample of SP-I was saved, and the remaining

fraction was used in the seventh step of purification. The

SP-II was applied to the affinity column similarly.

Purification of SP-II was completed with this step at a

final volume of 15 ml and total protein of 6 Ag.











Final steps of SP-I purification. SP-I was applied

once again to a DEAE column equilibrated in Epps-KOH buffer

as previously described. A 200-ml gradient from 0 to 0.6 M

KC1 was applied. The SP-I activity peak fractions were

pooled and dialyzed against Epps buffer. An aliquot was

saved, and 83 ml containing 0.162 mg protein was applied to

the affinity column for the final step of purification. The

completed purification yielded 0.07 mg SP-I protein in 54

ml.

Purification of arogenate dehydrocenase

ADH was purified by two methods: A) by a series of

steps of an established procedure which began with a 0 to

45% ammonium sulfate fractionation and ended with the NADP'

affinity column (8), or B) by the elution of ADH from a

Celite 545 column used to separate S-protein activities,

followed by HA column chromatography, DEAE column

chromatography, and the NADP+ affinity column chromatography

described above.

Purification of prephenate aminotransferase

PAT was purified by following an established procedure

(7), which began with a 45 to 75% of ammonium sulfate

saturation precipitation step, followed by a 700C treatment

to inactivate and then precipitate (by centrifugation)

contaminating aminotransferases, and ended with a specific

aminotransferase pyridoxamine phosphate (PMP) affinity

column.











PAGE

SDS gel electrophoresis

SDS gel electrophoresis was prepared by following the

procedure of Laemmli (51), with a 15% running gel and a 4%

stacking gel. A running buffer of Tris/glycine/SDS at pH

8.8 was used and the gel was run at constant voltage of 63 V

for about 8 h. The gel was stained for about 30 min in

Coomassie Brilliant Blue R250 dye, destined and viewed.

Silver staining

Silver staining of an SDS gel as described by Pharmacia

(Piscataway, NJ) was followed. Washes of methanol/acetic

acid and ethanol/acetic acid were included before incubation

of the gel in oxidizer, followed by addition of the silver

stain, then developer, and finally an acetic acid/water wash

to stop the reaction.

Native gel electrophoresis

A native PAGE was run according to the method of Shaw

and Prasad (74). Gel concentrations were 7% for the

stacking gel and 15% for the running gel. The running gel

was pre-electrophoresed to remove the ammonium persulfate,

and fluorescent light was used to solidify the stacking gel

which contained riboflavin. The gel was run at constant

voltage of 60 V for about 8 h.

Shikimate dehydrogenase activity stained gel

Optimal conditions for SDH activity stain on native

PAGE included 20 mg NADP*, 50 mg shikimate, 10 mg 3-(4,5-











dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide;

(thiazolyl blue), and 2 mg N-methyldibenzopyrazine methyl

sulfate (phenazine metasulfate) in 50 ml of 100 mM glycine

buffer (without glycerol) at pH 9. Gels were incubated at

370C for 10 to 20 min or at room temperature for 30 min to

an hour. These conditions were used unless otherwise stated

and were modified from the method presented by Davis (24).





Kinetic values

Shikimate dehydrogenase

Enzyme activities were assayed fluorometrically as

described for SP-I and SP-II. In the forward physiological

direction, either DHS or NADPH was held constant at

saturating levels while the remaining substrate

concentration was varied to obtain saturation curves. In

the reverse of physiological direction, SHK and NADP* were

assayed similarly. Double reciprocal plots of the substrate

saturation data were used to determine the Km for each

substrate.

Dehydroquinase

DQT activities were assayed spectrophotometrically as

described for SP-I and SP-II at varying concentrations of

DHQ to obtain saturation data, and then analyzed

appropriately as above to determine the Km for each protein.











Inhibition studies of S-proteins

Enzyme was incubated for 10 min with p-chloromercuri-

benzoate or with possible protectants {DTT, cysteine or (-

mercaptoethanol (PME)}. SDH was assayed at saturating

conditions and DQT was assayed at 0.1 mM DHQ. For

prevention experiments, DTT, cysteine, or PME was added

about 10 min before addition of substrates and PCMB, and

then rates were determined. For reversal experiments, PCMB

was added to enzyme and buffer for 10 min before assay with

substrates. After a rate (or no rate) was established, DTT

was added to the reaction mix. Concentrations are as stated

in Results.



Antibodies

Preparation of specific antibodies

Antibody preparations were produced by Kel-Farms

(Alachua, FL) with repeated injections of purified SP-I over

the course of several months until antibody titers, tested

by Ouchterlony double diffusion method against SDH, ADH or

PAT, were high enough to bleed the rabbits.

Purification of specific antibodies

Antibodies were purified following a BIO-RAD (Rockville

Center, NY) procedure, by passing each over Econo-Pac 10DG

columns for desalting and then passing through DEAE Affi-Gel

Blue Econo-Pac 10DG columns for serum IgG purification. E.

coli strain DH5a lysate was prepared and incubated with the











purified IgG. This step removed non-specific precipitant

bands observed in the double-diffusion experiments. Pre-

immune rabbit serum were purified similarly.

Antigen:antibody assay

Antigens (SP-I, ADH or PAT) from crude extracts or

extracts from various purification steps were combined 1:1

(v:v) (or as stated) with antibodies. Samples were

incubated at 370C for 10 min and returned to an ice bath,

then centrifuged (5 to 10 min) at full speed in a microfuge

to precipitate the completed antigen:antibody. The

supernatants were assayed for enzyme activities. Controls

were always performed with preimmune rabbit serum.

Ouchterlony assay

Ouchterlony plates were prepared with 1.5% agarose in

sodium barbital and sodium azide and wells were prepared at

precisely measured distances (66). Antibodies or antigens

were diluted with 0.9% NaCl whenever necessary and then

added to the wells. Plates were incubated at 370C

overnight.

Western blotting

Western blotting procedures from Sambrook et al. (68)

were followed, and bands from either SDS or native PAGE were

transferred to nitrocellulose filter paper. Membranes were

incubated in blocking solution alone and in blocking

solution containing diluted SP-I antibody for one h, washed,

incubated further with blocking buffer containing alkaline











phosphatase-linked goat-anti-rabbit antibody for 1 h, and

then incubated in STP buffer containing MgC21 and

substrates, nitroblue tetrazolium and the p-toluidine salt

of 5-bromo-4-chloro-3-indolyl phosphate. After 1 h,

membranes were viewed and photographed.

Antibody precipitation of DQT and SDH from C. sorokiniana

Antibody made to SP-I was incubated 1:1, 2:1, or 4:1

with Chlorella extract (controls included preimmune

antiserum and extract diluted with buffer). After 10 min at

370C, and centrifugation for 5 min at full speed in a

microfuge, preparations were assayed under standard

conditions for SDH and DQT activities.



cDNA cloning and sequencing

Tobacco leaf cDNA library

A cDNA library prepared from Nicotiana tabacum var.

SR1 tissue culture cells, inserted into pBluescript SK-

plasmids and packaged into Lambda ZAP II vectors, was

purchased from Stratagene, La Jolla, CA. E. coli XL1-Blue

(a host strain used for immunological screening of libraries

constructed in Lambda ZAP expression vectors) and E. coli

SOLR (a non-suppressing host strain for use after excision

of the pBluescript plasmid from the Lambda Zap vector) were

included. Helper phage, ExAssist, efficient for in vivo

excision of the plasmid from the Lambda Zap vector without

replication of the phage genome was also included.











Preparation of cDNA libraries for screening

The cDNA library was first titered, then mixed with the

freshly prepared host strain, XL1-Blue, plated with top agar

on about 80 LB plates at about 20,000 PFU (about 1.6 X 106

PFU/ desired clone) and incubated at 370C. Isopropylthio-3-

D-galactoside treated nitrocellulose filters were applied

about 6 to 8 h later and incubation was continued overnight.

Filters were removed and the immunological screening

procedure of Sambrook et al. (68) was followed.

Screening the cDNA library of Lambda ZAP II

Nitrocellulose filters removed from the plates were

washed, soaked in blocking buffer containing dry milk,

rocked gently in primary S-protein, ADH or PAT specific

antibody/blocking buffer and washed several times. The

filters were next exposed to secondary antibody, goat anti-

rabbit antiserum, washed as before and incubated with

substrates, nitroblue tetrazolium, 5-bromo-4-chloro-3-

indolyl phosphate and with 5 mM MgCl2. The filters were

kept in the dark and gently rocked for one to two hours.

Plaques corresponding to the small, deep blue spots on the

filters were cored from plates, dispensed into SM buffer

plus chloroform and stored at 40C for titering and plaque

purification.











Excision of Pbluescript from the Lambda ZAP II vector

Purified phage stocks were incubated with E. coli

strain XL1-Blue and ExAssist helper phage to excise the

pBluescript plasmid from the Lambda ZAP II vector for 2 to

2.5 h at 370C in 2XYT media (see Stratagene instruction

manual for details), heated to 700C and centrifuged. The

supernatant, containing the plasmid, was incubated with host

E. coli strain SOLR, spread on LB plates containing 50 mg

ml-1 ampicillin and incubated overnight at 370C. Colonies

from these plates were streaked on fresh LB-AMP plates for

immediate use and were also stored in DMSO for future use.

DNA purification

Plasmid DNA was purified by two methods. Method 1: The

pBluescript plasmids containing putative cDNA clones coding

for the S-protein, ADH or PAT were purified for sequencing

by the method of the DNA Sequencing Core Facility, ICBR,

University of FL. A colony from the E. coli SOLR strain

from the LB-AMP plate above was incubated overnight in 10 ml

of LB-AMP medium in a 50 ml tube for each clone sample. The

cultures were centrifuged and the pellets were resuspended

in GTE (68) buffer. Treatment with NaOH/SDS was followed by

neutralization with potassium acetate at pH 4.0, and

centrifugation was carried out to remove cellular debris.

The supernatants were treated with DNase-free RNase and

extracted twice with chloroform. DNA was precipitated with

isopropanol and washed with 70% ethanol. To precipitate the











plasmid DNA, the pellets were first dissolved in sterile

water, then 4M NaC1 and 13% PEGo000 was added, followed by

incubation on ice before centrifugation at 40C. The pellets

were rinsed in ethanol, dried under vacuum and resuspended

in sterile water. DNA concentrations were determined

spectrophotometrically at 260 nm and by agarose gel

electrophoresis with a known DNA standard. Method 2: The 10

ml cultures, as prepared above after centrifugation, were

treated with prepared solutions provided by Promega

(Madison, WI) in the Magic Miniprep DNA purification kit.

The DNA was passed through a mini-column and dissolved in

GTE (Promega instructions for preparation) buffer. This

rapid method is preferred for sequencing on a Li-Cor

sequencer available in the Department of Microbiology and

Cell Science.

Competent cells of E. coli strains

E. coli strains DH5a, AB1360 (aroD) and SK494 (aroE)

were grown overnight in 50-ml tubes and inoculated into 1-L

flasks containing 200 ml LB medium. The cells were

incubated at 370C in a shaker at 300 rpm for several hours

until a spectrophotometric reading at A600,, between 0.4 and

0.5 was reached. Cells were harvested by centrifugation in

sterile tubes after incubation on ice, treated with 100 mM

CaCl2 as described by Sambrook (68), resuspended into 100 mM

CaCl2 with 15% glycerol, aliquoted into microcentrifuge

tubes, dropped into liquid nitrogen and stored at -700C









41

until use. Controls were prepared by streaking auxotrophic

mutant strains on M9 medium plates +/- aromatic amino acids.

Transformation of plasmid DNA

Either pUC18, pGEM5zf(+), or pBluescript SK+ plasmids

were incubated with competent cells of DH5a on ice, heated

to 420C for 90 sec, cooled in an ice bath before addition of

SOC medium (68). The cells were then incubated at 370C for

1 h before spreading on LB-AMP plates. A colony from these

transformed cells was streaked onto a fresh LB-AMP plate.

Controls included spreading of competent cells without

plasmid or plasmid without insert onto LB-AMP plates.

Functional complementation

E. coli mutant strains, aroD (DQT-) and aroE (SDH-),

were prepared for transformation with the pBluescript

plasmids carrying cDNA clones and were plated on LB-AMP

plates. After growth, colonies were streaked onto M9

plates. Controls included nontransformed competent cells or

plasmid spread on LB-AMP plates, and competent mutant

strains spread on M9 plates +/- aromatic amino acids.

Initial sequencing of cloned cDNA

Purified pBluescript plasmid with cloned inserts were

submitted to the DNA Sequencing Core Facility, ICBR, UF, for

initial 5' and 3' end sequencing (from the T3 and T7

promoter sites).











Subcloninq cDNA for complete sequence analysis

Subcloning of cDNA inserted into pBluescript was

performed by use of appropriate restriction enzymes to

obtain fragments for sequencing both strands of the entire

cDNA. Plasmids used for fragment insertion {pUC18,

pGEM5zf(+), or pBluescript SK+} were incubated with the same

restriction enzyme(s), and were treated with calf intestinal

alkaline phosphatase (CIAP) (when only one RE was used) to

prevent rejoining of ends during ligation. Cloned cDNA

fragment samples and plasmid samples were loaded onto 1% DNA

agarose gels at 70V for about 1 h. Fragments and linear

plasmid DNA were excised and treated with the Gene Clean

system (Bio 101, LaJolla, CA), prior to ligation and

transformation.

Li-Cor sequencing of subcloned cDNA fragments

Plasmid DNA was denatured and reacted with forward or

reverse fluorescent primers, enzymes, 7-Deaza dGTP sequence

extending mix and NTPS according to the directions supplied

by Li-Cor (Lincoln, NE). Three il of each of four prepared

sample mixtures containing one of the four dideoxy

nucleotides was loaded onto wells of a urea polyacrylamide

gel set into the Li-Cor model 4000L sequencer. Parameters

were set in a computerized data collection file and the

sequencer was run overnight. The sequences were analyzed in

a data analysis computerized system.











N-terminal sequencing

About 1 pmol of purified SP-I protein was prepared for

N-terminal sequencing on SDS PAGE, followed by transfer of

the protein onto 3MM paper by electrophoresis. Mini-blot

system protocol and amino acid analysis by acid hydrolysis

was provided by the Protein Chemistry Core Facility,

Interdisciplinary Center of Biotechnology Research,

University of Florida.

Computer analysis

The GCG computer software package (26) was used to

analyze nucleotide and deduced amino acid sequences of

cloned cDNAs.



Biochemicals

Arogenate (90% pure) was isolated from a multiple

auxotroph of Neurospora crassa ATCC 36373 (42) or isolated

from a tyrosine auxotroph of Salmonella typhimurium (4).

Prephenate (85% pure) was prepared as the barium salt from

culture supernatants of the tyrosine auxotroph of S.

typhimurium and was converted to the potassium salt with

excess K2SO, prior to use. Dehydroquinate (90% pure) was

chemically synthesized by following the method of Haslam et

al. (37) using a platinum-catalyzed dehydrogenation of

quinic acid (about 90% pure). 2'5' ADP Sepharose-4B and AH-

Sepharose 4B were purchased from Pharmacia, Piscataway, NJ.

Bradford reagent was purchased from Bio-Rad, Rockville









44

Center, NY. DTT and hydroxylapatite were purchased from

Research Organics, Inc. Cleveland, OH. Shikimate, NADP,

NADPH, benzamidine, leupeptin, and PMSF were purchased from

Sigma Chemical Co., St. Louis, MO. All other chemicals were

purchased through Fisher Scientific Co., Orlando, FL.















CHAPTER III

PHYSIOLOGICAL PROFILE OF AROMATIC PATHWAY ENZYMES IN PLANTS



The activities of dehydroquinase and shikimate

dehydrogenase (of the common portion of aromatic

biosynthesis), and prephenate aminotransferase, arogenate

dehydratase and arogenate dehydrogenase (of the post-

prephenate portion of the pathway) were examined under

selected physiological conditions in the higher plant N.

silvestris and in the green alga Chlorella sorokiniana.

Prephenate aminotransferase from Chlorella exhibited several

of the striking characteristics of higher plant enzyme.

Arogenate dehydratase activities were followed in a

growth culture cycle of N. silvestris suspension cells

similarly as was followed for PAT and ADH (12).



Results

Specific activities from crude extracts

Specific activities have been compared for several

aromatic amino acid pathway enzymes of N. silvestris

cultured cells and from the green alga C. sorokiniana (Table

3-1). A constant ratio of about 5 for SDH and DQT activity

was found in N. silvestris, compared to a ratio of 1 for

crude extracts of C. sorokiniana. The activities of DQT and












SDH from Chlorella extract were unaffected when incubated

with SP-I N. silvestris specific antibody. Activity was not

found when QDH was assayed in N. silvestris and C.

sorokiniana crude extracts.





Table 3-1. Specific activities from N. silvestris and C.
sorokiniana aromatic pathway enzymes.

Specific Activity (nmol min' mg1 protein)


Enzyme N. silvestris C. sorokiniana

QDT 12.00 6.20
SDH 60.00 6.00
PAT 16.00 3.75
ADH/NADP 1.00 NF
ADH/NAD NF NF
PDH/NADP NF NF
PDH/NAD NF NF
ADT 0.6 NF
PDT NF NF

NF, not found; Crude extracts were used for assay.





Separation of activities

Column chromatography was used to separate the enzymes

of interest for this study from N. silvestris and C.

sorokiniana. DQT and SDH of the common pathway were not

separable in N. silvestris or in C. sorokiniana after two

steps of column chromatography (DE-52 and HA). Figure 3-1

shows a DEAE-cellulose chromatographic profile of

N. silvestris suspension cell extract with overlapping



















4J)
:j
OeH
0^
~44-Ir
0-
omu.
SciJ
CN
LnX
COC





4)
-H
ril
r-HE-1





04)
4-H
E




U-4
o




0H 0)
-rq








U,


4- a)
U0 0

44 rd




H

um -


tJ4 1)~
ro U





41i Q

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Wi o:






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v
w o co 0N co V
CM C q- d 0

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49

enzyme activities of PAT, ADH, ADT and SDH (DQT activity was

coincident with SDH activity; not shown). Although ADT and

ADH activities were not found in crude extracts of C.

sorokiniana, they were located in DEAE-cellulose fractions

at very low activities as shown in Fig. 3-2A. ADH was

specific for cofactor NADP'. Prephenate dehydratase and

prephenate dehydrogenase activities were not found in these

column fractions. The ADH and ADT activities overlapped

those of PAT, DQT, and SDH in the KC1 gradient fractions.

The coeluting DQT and SDH activity peak fractions, as shown

in Fig. 3-2B, were pooled, dialyzed and applied to an HA

column where they once again coeluted, this time in the wash

fraction, data not shown.

PAT in C. sorokiniana

Several of the unique properties earlier shown for

prephenate aminotransferase in N. silvestris suspension

cells (5) were studied in C. sorokiniana extracts. A high

temperature optimum of about 700C for PAT was also found in

the alga extract as shown in Fig. 3-3. In Table 3-2, it is

shown that PAT from Chlorella has a preference for GLU as

the amino acid donor in combination with the keto acid

substrate, PPA. As found in higher plants, ASP was utilized

at about 50% of the activity with GLU. Slight activity was

seen with TYR as the amino acid donor in C. sorokiniana but

not in N. silvestris.
































Fig. 3-2. DEAE-cellulose chromatography of aromatic
pathway enzymes from C. sorokiniana. Alga extract was
applied to a DE-52 column as described in Materials and
Methods. A) PAT (0), ADH (E), and ADT (A) eluted in the
KC1 gradient (-) and protein was monitored (...). B) SDH
(0) and DQT (0) eluted in the gradient.













5.5
DEAE-CELLULOSE
5.0

A
4.5 -

S4.0 -

3.5 -
-j
a
3.0

is.5 1.0 1.3

t 2.0 : 0.8 1.8











S.E.CEU.UOSE








70
0.5 0. ***-* : 02 0.4

0 -0

FRACTION NUMBER





100 OEAE-CELLULOSE




s0 B

70 -

so -

u 50 :". 1.0 1.8

40 0.8 1.

30 0.6 E 1.2

20 0.4 0.8

10 / **..- 0.2 0.4

0 0
0 20 40 80 80
FRACTION NUMBER









































t I


I I


0 20


TEMPERATURE (oC)


Fig.
prephenate
temperatures


3-3. Temperature optimum for activity of
aminotransferase in C. sorokiniana. The
range used was from 200C to 800C for 5 min.


I I











Table 3-2. Prephenate aminotransferase amino acid donor
specificity in C. sorokiniana.

L-Amino Acid Donor (10 mM) Specific Activity

GLU 16.0
ASP 8.0
TYR 2.0
LEU 0
ALA 0

aA concentration of 1 mM PPA was used in combination with
the amino acids. S. A. is nmol min- mg protein-


When C sorokiniana extract was incubated at 650C for 10

min and then assayed at 370C, activity with PPY as keto acid

substrate was lost, but activity with PPA remained constant.

At 700C for 20 min, however, about 60% of the PAT activity was

lost (Table 3-3).





Table 3-3. Prephenate aminotransferase activity after
thermal treatment in C. sorokinianaa.

Aminotransferase No 650C 700C 700C
Couple treatment 10 min 10 min 20 min

PPA/GLU 0.61 0.60 0.61 0.25
PPY/GLU 0.29 0 0 0

aReaction mixtures were incubated at 370C following thermal
treatment. Assays contained 1 mM PPA or 10 mM PPY, 10 mM GLU
and 0.05 mg protein.











ADT activities in cultured N. silvestris cells.

When N. silvestris stationary phase cells were diluted

into fresh medium, a substantial elevation of soluble

protein occurred which increased until day 2, leveled off

between days 2 and 3, and began a steady decline between

days 3 and 7 as shown in Fig. 3-4B. This rise ceased before

mid-exponential growth was reached. That soluble protein

synthesis was terminated before reaching a maximum was

indicated by the finding that EE cells achieved a two-fold

higher content of soluble protein. EE cells exhibited a

consistently higher soluble protein content of about 360 mg

g dry wt.-'.

During the transition of cell populations in lag phase

to exponential-phase growth, new synthesis of macromolecular

constituents must have occurred. Figure 3-5A shows that the

rise in ADT activity expressed as units of activity per g

dry wt., paralleled the rise in soluble protein between

subculture and day 2 (Fig. 3-4B). The rise in ADT activity

levels continued until day 3, whereas overall soluble

protein content plateaued between days 2 and 3. Thus,

specific activity or activity expressed on a cell basis

peaked at day 3. Since the specific activity of E cells and

EE cells are similar and since the ratio of soluble protein

to dry weight is greater in EE cells than in E cells, the

activity of ADT expressed as units per g dry wt was greater

in EE cells than in E cells. The decline in specific
































Fig. 3-4. Growth curve of N. silvestris in
suspension cultures. A) Cell growth monitored as cell
count or as dry weight. The physiological stages of
growth: lag (L), exponential (E) and stationary (S) are
indicated in each panel. B) Soluble protein content was
determined in samples harvested daily and was related to
the dry wt. and cell number. Comparable protein values
obtained with EE cells (average of ten determinations)
are indicated with stippled bars, the widths of which
indicate the range of variation.




































E

-J
LU






5x105





3x10s


350



z 150.
LU


o-
0

-J
.;J 50-
0
c,,


0 1 2 3 4 5 6


DAYS AFTER SUBCULTURE





























Fig. 3-5. Arogenate dehydratase activities followed
throughout a growth curve of N. silvestris. Activity was
followed in samples harvested daily throughout the growth
curve shown in Fig. 2-4. A) Activities expressed as
units/g dry wt., B) as units/cell, C) as specific
activity. The stippled bars indicate the corresponding
activities found in EE cells (average of 10
determinations), the width of the bars indicating the
range of variation. D) total cumulative units of
activity per culture. See Fig. 2-4 for meaning of L, E
and S.


















L E S
80o
80- ---"--~-1-.-----A
0 60-
r-
a 40






SL E S
16-


S12-

S fc 8 -
< ~ ,
UJ 4
)0
I-- 1 I I I
C
0 0.24-
>- z
I 3 0.20 -;;--
w i*-
0 0
Q 0.16-

< a 0.12
z E
a L 0E08L s
o -
m 0.04-
<
LE S





S12-
-J
8-
0
4


0 1 2 3 4 5 6 7

DAYS AFTER SUBCULTURE









59

activity (Fig. 3-5C) and units of activity per cell (Fig. 3-

5B) between days 3 and 5 (mid-to late-exponential growth

reflected a rate of enzyme synthesis that was slower than

the growth rate since total units of activity (Fig. 3-5D)

continued to increase during that time. From days 5 to 7

the decline in specific activity reflected a net loss of

enzyme activity.



Discussion

C. sorokiniana aromatic-pathway enzyme activities

appear to be somewhat lower but consistent with those found

in higher plants. The S-protein from N. silvestris must be

antigenically different from that of C. sorokiniana, since

the S-protein specific antibody did not precipitate the

protein from Chlorella. Specific antibodies made to PAT and

ADH are now available (see Chapter V) to test cross

reactions with these proteins from Chlorella.

Although the coincident DQT and SDH activity peak from

the DEAE column which eluted protein from C. sorokiniana

extract was only purified through two steps, it is

conceivable that further purification would yield a

bifunctional S-protein like that found in higher plants.

Activities from column fractions indicated that DQT activity

was about half that seen for SDH, whereas crude extracts

indicated that activities were about equal. Possible

explanations would be that (i) more than one protein is









60

present in Chlorella for DQT and/or SDH, (ii) some component

in crude extract inhibits the SDH activity or (iii) DHS, the

product of the reaction is being utilized by another

protein. The second possibility may also apply to ADT and

ADH since activity was not found in crude extracts but was

located in column fractions.

Most interesting, is the similarity of PAT in Chlorella

to that of higher plants, each having the high optimal

temperature for activity. At 650C, the aminotransferase(s)

using the PPY/GLU couple was inactivated. The high

specificity of Chlorella for the substrate couple of PPA and

GLU was also consistent with higher plant PAT (5, 6). From

these data, it is suggested that C. sorokiniana has an

aromatic biosynthetic pathway similar to that established

for higher plant chloroplasts. Supportive evidence would

include the isolation of Chlorella chloroplasts to determine

the location of the aromatic pathway enzymes. The isolation

and sequencing of cDNA clones coding for S-protein, PAT and

ADH (see Chapter VI) may be useful as probes to clone the

appropriate genes in Chlorella.

Arogenate dehydratase activities paralleled a similar

experiment with respect to the chloroplast isoenzyme of DAHP

synthase (Ds-Mn), where growth of cells and enzyme

activities where followed (32). Both enzymes rise in

exponential growth and decline in stationary phase of

growth. ADT exhibited an opposite pattern of activity to











three other aromatic pathway enzymes, SDH, PAT, and ADH.

The three enzymes are known to exist in the chloroplast of

higher plants, but the extent to which a fraction of each of

the total activities measured might be derived from the

cytosol is unknown. All three enzymes exhibited elevated

levels of activity in stationary phase and minimal levels of

activity in exponential-phase growth (11). Aromatic-pathway

enzymes may be sorting out into two different patterns of

expression for reasons yet to be elucidated. Alternatively,

since enrichment of Ds-Co (the cytosolic isoenzyme of DAHP

synthase), occurred in stationary phase, rising levels of

SDH, PAT and ADH in stationary phase may reflect the

expression of cytosolic isoenzymes. If so, the rise in

activity of cytosolic isoenzymes exceeds the decrease of the

plastidial isoenzyme counterparts. These data suggest that

while ADT is present in the chloroplast pathway, it may not

function in the cytosol.If so, perhaps prephenate

dehydratase of the alternative route (activity, not as yet

determined in plants, see Fig. 1-1), functions in the

cytosol to synthesize PHE that may be used extensively for

secondary metabolism.















CHAPTER IV

PURIFICATION OF PROTEINS
FROM THE AROMATIC AMINO ACID PATHWAY


This chapter includes the separation and purification

of the common pathway bifunctional DQT/SDH proteins from

Nicotiana silvestris suspension cells. Six to eight steps

of column chromatography, including a specific NADP'

affinity column have been used. Prephenate aminotransferase

and arogenate dehydrogenase were also purified by a series

of chromatographic steps.





Results

DOT/SDH purification

In crude extracts, activities for SDH and DQT (specific

activity of 61 and 13, respectively) were detected by

fluorometric or spectrophotometric assays. The enzyme(s)

are not stable at 40C after several days of storage, unless

glycerol and DTT are present. Under these conditions

activities remained stable to freeze/thaw, at either -20 or

-700C. Two peaks of activity for the bifunctional S-protein

were eluted from a Celite 545 column with an ammonium

sulfate gradient (Fig. 4-1). Elution of the major peak

began at about 50% of ammonium sulfate saturation and was

















E rl U) 41
: a) cl -1
r- r4d > 4-)
0 d -H (d
(d 4j)


-'- Lt -*p'

cuw~C cV
1 44 C0
rj) a) r .
4-J4..Q 0







0 -H 14
cp 1 Od
(LU) 0-
H 4J 0(d 0



cJ) r.,
(d ;j 0 a ~
04 t jf a



a) 0


'--4~u r
aro
4

144144 () 044.O
W a)~a)




0 r4 Q >4 1
0H rA 0



4.j) oD r
rx~ 0

'-4
0 Q Q








4. 44H 0 CO
t7a)0l-HU
0 -ri 04










0 0 D 4~
0 4)-ri4)
-rl 4.) 0 ra 4-)
E-4 (d 0 T

44 r
rO.~
>4l~
4 44 0a
m 5
04~





o m
444 (d0)"
U 0 M 04~~













S c "3 V Nic l Ou d


Co C4 Co o o o0 0



31Vd-1nS VinlNOVYViV lN30U3d

8 C 0 0 0 0
V-0


0 0 0 0 0 0 0
o (0 A C 0 00

L- u!u na.


0 0 o
c Coc


cO





8





0
0










U-





.




0
C,











almost completely removed from the column by the end of a

45% of ammonium sulfate saturation step. The major activity

peak fractions were completely separated from fractions

containing the minor peak of activity and eluted at about

25% of ammonium sulfate saturation. Fractions with DQT

activity were located coincidently with the two peak of SDH

activity. There were no column fractions containing only

one of these two activities. Fractions from each activity

peak were pooled and labeled SP-I for the major peak of

activity and SP-II for the minor peak of activity. Proteins

(SP-I and SP-II) were further purified similarly by

chromatography steps. It was calculated from this

chromatographic step that the major peak, SP-I, accounts for

90% or greater of the total activity in the crude extract.

The fold purifications calculated for the SP-I and SP-II

were based on this assumption. The proteins eluted in the

wash fractions of the CM columns in the second

chromatographic step of purification. Figure 4-2 shows

profiles of SP-I and SP-II elutions from the third step of

purification on DEAE-cellulose columns in Epps buffer at pH

8.6. Each protein peak eluted at about 0.3M KC1 in the

gradient and had coincident DQT and SDH activities. The

fourth chromatographic step was an HA column which eluted

each protein in the wash fractions. This step was followed

by another DEAE column, equilibrated at a lower pH of 7.2.

Each bifunctional protein eluted in the gradient of an NADP*































Fig. 4-2. Elution profiles of SP-I and SP-II from
DEAE- cellulose chromatography. The third step of
purification of the two S-proteins was a DEAE-cellulose
chromatographic column equilibrated in EPPS buffer (see
Materials and Methods for details). SDH (0) activity
remained coincident with DQT (0) activity for both SP-II
(panel A) and SP-I (panel B). The proteins began to elute
at about 0.25MKC1 of the gradient (solid line). Protein
profiles were followed at A280 n (...)














120

80

40


0 10 50 60 70 80 90
0 10 50 60 70 80 90


320

280


240

S200

E
0160

120

80


40


0


10 50 60 70 80 90
FRACTION NUMBER


0.4 -

0.2 -


0
CO
0.4 <
z
0.2 M
F-
0
O C
0 CL


















0.6

0.4 -


0.2 -


100 110


a

0.4 Z
0.2
0
cc
0 CL









68
specific 2'5'-Sepharose-4B affinity column at 1 AM NADP' and

18 mM NaCi (Fig. 4-3). Activities for DQT and SDH remained

coincident for each protein. The fold purification at this

step was 195 for SP-I and 918 for SP-II. Only SP-I was

purified further by repeating the DEAE-cellulose

chromatography step (Epps-KOH buffer), followed by a second

application on the affinity column. The final fold

purification was 1077 for the SP-I. Purification yields,

purities, specific activities and ratios of SDH/DQT

activities are shown in Table 4-1 and in Table 4-2.

Throughout the purification of SP-I and SP-II, the

ratio of shikimate dehydrogenase to dehydroquinase activity

was calculated to be within the range of 4.7 to 6.2 for SP-I

(at an average of about 5.1) and within a range of 4.4 to

5.4 for SP-II (at an average of about 4.8). Each protein was

concentrated on a PM-10 membrane to about 1 or 2 ml before

storage at -700 C.

Purification of ADH and PAT

When ADH was purified along with the SP-II, it was realized

that they eluted with overlapping activity peaks on the

Celite 545 column using the decreasing ammonium sulfate

gradient at about 25 to 20% of saturation as shown in Fig.

4-4. A critical step of separation occurred when SDH did

not bind to HA and ADH bound to HA (Fig. 4-5). Without the

inclusion of this step, the two enzymes co-purified

throughout the entire purification procedure, both eluting































Fig. 4-3. Elution profiles of S-proteins from an
NADP+ specific affinity column. A 2'5'-Sepharose-4B
column with a combined NADP+ (0-10 uM) and NaC1 (0-0.16M)
gradient was used as final steps of purification of the
S-proteins, each eluting at about 0.018M NaCl and luM
NADP. Both SDH (0) and DQT (0) activities coeluted. A)
step six of purification of SP-II protein. B) step eight
of purification of SP-I protein.


















A STEP 6 + 0. 0.12
80 AFFINITY CHROMATOGRAPHY 8
' SP-I z
40 4 z 0.06

r4
0 10 20 30 40 50 60

360 B STEP 8
AFFINITY CHROMATOGRAPHY

320 -
SP-I
280 -

240 -

.I 200 -

160 -
z
120 0.18
120 -

8- 0.12
80 -
z
40 4 -0.06



0 10 20 30 40 50 60 70 80
FRACTION NUMBER













Table 4-1. Purification of S1 protein from N.
silvestris suspension cultured cells.

SDH DQT
Total
Vol Protein Purity Yield Purity Yield Ratio
Step ml mg SA fold % SA fold % S/D

Crude 540 605 56 1 100 12 1 100 4.7
Celite 643 206 126 2 70 25 2 65 5.0
CM-52 720 49 412 7 55 94 8 59 4.4
DEAE-1 279 35 561 10 53 113 9 50 5.0
HA 618 8.7 1,515 27 36 257 22 28 5.9
DEAE-2 106 1.95 4,609 82 24 941 78 23 4.9
Aff-1 88 0.42 14,500 259 17 2,342 195 13 6.2
DEAE-3 83 0.16 33,878 605 15 7,365 614 15 4.6
Aff-2 54 0.07 65,877 1176 9 12,923 1077 12 5.1

S/D is shikimate dehydrogenase/dehydroquinase.






Table 4-2. Purification of S2 protein from N.
silvestris suspension cultured cells.

SDH DQT
Total
Vol Protein Purity Yield Purity Yield Ratio
Step ml mg SA fold % SA fold % S/D

Crude 540 605 5 1 100 1 1 100 4.6
Celite 348 97 26 5 68 5 5 64 5.0
CM-52 345 69 31 6 59 8 8 73 4.4
DEAE-1 77 31 66 13 55 14 14 53 5.1
HA 104 5 295 59 43 75 75 53 5.4
DEAE-2 31 1 757 151 34 187 187 34 4.1
Aff-1 15 0.006 4,590 918 7 880 880 7 5.2

S/D is shikimate dehydrogenase/dehydroquinate.





















100 100
CEUTE 545


80 80
E

o S2-Protein
60 E 60
c E ADH


S40 -. 4 -.... 2.0
40 40 .0

co
20 20 1.0




0 260 280 300 320 340 360
FRACTION NUMBER





Fig. 4-4. Celite 545 column chromatography overlap of
arogenate dehydrogenase and SP-II. Both SP-II (0) and ADH
(0) elute in the 45 to 0% ammonium sulfate gradient (solid
line) with overlapping activity peaks. Protein was
monitored at A2,, (...) .























140

120

100


40 -

20



0 20 40 60 80 100
FRACTION NUMBER







Fig. 4-5. Hydroxylapatite column chromatography
separation of arogenate dehydrogenase and SP-II. The SDH
activity (M) was found in the wash fractions and ADH (0)
eluted at about 120 mM KPO4.


500

400

300 0

200
E

100

0











in similar fractions from the NADP* affinity column.

Therefore, when ADH was purified by the earlier mentioned

procedure an HA column step was included to completely

remove SDH activity. Purification of PAT followed the

previously demonstrated procedure with a purification of

about 250-fold.





Discussion

The SP-I and SP-II proteins, well separated from each

other on the Celite 545 column, were each purified further

to about 1100- and 900-fold, respectively, by a series of

chromatographic steps. This complete separation was always

repeatable under similar conditions of the Celite 545 column

step (at least 12 times). SDH and DQT activities coeluted

at an activity ratio of about 5 throughout the entire

purification regimen for both bifunctional proteins. These

proteins may be isoenzymes from different compartments

chloroplastt and cytosol) or they may be isoenzymes residing

in different locations within the chloroplast. This second

possibility was reported for two DAHP synthase isoenzymes

(DS-1 and DS-2) presumably located in chloroplasts of

Arabidopsis (2). Further studies which may elucidate

differential properties of the two proteins and localization

studies should provide information with respect to possible

isoenzyme species and their location within the plant cell.









75

The purified SP-I and SP-II proteins were studied further to

characterize both the DQT and the SDH activities in (Chapter

V). The purified SP-I was used to produce specific antibody

against the protein (see Chapter VI) and for N-terminal

sequencing (see Chapter VII). Purification of PAT and ADH

was completed and then used to produce specific antibodies

to each protein for use as probes in cDNA cloning.















CHAPTER V

PROPERTIES OF THE S-PROTEINS



The main focus in my studies of the properties of the

two bifunctional proteins was to detect possible

differential properties which might suggest that these

proteins are isoenzymes. Molecular mass, pH and temperature

optima, thermal inactivation, and Km values for both

functional domains of the bifunctional proteins, SP-I and

SP-II, have been determined. Effects on activity of each

protein by various compounds, including PCMB, were also

studied.



RESULTS



Activity peaks on a Celite column

A Celite 545 column resolved about 6 peaks of SDH

activity when a more shallow gradient of ammonium sulfate

was applied than that used during the separation of the S-

proteins, Chapter IV (Fig. 5-1). Dehydroquinase activity

was coincident with SDH activities. Fractions of the six

peaks containing the enzyme activities were pooled

separately, avoiding overlapping fractions as much as

possible, and were labeled as shown in Fig. 5-1. The six

















X:- L) 040
41 WU 0)

-4 J
-r- L O

uuda
r.l

~~40 4J
Lna) 4J (
(U


41~
LA 4L





rA r)-I 0)
4J(

-4l a) 14~-4
r-4 0>

Ln




L44 44C

0-
4-1 r=o~ V







0-4 H)-

44


u-t4 j 0
::s4 4 4 )4
Ul 0




o a)~a,


4-4Hb)1
CU (U )m


0 x r=0
LA 1 0) $.q :
V M4J M a)










44 04 r-4 r=
0 r














02 r-40 (
.rA 0E











00 4-I
a) id tLn
riri as


M~ E rI -


i C) (d

u k o








78



o
cm

0












-CO











31 n -o LL
ic I



IC




.UwLu nd (*) HOS cv











pooled samples containing DQT/SDH activities were

concentrated and examined for possible differential

properties, substrate requirements for saturation with SHK

and NADP', alternative substrate specificities (i.e., NAD+

and QA), temperature optimum, thermal inactivation, SDS PAGE

and activity-stained native PAGE. No significant

differences were found between the overlapping peak samples

of I to V (data is not shown). All six protein samples were

specific for NADP* and SHK when assayed for SDH activity.

Pool sample IV (or perhaps I through V) corresponds to SP-I,

and Peak VI corresponded to SP-II. SP-I and SP-II exhibited

similar properties except for M,, Km and activity stained

gels.

Molecular Mass

A difference was seen in the molecular mass of SP-I and

SP-II as shown in Fig. 5-2. SP-I migrated slightly ahead of

SP-II on SDS gels (A) and on a silver stained SDS gels (B).

Both gels showed bands (or set of bands) at about 59 kD and

40 kD for purified SP-I, and bands at about 61 kD for

purified SP-II. A band at 42 kD can be detected when higher

concentration of SP-II was used in these experiments. The

silver stained gel offered higher resolution and indicated

that the affinity purified SP-I also had a set of bands at

about 29 kD. Native Mr was determined on a Sephadex G-200

column,( Fig. 5-3, panels A and B), to be about 59 kD and 62

kD for SP-I and SP-II, respectively. In crude extract






















Fig. 5-2. Molecular weight determinations by SDS
PAGE and by silver stain of the two S-proteins. A) The
SDS gel shows 2 bands for SP-I (584 ng) at molecular
weights of about 60,000 and 40,000 in lane 3. Lane 5 has
a single detectable band at a molecular weight of about
62,000 for the SP-II (155 ng). Lanes 1 and 7 are
molecular weight markers with MW as indicated. B) The
silver staining technique was also used to determine
molecular weights of the S-proteins. Lanes 1, 9, and 16
show molecular weight markers. Lanes 2 through 7
represent SP-I and lanes 11 through 14 represent SP-II at
various stages of purification as follows:

Lane Protein purification step Molecular weight(s)
2 S1 25ul Affinity pure 60,000 40,000
3 S1 10ul Affinity pure 60,000 40,000
4 S1 25ul DEAE-cellulose 60,000 40,000 29,000
5 S1 10ul DEAE-cellulose 60,000 40,000 29,000
6 S1 10ul Affinity pure (AB) 60,000 40,000 29,000
7 S1 25ul Affinity pure (AB) 60,000 40,000 29,000
11 S2 25ul Affinity pure (1) 62,000
12 S2 10ul Affinity pure (1) 62,000
13 S2 25ul Affinity pure (2) 62,000
14 S2 10ul Affinity pure (2) 62,000

(AB) is purified enzyme from several runs that were
combined, passed through the affinity column a third time
and concentrated. For SP-II the (1) represents the
purified protein after the first pass through the
affinity column and (2) represents combined column runs
of SP-II passed a second time through the affinity column
and concentrated.


















A
1 3 5 7

-- 94,000
67,000
43,000

S 30,000

20,100
4 14.400
-U


B
1 2 3 4 5 6 7 8 910111213141516

94,000
67,000
43,000


30,000


20,100


14,400






























Fig. 5-3. Gel filtration chromatography of S-
proteins. A) Concentrated crude extract () pool IV
(SP-I) (0) and pool VI (SP-II) (A) (from the shallow
gradient Celite 545 column) were passed through a
Sephadex G-200 column. The dotted profile represents the
three marker enzymes (alcohol dehydrogenase, BSA and
carbonic anhydrase). B) A molecular weight curve based
on the column profile from A suggests the molecular
weights for the SP-I to be about 59,000 and about 62,000
for SP-II. Crude extract S-protein is suggested to be
about 63,000.












83






A ADH BSA CAD
I *I I

0.15- Sephadex G-200 30




o 0.10- 20
0 / .20





0.05. 10





0 200 220 240 260 280 300 320 340 360 380 400 420
FRACTIONS
3 -
B
2 ADH



BSA
1i SP-II
-- 9
8 -
7-
5 e/ --- SP-I
s S-Proteins

0
O CAD

S3
o

2 -



1 --- I ------------ 1 ----
100 200 300 400
VOLUME (ml)











(containing all fractions of the S-protein), the

bifunctional protein was determined to have only one native

molecular weight of about 63 kD.

Activity stained gels

In order to optimize conditions for a native PAGE

activity stain gel, various combinations of substrate and

dyes were studied with concentrated crude extract, data not

shown. All activity stained gels were assayed at optimal

conditions as described in Methods (Chapter II). Activity

stained bands required both substrate, SHK and NADP for

visibility. Crude extract revealed 3 bands on native gels

after application of the activity stain (Fig. 5-4). In Fig.

5-5, purified SP-I and SP-II had 2 activity stained bands

each. SP-II migrated at a faster rate than did SP-I.

pH and temperature optima of S-proteins

Both functional domains of the two bifunctional

proteins were studied with respect to pH optima (Fig. 5-6).

SDH activity was followed in a pH range of 6.5 to 10 in 200

mM Bis Trispropane buffer and showed optimal activity at pH

9.0 for both proteins. DQT, assayed by the coupled assay

method had very low activities when the same buffer was used

over the same pH range for both SP-I and SP-II. A pH range

from 6.1 to 9.5 in potassium phosphate, Epps or glycine

buffers, each at 100 mM resulted in higher activities and

showed optimal activity at pH 7.25 for both proteins. When

the three buffers above were used for the SDH assay there
































Fig. 5-4. Optimal PAGE activity staining for SDH
in crude extract. An activity stain of a native
polyacrylimide gel was done at optimal conditions when
concentrated crude extract of 360 ug was applied to the
gel. Three bands are shown after incubation in
substrates at concentrations for optimal activity.








































,.
.r~~. 4.
Ir
r



Ii


ammmmm--





-mommem-





M-NNON--


!U:Ar T!;: -- :..































Fig. 5-5. PAGE activity staining of purified SP-I
and SP-II. SP-I was added to lanes 2 and 3, and SP-II
was added to lanes 1 and 4. Activity staining was done
under optimal conditions. A) After incubation for about
15 min, SP-I showed two bands of activity in both lanes.
SP-II, at lower concentrations, showed two bands in lane
4, each migrated faster than the SP-I bands. B) The gel
was incubated further for about 15 min. The SP-II in lane
1 was then visible. C) After incubation for 1 h, 2 bands
were more pronounced for all four lanes.




Full Text
MOLECULAR CHARACTERIZATION OF A BIFUNCTIONAL ENZYME
(DEHYDROQUINATE DEHYDRATASE/SHIKIMATE DEHYDROGENASE)
AND THE POST-PREPHENATE PATHWAY ENZYMES NEEDED FOR
PHENYLALANINE AND TYROSINE BIOSYNTHESIS IN NICOTIANA SPP.
BY
CAROL ANN BONNER
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
1994

ACKNOWLEDGEMENTS
Many thanks to Roy A. Jensen for the inspiration and
guidance throughout my studies for this dissertation. I
thank, also, my committee members, Dr. Richard P. Boyce from
the Department of Biochemistry and Molecular Biology; and
from the Department of Microbiology and Cell Science, Dr.
Lonnie 0. Ingram, Dr. Robert R. Schmidt and Dr. Keelnatham
T. Shanmugam for their support and encouragement in
completion of this dissertation. Special thanks to all the
members of our lab who have been a great source of help to
me during these studies. Firstly, to Randy Fischer and
Premila Rao, my most grateful thanks for their help and
encouragement in so many ways from the very beginning of my
studies. Thanks to Astride Rodrigues, Jackie Miller,
Michelle Miller for all the work in the tissue culture lab.
Most special thanks to Tianhui Xia, Genshi Zhao, Prem
Subramaniam, Wei Gu and Jain Song for all the discussion and
work we have done together. I would also like to thank the
other members of the microbiology and cell science
department who have helped me with their knowledge and
advice, especially Philip Miller, for all the information
and insight that he gave me with respect to the molecular
biology portion of my dissertation.
11

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF FIGURES v
LIST OF TABLES viii
ABBREVIATIONS ix
ABSTRACT xi
CHAPTERS
ILITERATURE REVIEW AND RATIONALE 1
Aromatic Amino Acid Biosynthesis in Plants.. 1
Rational for Focus upon DQT and SDH 8
Shikimate biosynthesis and quinate
catabolism 9
Multiple Species of Shikimate Dehydrogenase. 12
The Post-Prephenate Pathway in Plants 16
IIMATERIALS AND METHODS 19
Organisms and Extract Preparation 19
Enzyme Assays 23
Separation of Pathway Enzymes by
Chromatography 27
Purification of Pathway Proteins 29
Polyacrylamide Gel Electrophoresis 33
Kinetic Values 34
Inhibition studies 35
Antibodies 35
cDNA cloning and sequencing 3 7
Biochemicals 43
IIIPHYSIOLOGICAL PROFILE OF AROMATIC PATHWAY
ENZYMES IN PLANTS 4 5
Results 45
Discussion 59
iii

IVPURIFICATION OF PROTEINS FROM THE AROMATIC
AMINO ACID PATHWAY 62
Results 62
Discussion 53
V PROPERTIES OF THE S-PROTEINS 76
Results 76
Discussion 104
VISPECIFIC ANTIBODY TO THE BIFUNCTIONAL
S-PROTEIN AND TO TWO POST-PREPHENATE
PROTEINS 108
Results 108
Discussion 116
VIICLONING cDNAS ENCODING THE BIFUNCTIONAL
S-PROTEIN, ADH AND PAT 118
Results 118
Discussion 132
SUMMARY 14 8
REFERENCES 14 9
BIOGRAPHICAL SKETCH 157
IV

LIST OF FIGURES
Figure Page
1-1 Aromatic amino acid biosynthetic pathway 3
1-2 Multiple compartmentation of pathways in plant
cells 7
1-3 Quinate catabolism and aromatic amino acid
biosynthesis 11
3-1 DEAE-cellulose chromatography of aromatic pathway
enzymes from N. silvestris 48
3-2 DEAE-Cellulose chromatography of aromatic pathway
enzymes from C. sorokiniana 51
3-3 Temperature optimum for activity of prephenate
aminotransferase in Chlorella sorokiniana 52
3-4 Growth curve of N. silvestris in suspension
cultures 56
3-5 Arogenate dehydratase activities followed
throughout a growth curve of N. silvestris 58
4-1 Elution profiles of SP-I and SP-II from Celite
545 column chromatography 64
4-2 Elution profiles of SP-I and SP-II from DEAE-
cellulose chromatography 67
4-3 Elution profiles of S-proteins from an NADP+
specific affinity column 70
4-4 Celite 545 column chromatography overlap of
arogenate dehydrogenase and SP-II 72
4-5 Hydroxylapatite column chromatography separation
of arogenate dehydrogenase and SP-II 73
5-1 Elution profile of SDH activities from a Celite
545 column run with a shallow gradient 78
5-2 Molecular weight determinations by SDS PAGE and
by silver stain gels of the two S-proteins 81
v

5-3 Gel filtration chromatography of S-proteins 83
5-4 Optimal PAGE activity staining for SDH in crude
extract 86
5-5 PAGE activity staining of purified SI- and 32-
proteins 88
5-6 S-proteins pH optima of optima for activity 90
5-7 Temperature optima for S-proteins 93
5-8 Thermal inactivation of S-proteins 95
5-9 Substrate protection against thermal
inactivation of S-proteins 97
5-10 Saturation curves and double reciprocal plots
for SP-1 100
5-11 Saturation curves and double reciprocal plots
for SP-II 102
6-1 Precipitation of S-proteins with SP-I specific
antibody on Ouchterlony plates 110
6-2 Effect of SP-I specific antibody on SP-I and
SP-II activities 113
6-3 Comparison of Western blot and activity stained
gels 115
7-1 Agarose gels of cDNA encoding aromatic pathway
proteins 121
7-2 Preliminary alignment of translated sequences of
clone SP3 with established DQT sequences 124
7-3 Sequencing strategy for cDNA encoding the
bifunctional protein 126
7-4 Agarose gels of SP3 cDNA fragments during
stages of subcloning 128
7-5 Nucleotide and deduced amino acid sequence of
cDNA coding for the bifunctional S-protein 131
7-6 Multiple amino acid alignment of the AroD
domain of AroD*E with its homologues 135
7-7 Dendrogram of dehydroquinase homologues 13 7
vi

7-8 Multiple amino acid alignment of the shikimate
dehydrogenase domain of AroD»E and its
homologues 141
7-9 Dendrogram for shikimate dehydrogenase
homologues 143
Vil

LIST OF TABLES
Table Page
1-1 Molecular mass and Km values for DQT and SDH 15
3-1 Specific activities from N. silvestris and C.
sorokiniana aromatic pathway enzymes 4 6
3-2 Prephenate aminotransferase amino acid donor
specificity in C. sorokiniana 53
3-3 Prephenate aminotransferase activity after
thermal treatment in C. sorokiniana 53
4-1 Purification of SP-I from N. silvestris
suspension cultured cells 71
4-2 Purification of SP-II from N. silvestris
suspension cultured cells 71
5-1 Kinetic parameters for SP-I and SP-II 103
5-2 Effect of PCMB on enzyme activity 104
6-1 S-protein antibody effects on N. silvestris and
E. coli extracts 116
7-1 SDH/DQT activities in transformed aroD and aroE
mutant strains of E. coli 122
7-2 Pairwise comparisons of S-protein with its
homologues 144
7-3 NAD(P)H binding motif of enzymes 145
7-4 Transit peptides of aromatic pathway proteins
and other plant proteins 143
viii

ABBREVIATIONS
ADH
ADT
AGN
aKG
AMP
ANS
ANS-1
jSME
BSA
CHA
CM
CM-52
CM-1
CM-11
DAHP
DAHPS
DE52
DHQ
DHS
DQT
Ds-Co
Ds-Mn
DTT
E-cells
EDTA
EE-cells
EPPS
EPSP
E4P
HA
HPLC
HPP
ICBR
IPTG
KP04
LB
LB-AMP
LES
MTT
NADP
NTPS
arogenate dehydrogenase
arogenate dehydratase
L-arogenate
a-ketoglutarate
ampicillin
anthranilate synthase
aneuploid N. silvestris suspension cell line
beta mercaptoethanol
bovine serum albumin
chorismate
chorismate mutase
carboxymethyl cellulose-52 cation exchange column
chloroplast chorismate mutase
cytosolic chorismate mutase
3-deoxy-D-arabino-heptulosonate 7-phosphate
DAHP synthase, enzyme 1 of aromatic pathway
(DEAE) diethylaminoethyl cellulose-52 anion
exchange column
dehydroquinate
dehydroshikimate
dehydroquinate dehydratase or dehydroquinase
cytosolic DAHP synthase of plants
chloroplast DAHP synthase of plants
dithiothreitol
cells of about two generations of exponential
growth
ethylenediaminetetraacetate
cells in continuous exponential growth
(N-[2-Hydroxyethyl]piperazine-N'-[3-
propanesulfonic acid];HPPS)
5-enolpyruvylshikimate-3-phosphate
erythrose 4-phosphate
hydroxylapetite column
high performance liquid chromatography
4-hydroxyphenylpyruvate
Interdisciplinary Center of Biotechnology Research
isopropylthio-jS-D-galactoside
potassium phosphate buffer
Luria-Bertaini medium
LB plus ampicillin
lag, exponential and stationary phases of growth
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium-bromide; (thiazolyl blue)
nicotinamide adenine dinucleotide
nucleotide triphosphates
ix

OAA oxaloacetate
OPA orthopthalaldyhyde
PAGE polyacrylamide gel electrophoresis
PAT prephenate aminotransferase
PCA protocatechuic acid
PCMB p-chloromercuribenzoate
PDH prephenate dehydrogenase
PDT prephenate dehydratase
PEGaooo polyethylene glycol
PEP phosphoenolpyruvate
PLP pyridoxal 5'-phosphate
PMS N-methyldibenzopyrazine methylsulfate (phenazine
metasulfate)
PMSF phenylmethylsulfonyl fluoride
PPA prephenate
PPY phenylpyruvate
PCA protocatechuic acid
QA quinic acid
QDH quinate dehydrogenase
QDT quinate dehydratase
RE restriction enzymes
SA specific activity
S-protein shikimate-bifunctional DQT/SDH protein
SDH shikimate dehydrogenase
SDS sodium dodecyl sulfate
SP-I (Sl-protein) bifunctional protein (DQT/SDH), major
peak from Celite 545 chromatography separation
SP-II (S2-protein) bifunctional protein (DQT/SDH), minor
peak from Celite 545 chromatography separation
SHK shikimate

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
MOLECULAR CHARACTERIZATION OF A BIFUNCTIONAL ENZYME
(DEHYDROQUINATE DEHYDRATASE/SHIKIMATE DEHYDROGENASE)
AND THE POST-PREPHENATE PATHWAY ENZYMES NEEDED FOR
PHENYLALANINE AND TYROSINE BIOSYNTHESIS IN NICOTIANA SPP.
By
Carol Ann Bonner
April 1994
Chairman: Roy A. Jensen
Major Department: Microbiology and Cell Science
Two bifunctional proteins containing catalytic domains
for dehydroquinate dehydratase (dehydroquinase) and
shikimate dehydrogenase were separated from extracts of
Nicotiana silvestris suspension cells by use of a decreasing
ammonium sulfate gradient on a Celite 545 column. The two
apparent isoenzymes, denoted as SP-I and SP-II, were
purified 1000- and 800-fold, respectively. Higher molecular
mass values were obtained for SP-II than for SP-I on the
criteria of sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis (PAGE) and gel filtration chromatography.
Each protein exhibited multiple bands on SDS-PAGE and on
native-PAGE monitored with a specific shikimate
xi

dehydrogenase activity stain. Temperature and pH optima for
catalysis were similar for each protein, as were the
temperatures of inactivation. However, Km values for
shikimate were 0.80 and 0.36 mM for SP-I and SP-II,
respectively. Antibody raised against SP-I cross-reacted
with SP-II, but not with Escherichia coli proteins
possessing the corresponding activities. Antibody screening
of a cDNA library of Nicotiana tabacum was used to isolate
clones expressing the S-protein. Functional complementation
of aroD and aroE E. coli auxotrophs transformed with
plasmids carrying cloned cDNA were successful. Activities
for each enzyme were 15-fold greater in the transformed
mutant strains than in the wild type strain of E. coli.
Analysis of the amino acid sequence deduced from the cloned
cDNA sequence revealed homology with the appropriate
functional domains of the pentafunctional Arol protein of
Saccharomyces cerevisiae and with the monofunctional AroD
and AroE proteins of E. coli. The post-prephenate pathway
enzymes were also studied in Nicotiana silvestris suspension
cells. Levels of arogenate dehydratase activity changed
throughout a growth cycle in suspension cells. Using
specific antibodies, several putative cDNA clones encoding
prephenate aminotransferase or arogenate dehydrogenase were
isolated for further study. The base of comparative
enzymology in higher plants for the aforementioned enzymes
was extended to the unicellular alga, Chlorella sorokiniana.
XI1

Prephenate aminotransferase of Chlorella exhibited the
striking high-temperature activity optimum and specificity
for prephenate that is typical of higher plants.

CHAPTER I
LITERATURE REVIEW AND RATIONALE
Aromatic Amino Acid Biosynthesis in Plants
In higher plants, the three aromatic amino acids (L-
phenylalanine, L-tyrosine and L-tryptophan) are not only
required for protein synthesis and primary metabolite
synthesis (such as the hormone, indole acetic acid and the
structural component of woody plants, lignin), but also must
be available as starting substrates for a vast array of
secondary metabolites including alkaloids, coumarins,
isoflavones, and tannins (2, 78, 79). Intermediates of the
pathway are also precursors for synthesis of other essential
metabolites in plant cells. The vitamin-like derivatives,
folic acid and ubiquinone, are synthesized from chorismate,
the first branch point intermediate in the aromatic pathway.
Protocatechuic acid is derived from the common pathway
intermediate, dehydroshikimate (Fig. 1). It has been
estimated that up to 60% of plant carbon flows through the
aromatic amino acid pathway (40). Humans as well as other
higher forms of life depend heavily on plants to provide the
three essential aromatic amino acids, and knowledge of
aromatic biosynthesis in higher plants is fundamentally
1

Fig. 1-1. Aromatic amino acid biosynthetic pathway.
The common portion of the biosynthetic pathway leading to
the branch point intermediate, chorismate, begins with
DAHP synthase [1] catalizing the condensation of
erythrose 4-phosphate and phosphoenolpyruvate and
consists of seven steps. These steps consist of
dehydroquinate synthase [2] , dehydroquinate dehydratase
[3], shikimate dehydrogenase [4], shikimate kinase [5],
EPSP synthase [6] , and chorismate synthase [7] .
Chorismate, at the branchpoint of the pathway is utilized
in one direction to yield L-tryptophan (steps 8-12) and
in another direction by chorismate mutase [13] to produce
prephenate, another branch point intermediate. Organisms
in nature use different routings from prephenate. Higher
plant chloroplasts proceed through prephenate
aminotransferase [14] and arogenate dehydratase [15] to
form PHE, or through prephenate aminotransferase and
arogenate dehydrogenase [16] to form TYR. In other
organisms a second route proceeds through prephenate
dehydratase [19] , then through aromatic (or PHE)
aminotransferase [20] to synthesize PHE, or through
prephenate dehydrogenase [17] and through aromatic (or
TYR) aminotransferase [18] to synthesize TYR.

z

4
important. Figure 1 shows a composite schematic of the
biosynthetic steps of the common portion of the pathway and
of the three divergent amino acid branches.
It has been established that a complete aromatic
biosynthetic pathway is present in the chloroplasts of plant
cells (2, 41). The common portion of the pathway begins
with the condensation of erythrose 4-phosphate and
phosphoenol pyruvate to form 3-deoxy-D-arabino-heptulosonate
7-phosphate by DAHP synthase and continues with six more
enzymatic steps to form CHA, the major branchpoint
intermediate. From CHA, the pathway branches to form L-
tryptophan via anthranilate synthase (ANS, enzyme 8) and to
L-phenylalanine or L-tyrosine via chorismate mutase (CM,
enzyme 13). From prephenate, the product of the CM
reaction, different pathway variations have been shown in
nature to complete the synthesis of PHE and TYR (16). In
plant chloroplasts, the post-prephenate pathway begins with
the transamination of PPA to yield L-arogenate by prephenate
aminotransferase (PAT, enzyme 14). Finally, AGN is either
converted to PHE by arogenate dehydratase (ADT, enzyme 15)
or to TYR by arogenate dehydrogenase (ADH, enzyme 16). This
pathway is illustrated with dashed arrows in Fig. 1.
The basic hypothesis underlying my studies is that a
second pathway for aromatic amino acid biosynthesis exists
in the cytosol compartment of higher plants. The supportive
evidence is that DAHP synthase (the early-pathway enzyme)

5
(32), CM (the mid-pathway enzyme) (23) and ANS (the initial
TRP-branch enzyme) (15) are present as pairs of separately
compartmented isoenzymes (Fig. 1-2). It is also known that
starting substrates for aromatic biosynthesis (E4P and PEP)
are synthesized by duplicate pathways of carbohydrate
metabolism located in both the plastid and cytosolic
compartments (Fig. 1-2). Since most secondary metabolites
are synthesized in the cytosol of plant cells (39) (Fig. 1-
2), it seems likely that an aromatic pathway should be
available in the cytosol to produce aromatic amino acids not
only for protein synthesis but as starting substrates for
secondary metabolism as well.
Cytosolic isoenzymes of DAHP synthase and chorismate
mutase (Ds-Co and CM-II, respectively) are markedly
different from Ds-Mn and CM-I (the corresponding plastid-
localized isoenzymes) in both catalytic and regulatory
properties (32, 23). Such differences and indeed a lack of
homology would be consistent with the endosymbiotic
hypothesis of organelle evolution (55). If cytosolic
species of other pathway steps are present in plant cells,
it would not be surprising to find them to have equally
divergent properties that reflect their xenologous origin at
the time of endosymbiosis. Thus, the alternative post-
prephenate steps shown in Fig. 1 (solid arrows) might be
used in the cytosol. From this perspective it seems
unlikely that dehydroquinase and shikimate dehydrogenase

Fig. 1-2. Multiple compartmentation of pathways in plant cells. Glycolysis
and the pentose phosphate pathways are shown in both the chloroplast and the
cytosol compartments of higher plants. An intact aromatic amino acid pathway is
shown in the chloroplast. A hypothetical aromatic amino acid pathway is shown in
the cytosol. Three chloroplast isoenzymes of aromatic biosynthesis (a) and the
corresponding cytosolic isoenzymes (a) are shown. Numerous metabolites, known to
be synthesized in the cytosol from aromatic amino acids, PHE, TYR and TRP, are
given.

CYANOOEN1C
QLYC0S4DES
ANTHRAQUtNONES 4
CHLOROPLAST
'CYTOPLASM
CARBOXY-PHE ;
m-CAR BOXY • TYR
\ / :
ItochorismaU
0IUCO4» • »
lr° 1
. B7P
Oxklatív* PP Pathway
l A
•« cma-{cv-eJ— ppa
1 y
EXTENSIN
ROSMARIMC
ACID
["^I
<32P>
INDOLE ALKALOIDS
^
• \\ PHYTOALEXINS
» \"'v
\ 'OFLAVONOIDS
^ LIGNIN
COLIMAR INS
-]

8
would co-exist as a bifunctional protein, as they do in
plastids. Alternative possibilities would be that they are
monofunctional species, or that they are domains of a large
pentafunctional protein like that present in fungi (21).
That the enzymes catalyzing the third and fourth steps
in the common pathway of plastids are bifunctional in higher
plants has been documented (14, 61). In E. coli, DQT and
SDH are each found as monofunctional proteins (18,19).
Yeast and fungi produce the arom protein (21), a
pentafunctional protein bearing functional domains
corresponding to steps 2 through 6 of aromatic amino acid
biosynthesis (Fig. 1-1). It is interesting that when the
arom pentafunctional protein of Neurospora crassa is
subjected to limited proteolysis, the DQT/SDH domains were
retained as an intact and functional fragment (77).
Rationale for Focus upon DOT and SDH
The rationale for choice of the DQT/SDH protein (or S-
protein) for study was as follows, (i) SDH is easily
assayed, has very high activity and the substrates are
readily available. (ii) The S-protein is in the middle of
the common portion of the pathway and provides a link from
the early-pathway isoenzyme pair of DAHP synthase to the
second known mid-pathway pair of isoenzymes of chorismate
mutase. (iii) Two separable shikimate dehydrogenases had
been demonstrated by Rothe et al. (67) in pea seedlings, and

9
my preliminary experiments had shown this result to be
repeatable in N. silvestris. (iv) Distinctly different
properties of the isoenzyme pairs for DAHP synthase,
chorismate mutase, and anthranilate synthase have been most
useful to demonstrate the separate spatial locations. Since
DQT and SDH were known to coexist as a plastid-localized
bifunctional protein, I thought it likely that the
corresponding cytosolic enzymes would be monofunctional. If
so, this would be a type of differential property that might
allow me to distinguish the two during cell fractionation
studies.
Shikimate biosynthesis and guinate catabolism
In monocots, such as Zea mays, there are two
dehydroquinases, one is bifunctional with SDH (specific for
NADP+) and the other is either bifunctional or stably
complexed with a NAD+ specific quinate dehydrogenase (36).
The pathway steps to quinic acid in plants have not yet been
rigorously established and may originate from E4P and PEP
via DHQ, or from unknown pathways. Quinate has been shown
to accumulate in plant vacuoles and to account for at least
10%, if not more, of the dry weight of certain plant tissues
(2, V. I. Ossipov, personal communications). The quinate
catabolic system from fungi (34,35) overlaps the
biosynthetic shikimate pathway with several analogous
dehydrogenase and dehydratase reactions (Fig. 1-3). An

Fig. 1-3. Quínate catabolism and aromatic amino acid biosynthesis. Aromatic
amino acid biosynthesis is shown by the thin horizontal arrows. The quínate
catabolic system is shown by the heavy arrows starting with NAD+ specific quínate
dehydrogenase, dehydroquinate dehydratase and dehydroshikimate dehydratase to form
protocatechuic acid. The remaining heavy arrow is the NAD+/QDH which also uses
SHK. The heavy dot between the thin and thick arrow emphasizes the overlapping
step common to biosynthesis and catabolism, ie, the biosynthetic dehydroquinase and
the inducible catabolic dehydroquinase. Biosynthetic NADPH specific QDH (the thin
vertical arrow) may use DHQ to synthesize QA, or may be used in an unknown route
for QA biosynthesis shown by the short dashed arrow. The long dashed arrow show
quínate dehydratase, an activity reported in some plants.

?
MO. COOM
B4P
♦
PBP
AROMATIC
AMINO
ACIDS
V
CARBON
BOILDINQ
BLOCKS

12
inducible catabolic dehydroquinase takes DHQ that comes
directly from quinic acid (by a specific NAD+ quinate
dehydrogenase) to dehydroshikimate which is converted to PCA
and on to carbon building blocks generated by the TCA cycle.
The dehydroquinase functional domain of the pentafunctional
arom protein of yeast and fungi channels DHQ formed from
DAHP to EPSP, thus bypassing PCA formation and avoiding
futile cycling (17, 21, 28). Both a NADPH specific QDH (V.
I. Ossipov, personal communication) and a quinate
dehydratase (V.I. Ossipov, personal communication) have
recently been shown. The NAD+ specific QDH from E. nidulans
also will utilize SHK (34). These two systems offer some
interesting possibilities for overlapping metabolism in
plants. For example, it is conceivable that a QDT might
convert quinate to SHK for synthesis of aromatic amino acids
(Fig. 1-3) to be utilized as precursors for the synthesis of
lignin and other compounds.
Multiple Species of Shikimate Dehydrogenase
Shikimate dehydrogenase has been a popular enzyme
choice for a variety of isoenzyme studies and multiple
activities have been described on many occasions.
The bifunctional DQT/SDH protein (NADP+ specific) was
examined in dicots (50). No evidence of a separate DQT or
SDH has been determined, thus far.

13
Cofactor specificity. Rothe et al. (67) separated two
SDH (NADP*) activities from pea seedlings on a Celite 545
column with an ammonium sulfate gradient. Whether DQT
activity coeluted with one or both of the SDH activities was
not examined. Shikimate dehydrogenase was shown to be
located in the stroma of spinach chloroplasts and was
partially purified (30). Mousdale et al. (61) purified a
bifunctional DQT/SDH (NADP* specific) from pea seedling
chloroplasts and also showed by chromatofocusing that there
were two chloroplast isoenzymes and one unstable isoenzyme,
probably located in the cytosol. Two forms of nondissoc-
iable DQT/SDH (NADP*) activities and one of QDH (NAD*) were
found in Phaseolus mungo seedlings by Minamikawa (58).
Koshiba (49) also found two forms of DQT/SDH (NADP*)
activities from Phaseolus mungo seedlings. In 1988, Ogawa
and Tateoka (64) reported one SDH (NADP*) specific activity
and two SDH (NAD+) specific activities (DQT apparently was
not assayed in these fractions) from Phaseolus mungo. This
is the first description of NAD* specific SDH activities and
is surprising since the same methodology for separation was
used by the three groups and each checked for specificity of
cofactors. A recent study of QDH in Phaseolus mungo by Kang
and Scheibe (44) found that the enzyme could use NADP* as
well as NAD*. When SHK was added to the reaction mix
containing quinic acid and NAD*, some inhibition was found.
Apparently SHK was not tried as a substrate for QDH. If QDH

14
also functions as a SDH, competition for two products
(dehydroquinate and dehydroshikimate) would occur and this
could account for the inhibition seen with SHK added to the
reaction mix. This might also explain the earlier report of
the NAD* specific SDH activity by Ogawa and Tateoka (64), if
the enzyme was actually a nonspecific QDH. The qa gene
cluster of Neurospora crassa encodes a functional domain for
a QDH (NADH specific) that will also utilize SHK/NADH (34).
Properties. The range of molecular mass for DQT/SDH
(or SDH, where DQT was not studied) is from 44,000 to
73,000. It is not clear as to whether the lower Mr of
44,000 might represent a monofunctional SDH. SDH activity
is usually assayed in the reverse of the physiological
direction with the substrates, SHK and NADP+, rather than
the forward direction with DHS and NADPH:
NADPH
Dehydroshikimate <=
^=> Shikimate
NADP*
Values of Km have been determined and vary from 0.060 to 1.3
mM for SHK, from 0.010 to 0.10 for NADP*, and from 0.130 to
0.180 mM DHQ. Table 1-1 gives estimates of Mr and Km values
for DQT and SDH from several plant species, as well as Mr
from E. coli and from S. cerevisiae.
Inhibition studies with HgCl2, p-chloromercuri benzoate
and oxidized glutathione have suggested that SDH

15
Table 1-1
.. Molecular mass and
Km values
of
DQT and SDH.
Source
Substrate
Km (mM)
Mra
Reference
Sweet potato
SHK
1.300
48
NADP
0.100
Mung bean 1
DHQ
0.130
57,000
(GE)
49
SHK
0.220
NADP
0.025
Mung bean 2
DHQ
0.180
57,000
(GE)
49
SHK
0.910
NADP
0.025
Tomato
SHK
0.038
73,000
(GF)
52
Pea
SHK
0.690
59,000
61
NADP
0.013
Corn 1
55,000
(GE)
13
Corn 2
49,000
(GE)
Spinach
67,000
(GF)
30
59,000
(GE)
Cucumber
SHK
0.060
44,000
(GF)
53
NADP
0.010
Radish
SHK
0.860
60,000
(GF)
71
NADP
0.090
Spinach
SHK
0.480
44,000
(GF)
71
NADP
0.032
49,000
(GE)
E. coli
56,000
(AA)
28b
S. cerevisiae
—
68,000
(AA)
77
aMolecular mass estimated by gel filtration (GF), by PAGE
(GE) or calculated from the deduced amino acid sequence
(AA) .
bMolecular mass of monofunctional DQT and SDH were combined.
sulfhydryl groups act as functional groups in the catalysis
of the enzyme and that partial reversal of inhibition is
accomplished by thiols (48, 53). Aromatic compounds,
metals, and salts have been studied with respect to effect
on DQT and SDH activities. Protocatechuic acid was found to
be a competitive inhibitor of SDH activity in tomato (52,)
and Cl' inhibited DQT activity competitively in pea (61).
Ca2+ stimulated SDH activity in Brassica rapa (71) .

16
Molecular genetics. Plant genes encoding DQT or SDH
have not been sequenced, but the monocistronic aroD and aroE
genes have been cloned and sequenced in E. coli (1, 27).
Amino acid sequences are also available for discrete domains
encoding these activities in S. cerevisiae (28) and
Aspergillus nidulans (now known as Emericella nidulans) (17)
as part of the pentafunctional arom gene (aroM) . The E. coli
amino acid sequences, aroD (DQT) and aroE (SDH) exhibit only
have about 25 and 21% identity, respectively, with the
corresponding functional domains of the yeast arom protein.
In higher plants, aromatic-pathway genes thus far
cloned and sequenced are limited to DAHP synthase (29),
shikimate kinase (70), EPSP synthase (73), and chorismate
synthase (69) of the common portion of the pathway.
Anthranilate synthase (63) and tryptophan synthase subunit
(3) of the tryptophan branch of the pathway have been cloned
and sequenced.
The Post-Prephenate Pathway in Plants
The dual-pathway hypothesis of aromatic amino acid
biosynthesis specifies that an intact post-prephenate
pathway exists in the cytosol in addition to that already
demonstrated in the plastid compartment (41). If a separate
cytosolic pathway functions: (i) separate genes encoding
isoenzyme counterparts of the plastid-localized enzymes may
exist, (ii) separate genes encoding alternative enzymes

17
steps may exist, or (iii) the unprocessed preprotein
precursor of the plastid enzymes may function in the
cytosol.
Preohenate aminotransferase. PAT of higher plants was
first characterized in partially purified extracts,
separated from aromatic aminotransferases, and shown to be
unusually specific for its substrate, prephenate. Activity
was optimal at 70°C for PAT, a temperature which inactivated
other interfering aminotransferases (5). PAT was purified
about 1000-fold (7), and localization studies showed the
enzyme to be located in the chloroplast (Bonner and Jensen,
unpublished data, 1985; 41, 76). A simple, inexpensive
spectrophotometric assay was developed for PAT by following
the increase in oxaloacetate when the L-aspartate/a-
ketoglutarate couple was used (6). In the same study, the
ASP/aKG couple was used with native PAGE activity stained
gels to relate the position of PAT with respect to other
aminotransferases and to show the disappearance of heat-
inactivated aminotransferases.
Arocrenate dehydrogenase. Partially purified ADH was
studied in N. silvestris with respect to allosteric
regulation by TYR by Gaines et al. (31). ADH was partially
purified and regulatory properties were determined in
Sorghum bicolor (22). The enzyme was purified about 1000-
fold (8). PAT, ADH and SDH activities were followed
throughout a growth cycle in N. silvestris suspension cells

18
(12) and after wounding of potato (59).
Arogenate dehydratase. ADT activity was first
determined in N. silvestris cells by Jung et al. (43). The
enzyme was localized in spinach chloroplasts and allosteric
regulation (activation by TYR and inhibition by PHE) was
studied (43). Siehl and Conn (75) partially purified ADT
from Sorghum bicolor and studied some kinetic and regulatory
properties.
Rationale for focus upon the post-prephenate enzymes.
The rationale in choosing to study some aspects of the post-
prephenate enzymes for this dissertation is my long-term
objective to follow up earlier work with molecular-genetic
approaches. Studies of the individual enzymes may shed some
light as to the multiplicity of compartmentation. The
merits are as follows. A) Prephenate aminotransferase (i)
has been characterized and a well-developed HPLC assay
exists (5), (ii) has important biotechnological application
due to its unique aminotransferase properties and the value
of its product, L-arogenate, and (iii) can be purified by an
established procedure (7). B) Arogenate dehydrogenase (i)
has an established purification methodology (8) for
providing purified protein for antibody production that may
be used to probe cDNA libraries, and (ii) is easily assayed
fluorometrically. C) Arogenate dehydratase has an
established HPLC assay (43), but is by far the most
difficult of the three enzymes to assay and purify.

19
CHAPTER II
MATERIALS AND METHODS
Organisms and extract preparations
Eukaryotes
Nicotiana silvestris Speg. et Comes suspension cultured
cells originally isolated from haploid leaf material have
been maintained in our laboratory for about twelve years and
are well described physiologically and biochemically (10,
11, 12). Cell populations are subcultured in stationary
phase (7 day), diluted fivefold into fresh Murashige and
Skoog medium (62), and grown under specified conditions in
controlled growth chambers (10). For these experiments
(unless otherwise stated) seven-day old cells were harvested
by filtering through Miracloth (Calbiochem, LaJolla, CA),
rinsed with nanopure water, ground in liquid nitrogen in a
Waring blender and stored at -70°C. until use.
C. sorokiniana cells cultured as described by Meridith
and Schmidt (57) were kindly provided by Dr. Robert Schmidt.
Bacterial strains and plasmids
E. coli strains used for transformation include DH5a,
AB1360 aroD (DQT') and SK494 aroE (SDH") . Plasmids used for
subcloning were pUC18, pGEM5Zf(+) or pBluescript SK+.

20
Preparation of N. silvestris extract
Crude extracts for assay. Frozen, ground N. silvestris
cells were suspended (1:1) in 100 mM KP04 buffer at pH 7.3
containing 20% glycerol, 1.0 mM phenylmethylsulfonyl
fluoride and 1.0 mM dithiothreitol, centrifuged at 35,000 x
g at 4°C for 20 min. Extracts were treated to 80% of
saturation with ammonium sulfate, centrifuged as above,
followed by resuspension of pellets in EPPS-KOH buffer (pH
8.6) with all of the above components and dialyzed with four
changes of buffer until ammonia could not be detected by
HPLC. Extracts not treated with ammonium sulfate were
desalted using PD10 columns in Epps-KOH buffer as above or
dialyzed overnight with 4 changes of the Epps-KOH buffer.
Extracts for purification of ADH and PAT. Five-day N.
silvestris suspension cells were used in the purification of
ADH and PAT. Crude extracts were prepared as described
above and also included 0.1 mM pyridoxal 5' phosphate and
1.0 mM EDTA in order to stabilize PAT (5).
Extracts for S-protein purification. For purification
of the S-proteins, frozen, ground seven-day N. silvestris
suspension cells (300 g) were suspended into 300 ml of 100
mM KP04 buffer, pH 7.8, containing 20% glycerol, 1.0 mM DTT,
1.0 mM benzamidine, 0.2 mM PMSF and 0.05 mM leupeptin. A
sample was immediately desalted using a PD10 column,

21
equilibrated with 50 mAf EPPS-KOH buffer at pH 8.6,
containing the same components as above and labeled as crude
extract.
Preparation of cultured cells for assay of ADH levels
The isogenic suspension cell-culture line of N.
silvestris was grown in continuous light with gentle shaking
at 150 rev/min. The growth properties of a N. silvestris
suspension cell line (ANS-1) in lag, exponential, and
stationary phases of growth have been characterized
throughout a conventional growth cycle (referred to as E
cells) and for cells which have been maintained in
exponential growth for more than 10 generations, EE cells
(10). For routine subculture, 80 ml of suspension-culture
medium were inoculated with 20 ml of 7-day stationary-phase
cells in 500-ml Erlenmeyer flasks. EE cells (2 g wet
weight) were transferred to fresh medium on day 7 when cells
reached about 1 X 106 cells ml'1. Daily samples were
harvested using Miracloth for filtration, washed with
distilled water, ground and frozen in liquid nitrogen, and
stored at -70°C until extract preparation. Daily sampling
was also done to determine a cell count by mixing 1 ml of a
50 ml culture with 12% chromium trioxide (helps to separate
cell clumps and visualize cells more easily). Samples were
heated to 70°C for 2 to 5 min before counting in a Fuchs-
Rosenthal counting chamber at 20x magnification. The
remaining 49 ml of culture were filtered and cells were

22
dried on preweighed filters made from Miracloth in an oven
at 50°C and were weighed daily until constant dry weight
values were obtained. From these two methods, growth curves
were determined.
Preparation of C. sorokiniana extracts
Frozen cell pellets of C. sorokiniana were suspended
into 50 mM PIPES buffer, pH 7.3, containing 1.0 mM DTT, 1.0
mM PMSF, 35 mM KCl, and were broken by 2 passes through a
French Pressure Cell at 20,000 psi. The slurry was
centrifuged at 18,000 x g, at 4°C for 15 min, and the
supernatant was next centrifuged at 150,000 x g for 1 h at
4°C. Extracts were either desalted and used for column
chromatography, or dialyzed with 50 mM EPPS-KOH buffer with
all protective components as listed above for use in enzyme
assays. (Pipes buffer plus KCl was used in place of EPPS
buffer for column chromatography in order to separate
isoenzymes of chorismate mutase for another study.)
Glycerol and PLP were added to column fractions to protect
several of the enzymes to be assayed (PAT and ADH).
Preparation of bacterial extracts
The mutant E. coli strains (aroD and aroE) carrying the
cDNA clones encoding functional proteins (DQT or SDH,
respectively) were grown in 200 ml of LB/Amp media, washed
and resuspended in KP04 buffer containing 20% glycerol and 1
mM DTT at pH 7.6, and centrifuged at 150,000 x g, at 4°C for

23
1 h. The supernatants were desalted by passage through
Sephadex G-25 columns before fluorometric assay.
Enzyme assays
Aminotransferases
Prephenate aminotransferase. Prephenate
aminotransferase was usually incubated with concentrations
of 20 mAf L-glutamate and 0.8 mAf prephenate (unless otherwise
stated) , incubated at 37°C for 10 min (or as otherwise
stated) and then assayed by HPLC as an orthopthalaldyhyde
derivative in a 60% methanol/40% of 20 mAf KP04 buffer
system. Peaks of PHE were quantified before and after
acidification of the product of the reaction, AGN, in IN HC1
for 10 min at 37°C.
Aminotransferase couples. Other aminotransferase
assays included an amino acid donor (ASP, TYR, LEU or ALA)
and keto acid (PPY) at concentrations as stated and were
assayed similarly by HPLC, before and after acidification
when applicable. Controls included samples without amino
acid donor or keto acid.
Arogenate dehydratase
Arogenate dehydratase was assayed according to Jung et
al. (43) by measurement of OPA derivatives following HPLC
where the peak of AGN formed were monitored before and after
acidification to PHE. Concentration of 0.5 mAf AGN, 0.25 mAf
TYR (activator), 50 mAf EPPS-KOH buffer at pH 8.6 and enzyme

24
were included in the reaction sample of 100 /! 1 that were
incubated at 32°C for 30 min. Controls included samples
without AGN or enzyme.
Dehvdroauinase
Dehydroquinase activity was assayed spectro-
photometrically at 234 nm at room temperature (24°C) by
following the rate of increase of product, dehydroshikimate,
when dehydroquinate was provided as substrate. Unless
otherwise stated, a reaction sample included 0.5 mM DHQ,
enzyme and 50 mM EPPS-KOH buffer at pH 8.6. An extinction
coefficient of 12,000 was used to calculate activity (37).
DQT was also assayed fluorometrically via a coupled assay
with SDH. DHS formed from DHQ by the dehydratase reaction
was reduced with NADPH (cofactor for SDH) and the rate of
NADPH disappearance was followed. Reaction samples
contained 0.5 mM DHQ, 0.2 mM NADPH in 50 mM EPPS-KOH at pH
8.6 for column fractions or 50 mM Bis-tris propane buffer,
pH 7.5, for other studies as stated. Controls were carried
out for each enzyme assayed by adding only one substrate for
about three to five min before adding the second substrate.
Dehydrogenases
Dehydrogenase reactions were usually assayed
fluorometrically at room temperature (24°C) with cofactor
NAD(P)+ for 1 to 2 min before addition of substrate, and the
rate of formation of NAD(P)H was monitored (340 nm emission

25
and 460 nm excitation). Unless otherwise stated, a total
vol of 400 /¿I contained substrates and 50 mM EPPS-KOH buffer
at pH 8.6. The rate is expressed as fluorometric units per
min (FU min-1) . Activities were calculated as nmol min-1 by
relating experimental values to a standard curve obtained
with authentic NADPH. Any background rate seen when
cofactor was present alone in reaction sample (only in some
crude extracts) was subtracted from the rate obtained from
reaction sample containing both substrates to give an
appropriate correction. A spectrophotometric assay was also
used occasionally by following the rate of formation of
NAD (P) H at A340 nm.
Arogenate dehydrogenase. Arogenate dehydrogenase
activity was determined when 0.5 mAf AGN was added to the
reaction assay after 0.5 mAf NADP* had been added or as
otherwise stated. When appropriate, NAD* was added as
cofactor to the reaction mix.
Preohenate dehydrogenase. Prephenate dehydrogenase
activity was assayed with 0.5 mAf NADP* (or NAD*) and 1.0 mAf
PPA.
Shikimate dehydrogenase. Shikimate dehydrogenase
activity was usually assayed in the reverse-of-physiological
direction with 1.0 mAf NADP* and 4.0 mAf SHK, unless otherwise
stated. When appropriate, NAD* was added to the reaction
sample. In the forward direction, SDH was assayed with

26
concentrations of 1.0 mM NADPH and 1.0 mM DHS or at
concentrations otherwise stated.
Ouinate dehydrogenase. Quinate dehydrogenase was
assayed with 1.0 mM quinate and 1.0 mM NAD* or NADP* in 100
mM glycine buffer at pH 9.5.
Prephenate dehydratase
Prephenate dehydratase was assayed by incubation of PPA
and enzyme in 50 mM EPPS buffer (total vol of 100 /il) at
37°C for 10 to 20 min before addition of 100 /xl IN HCl
followed by further incubation at 37°C for 10 min. Controls
included PPA or enzyme with buffer only. Activity was
calculated from spectrophotometric readings at A320 nm and an
extinction coefficient of 17,500.
Protein estimations
Protein concentrations were estimated by the method of
Bradford (14) with BSA as a standard. A micro-assay was
used, as described, to estimate highly purified protein
concentrations.
Definition of activity and specific activity
Activity for all enzyme reactions in this study are
defined as nmol min'1 for the stated amount of protein added
into a standard assay volume. Specific activity is defined
as nmol min'1 mg protein'1.

27
Separation of pathway enzymes by chromatography
Column chromatography of N. silvestris extracts
A DEAE-cellulose column for separation of enzymes of
aromatic biosynthesis was prepared similarly as described
under purification. A 100 mg amount of protein was applied
to a bed volume of 102 ml of a DEAE column equilibrated in
50 mW KP04 at pH 7.5. After washing the column with about 3
column volumes of buffer, a 600 ml gradient from 0 to 0.4 M
KCl (KP04 buffer) was applied.
Column chromatography of C. sorokiniana extracts
A DEAE-cellulose column (20 ml) was prepared,
equilibrated in Pipes buffer (above), and about 29 mg of
protein from Chlorella were applied. The column was washed
with about 40 ml of the same buffer before a 35 mM to 500 mM
KCl gradient was added. Finally, 1.0 M KCl/Pipes buffer was
added to remove residual protein from the column.
Appropriate fractions with DQT and SDH activities in the
gradient were pooled and dialyzed against 5 mAf KP04 buffer,
20% glycerol, and 1 mM DTT at pH 7.2. An HA column (10 ml)
was prepared with the 5 mM KP04 buffer, pH 7.2 . A 15-ml
sample from above was applied to the HA column and
activities for DQT and SDH eluted in the wash fractions.
Celite 545 (shallow-gradient application of Fig, 5-1)
A 200-g amount of ground, frozen 7-day suspension cells
was suspended in 200 ml of KP04 buffer, 7.2 pH, containing
protectants and centrifuged as described above. A 3-ml

28
amount was removed for dialysis in Epps-KOH buffer and used
as a crude extract. Ammonium sulfate was added to give 75%
of saturation. From the slurry, 70 ml were removed and
prepared for use as a concentrated crude extract. To the
remaining slurry, 40 g of Celite 545 was added, stirred,
poured into a column (90 ml vol) and the eluate collected
into a cylinder. The column was washed with 65% (NH4)2S04 of
saturation in Epps-KOH buffer (about 180 ml) and 8-ml
volumes were collected. Buffer with (NH4)2S04 at 55% of
saturation (120 ml) was added, followed by a shallow
gradient of 600 ml from 55% to 45% of (NH4)2S04 saturation in
Epps-KOH buffer. A step gradient of 180 ml of buffer at 45%
of (NH4)2S04 saturation was applied before the second
gradient of 45% to 0 of (NH4)2S04 saturation (400 ml) was
added. A final wash of 120 ml of Epps-KOH buffer completed
the elution schedule.
Gel filtration
A Sephadex G-200 gel filtration column was used to
determine the native molecular weight of the S-proteins.
The column was equilibrated with 50 mM Epps buffer, pH 8.6
and DTT. Three ml of concentrated crude extract, and
concentrated SP-I or SP-II combined with 0.1 M sucrose were
applied individually to the same column (column vol of 471
ml, void vol of 119.4 ml). Protein standards of 5 mg each,

29
alcohol dehydrogenase (150,000 D), BSA (66,000 D) and
carbonic anhydrase (29,000 D) plus sucrose were applied to
the column.
Purification of pathway proteins
Purification of the S-proteins
Crude extract of 540 ml was brought to 60% of ammonium
sulfate saturation at 4°C while stirring and was used for
the purification of the S-proteins.
Celite 545 chromatography. After stirring the 60% of
ammonium sulfate saturation slurry for 30 min, 70 g of
Celite 545 was added, with continued stirring for about 20
min. The slurry containing 605 mg protein was applied to a
column and the eluate collected into a large cylinder. The
column volume of 174 ml was washed with 60% of(NH4)2S04
saturation-Epps-KOH buffer containing 20% glycerol and 1.0
mM DTT at pH 8.6 and was collected in a separate cylinder.
No activity was found in these eluates, although the protein
level was very high. A 500-ml gradient from 60 to 40% of
(NH4)2S04 saturation in the same buffer was applied and 8-ml
fractions were collected. Next a 40% of (NH4)2S04
saturation-Epps-KOH buffer of about 130 ml was applied. A
second 400 ml gradient of 40 to 0% of (NH4)2S04 saturation-
Epps-KOH buffer was applied, followed by a wash of buffer
without (NH4)2S04. Two peaks of SDH/DQT activities were
located, pooled individually, and dialyzed against the Epps

30
buffer as above. Samples of each were saved for assay, and
the remainder of each pool was used in the second
purification step (643 ml, 206 mg of SP-I protein and 348
ml, 97 mg of SP-II protein).
CM-cellulose chromatography. CM cation exchanger
columns were equilibrated with 50 mM Epps-KOH, 20% glycerol
and 1 mM DTT at pH 8.6. SP-I was applied to a bed volume of
123 ml and SP-II was applied to a bed volume of 80 ml. Both
SP-I and SP-II eluted in wash fractions and a small portion
was saved for assay. The remaining portions of the
preparations were used in the third step of purification.
DEAE-cellulose-52 chromatography. Two DEAE-52 anion
exchanger columns were prepared and equilibrated in the same
Epps-KOH buffer as the preceding step. Proteins were
applied, SP-I to bed volume of 107 ml and SP-II to a bed
volume of 72 ml, and washed with the same buffer. A 400-ml
(SP-I) or 300-ml (SP-II) gradient from 0 to 0.6 mM KCl was
applied to the columns. Fractions of about 6.5 ml were
collected. Single peaks of activity, each eluting at about
0.3 mM KCL were individually pooled and dialyzed against 5
mM KP04 buffer containing 20% glycerol and 1 mM DTT at pH
7.3. Samples were saved for assay, and the remainder of
each protein was used in the fourth step of purification.
Hvdroxvlapatite column chromatography. SP-I and SP-II
were applied to HA columns equilibrated in the KP04 buffer
of the previous step (100 ml and 50 ml bed volumes,

31
respectively). Each protein eluted in the wash and was
applied to the fifth step of purification after samples were
removed for assay.
DEAE KPO„ chromatography. DEAE columns were
equilibrated in 50 mM KP04 buffer at pH 7.3, before applying
the proteins from the previous step. Gradients up to 0.3 M
KCl were applied to each column. Single peaks of each
protein were eluted, pooled and dialyzed into 50 mM Epps-KOH
containing 20% glycerol and 1 mM DTT at pH 8.6. Samples
were saved, and the remaining proteins were subjected to the
sixth step of purification.
2'5'ADP Sepharose-4B affinity chromatography. An
NADP+-specific 2'5'ADP Sepharose-4B affinity column was
prepared and equilibrated with the Epps-KOH buffer. The
SP-I sample was applied to the column and washed with
starting buffer before a 300-ml gradient from 0 to 0.18 M
NaCl and 0 to 12 @8,25 /¿M NADP* was applied. The protein
eluted at about 4 fiM NADP+ in the gradient. The SP-I
Activity peak was pooled and dialyzed into the Epps-KOH
buffer. A sample of SP-I was saved, and the remaining
fraction was used in the seventh step of purification. The
SP-II was applied to the affinity column similarly.
Purification of SP-II was completed with this step at a
final volume of 15 ml and total protein of 6 ¿ig.

32
Final steps of SP-I purification. SP-I was applied
once again to a DEAE column equilibrated in Epps-KOH buffer
as previously described. A 200-ml gradient from 0 to 0.6 M
KC1 was applied. The SP-I activity peak fractions were
pooled and dialyzed against Epps buffer. An aliquot was
saved, and 83 ml containing 0.162 mg protein was applied to
the affinity column for the final step of purification. The
completed purification yielded 0.07 mg SP-I protein in 54
ml.
Purification of arogenate dehydrogenase
ADH was purified by two methods: A) by a series of
steps of an established procedure which began with a 0 to
45% ammonium sulfate fractionation and ended with the NADP+
affinity column (8), or B) by the elution of ADH from a
Celite 545 column used to separate S-protein activities,
followed by HA column chromatography, DEAE column
chromatography, and the NADP+ affinity column chromatography
described above.
Purification of preohenate aminotransferase
PAT was purified by following an established procedure
(7), which began with a 45 to 75% of ammonium sulfate
saturation precipitation step, followed by a 70°C treatment
to inactivate and then precipitate (by centrifugation)
contaminating aminotransferases, and ended with a specific
aminotransferase pyridoxamine phosphate (PMP) affinity
column.

33
PAGE
SDS gel electrophoresis
SDS gel electrophoresis was prepared by following the
procedure of Laemmli (51), with a 15% running gel and a 4%
stacking gel. A running buffer of Tris/glycine/SDS at pH
8.8 was used and the gel was run at constant voltage of 63 V
for about 8 h. The gel was stained for about 30 min in
Coomassie Brilliant Blue R250 dye, destained and viewed.
Silver staining
Silver staining of an SDS gel as described by Pharmacia
(Piscataway, NJ) was followed. Washes of methanol/acetic
acid and ethanol/acetic acid were included before incubation
of the gel in oxidizer, followed by addition of the silver
stain, then developer, and finally an acetic acid/water wash
to stop the reaction.
Native gel electrophoresis
A native PAGE was run according to the method of Shaw
and Prasad (74). Gel concentrations were 7% for the
stacking gel and 15% for the running gel. The running gel
was pre-electrophoresed to remove the ammonium persulfate,
and fluorescent light was used to solidify the stacking gel
which contained riboflavin. The gel was run at constant
voltage of 60 V for about 8 h.
Shikimate dehydrogenase activity stained gel
Optimal conditions for SDH activity stain on native
PAGE included 20 mg NADP*, 50 mg shikimate, 10 mg 3-(4,5-

34
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide;
(thiazolyl blue), and 2 mg N-methyldibenzopyrazine methyl
sulfate (phenazine metasulfate) in 50 ml of 100 mM glycine
buffer (without glycerol) at pH 9. Gels were incubated at
37°C for 10 to 20 min or at room temperature for 30 min to
an hour. These conditions were used unless otherwise stated
and were modified from the method presented by Davis (24).
Kinetic values
Shikimate dehydrogenase
Enzyme activities were assayed fluorometrically as
described for SP-I and SP-II. In the forward physiological
direction, either DHS or NADPH was held constant at
saturating levels while the remaining substrate
concentration was varied to obtain saturation curves. In
the reverse of physiological direction, SHK and NADP+ were
assayed similarly. Double reciprocal plots of the substrate
saturation data were used to determine the Km for each
substrate.
Dehvdroauinase
DQT activities were assayed spectrophotometrically as
described for SP-I and SP-II at varying concentrations of
DHQ to obtain saturation data, and then analyzed
appropriately as above to determine the Km for each protein.

35
Inhibition studies of S-proteins
Enzyme was incubated for 10 min with p-chloromercuri-
benzoate or with possible protectants {DTT, cysteine or /3-
mercaptoethanol (/3ME) } . SDH was assayed at saturating
conditions and DQT was assayed at 0.1 mM DHQ. For
prevention experiments, DTT, cysteine, or /8ME was added
about 10 min before addition of substrates and PCMB, and
then rates were determined. For reversal experiments, PCMB
was added to enzyme and buffer for 10 min before assay with
substrates. After a rate (or no rate) was established, DTT
was added to the reaction mix. Concentrations are as stated
in Results.
Antibodies
Preparation of specific antibodies
Antibody preparations were produced by Kel-Farms
(Alachua, FL) with repeated injections of purified SP-I over
the course of several months until antibody titers, tested
by Ouchterlony double diffusion method against SDH, ADH or
PAT, were high enough to bleed the rabbits.
Purification of specific antibodies
Antibodies were purified following a BIO-RAD (Rockville
Center, NY) procedure, by passing each over Econo-Pac 10DG
columns for desalting and then passing through DEAE Affi-Gel
Blue Econo-Pac 10DG columns for serum IgG purification. E.
coli strain DH5a lysate was prepared and incubated with the

36
purified IgG. This step removed non-specific precipitant
bands observed in the double-diffusion experiments. Pre-
immune rabbit serum were purified similarly.
Antigen:antibody assay
Antigens (SP-I, ADH or PAT) from crude extracts or
extracts from various purification steps were combined 1:1
(v:v) (or as stated) with antibodies. Samples were
incubated at 3 7°C for 10 min and returned to an ice bath,
then centrifuged (5 to 10 min) at full speed in a microfuge
to precipitate the complexed antigen:antibody. The
supernatants were assayed for enzyme activities. Controls
were always performed with preimmune rabbit serum.
Ouchterlonv assay
Ouchterlony plates were prepared with 1.5% agarose in
sodium barbital and sodium azide and wells were prepared at
precisely measured distances (66) . Antibodies or antigens
were diluted with 0.9% NaCl whenever necessary and then
added to the wells. Plates were incubated at 37°C
overnight.
Western blotting
Western blotting procedures from Sambrook et al. (68)
were followed, and bands from either SDS or native PAGE were
transferred to nitrocellulose filter paper. Membranes were
incubated in blocking solution alone and in blocking
solution containing diluted SP-I antibody for one h, washed,
incubated further with blocking buffer containing alkaline

37
phosphatase-linked goat-anti-rabbit antibody for 1 h, and
then incubated in STP buffer containing MgCl2 and
substrates, nitroblue tetrazolium and the p-toluidine salt
of 5-bromo-4-chloro-3-indolyl phosphate. After 1 h,
membranes were viewed and photographed.
Antibody precipitation of DOT and SDH from C. sorokiniana
Antibody made to SP-I was incubated 1:1, 2:1, or 4:1
with Chlorella extract (controls included preimmune
antiserum and extract diluted with buffer). After 10 min at
37°C, and centrifugation for 5 min at full speed in a
microfuge, preparations were assayed under standard
conditions for SDH and DQT activities.
cDNA cloning and sequencing
Tobacco leaf cDNA library
A cDNA library prepared from Nicotiana tabacum var.
SRI tissue culture cells, inserted into pBluescript SK-
plasmids and packaged into Lambda ZAP II vectors, was
purchased from Stratagene, La Jolla, CA. E. coli XLl-Blue
(a host strain used for immunological screening of libraries
constructed in Lambda ZAP expression vectors) and E. coli
SOLR (a non-suppressing host strain for use after excision
of the pBluescript plasmid from the Lambda Zap vector) were
included. Helper phage, ExAssist, efficient for in vivo
excision of the plasmid from the Lambda Zap vector without
replication of the phage genome was also included.

38
Preparation of cDNA libraries for screening
The cDNA library was first titered, then mixed with the
freshly prepared host strain, XLl-Blue, plated with top agar
on about 80 LB plates at about 20,000 PFU (about 1.6 X 106
PFU/ desired clone) and incubated at 37°C. Isopropylthio-/?-
D-galactoside treated nitrocellulose filters were applied
about 6 to 8 h later and incubation was continued overnight.
Filters were removed and the immunological screening
procedure of Sambrook et al. (68) was followed.
Screening the cDNA library of Lambda ZAP II
Nitrocellulose filters removed from the plates were
washed, soaked in blocking buffer containing dry milk,
rocked gently in primary S-protein, ADH or PAT specific
antibody/blocking buffer and washed several times. The
filters were next exposed to secondary antibody, goat anti¬
rabbit antiserum, washed as before and incubated with
substrates, nitroblue tetrazolium, 5-bromo-4-chloro-3-
indolyl phosphate and with 5 mM MgCl2. The filters were
kept in the dark and gently rocked for one to two hours.
Plaques corresponding to the small, deep blue spots on the
filters were cored from plates, dispensed into SM buffer
plus chloroform and stored at 4°C for titering and plaque
purification.

39
Excision of Pbluescript from the Lambda ZAP II vector
Purified phage stocks were incubated with E. coli
strain XLl-Blue and ExAssist helper phage to excise the
pBluescript plasmid from the Lambda ZAP II vector for 2 to
2.5 h at 37°C in 2XYT media (see Stratagene instruction
manual for details), heated to 70°C and centrifuged. The
supernatant, containing the plasmid, was incubated with host
E. coli strain SOLR, spread on LB plates containing 50 mg
ml"1 ampicillin and incubated overnight at 37°C. Colonies
from these plates were streaked on fresh LB-AMP plates for
immediate use and were also stored in DMSO for future use.
DNA purification
Plasmid DNA was purified by two methods. Method 1: The
pBluescript plasmids containing putative cDNA clones coding
for the S-protein, ADH or PAT were purified for sequencing
by the method of the DNA Sequencing Core Facility, ICBR,
University of FL. A colony from the E. coli SOLR strain
from the LB-AMP plate above was incubated overnight in 10 ml
of LB-AMP medium in a 50 ml tube for each clone sample. The
cultures were centrifuged and the pellets were resuspended
in GTE (68) buffer. Treatment with NaOH/SDS was followed by
neutralization with potassium acetate at pH 4.0, and
centrifugation was carried out to remove cellular debris.
The supernatants were treated with DNase-free RNase and
extracted twice with chloroform. DNA was precipitated with
isopropanol and washed with 70% ethanol. To precipitate the

40
plasmid DNA, the pellets were first dissolved in sterile
water, then 4M NaCl and 13% PEG8000 was added, followed by
incubation on ice before centrifugation at 4°C. The pellets
were rinsed in ethanol, dried under vacuum and resuspended
in sterile water. DNA concentrations were determined
spectrophotometrically at 260 nm and by agarose gel
electrophoresis with a known DNA standard. Method 2: The 10
ml cultures, as prepared above after centrifugation, were
treated with prepared solutions provided by Promega
(Madison, WI) in the Magic Miniprep DNA purification kit.
The DNA was passed through a mini-column and dissolved in
GTE (Promega instructions for preparation) buffer. This
rapid method is preferred for sequencing on a Li-Cor
sequencer available in the Department of Microbiology and
Cell Science.
Competent cells of E. coli strains
E. coli strains DH5Q1, AB1360 (aroD) and SK494 (aroE)
were grown overnight in 50-ml tubes and inoculated into 1-L
flasks containing 200 ml LB medium. The cells were
incubated at 37°C in a shaker at 3 00 rpm for several hours
until a spectrophotometric reading at A600 nm between 0.4 and
0.5 was reached. Cells were harvested by centrifugation in
sterile tubes after incubation on ice, treated with 100 mM
CaCl2 as described by Sambrook (68), resuspended into 100 mM
CaCl2 with 15% glycerol, aliquoted into microcentrifuge
tubes, dropped into liquid nitrogen and stored at -70°C

41
until use. Controls were prepared by streaking auxotrophic
mutant strains on M9 medium plates + /- aromatic amino acids.
Transformation of plasmid DNA
Either pUC18, pGEM5zf(+), or pBluescript SK+ plasmids
were incubated with competent cells of DH5a on ice, heated
to 42°C for 90 sec, cooled in an ice bath before addition of
SOC medium (68). The cells were then incubated at 37°C for
1 h before spreading on LB-AMP plates. A colony from these
transformed cells was streaked onto a fresh LB-AMP plate.
Controls included spreading of competent cells without
plasmid or plasmid without insert onto LB-AMP plates.
Functional complementation
E. coli mutant strains, aroD (DQT') and aroE (SDH') ,
were prepared for transformation with the pBluescript
plasmids carrying cDNA clones and were plated on LB-AMP
plates. After growth, colonies were streaked onto M9
plates. Controls included nontransformed competent cells or
plasmid spread on LB-AMP plates, and competent mutant
strains spread on M9 plates +/- aromatic amino acids.
Initial sequencing of cloned cDNA
Purified pBluescript plasmid with cloned inserts were
submitted to the DNA Sequencing Core Facility, ICBR, UF, for
initial 5' and 3' end sequencing (from the T3 and T7
promoter sites).

42
Subclonincr cDNA for complete sequence analysis
Subcloning of cDNA inserted into pBluescript was
performed by use of appropriate restriction enzymes to
obtain fragments for sequencing both strands of the entire
cDNA. Plasmids used for fragment insertion {pUC18,
pGEM5zf(+), or pBluescript SK+} were incubated with the same
restriction enzyme(s), and were treated with calf intestinal
alkaline phosphatase (CIAP) (when only one RE was used) to
prevent rejoining of ends during ligation. Cloned cDNA
fragment samples and plasmid samples were loaded onto 1% DNA
agarose gels at 70V for about 1 h. Fragments and linear
plasmid DNA were excised and treated with the Gene Clean
system (Bio 101, LaJolla, CA), prior to ligation and
transformation.
Li-Cor sequencing of subcloned cDNA fragments
Plasmid DNA was denatured and reacted with forward or
reverse fluorescent primers, enzymes, 7-Deaza dGTP sequence
extending mix and NTPS according to the directions supplied
by Li-Cor (Lincoln, NE) . Three fil of each of four prepared
sample mixtures containing one of the four dideoxy
nucleotides was loaded onto wells of a urea polyacrylamide
gel set into the Li-Cor model 4000L sequencer. Parameters
were set in a computerized data collection file and the
sequencer was run overnight. The sequences were analyzed in
a data analysis computerized system.

43
N-terminal sequencing
About 1 pmol of purified SP-I protein was prepared for
N-terminal sequencing on SDS PAGE, followed by transfer of
the protein onto 3MM paper by electrophoresis. Mini-blot
system protocol and amino acid analysis by acid hydrolysis
was provided by the Protein Chemistry Core Facility,
Interdisciplinary Center of Biotechnology Research,
University of Florida.
Computer analysis
The GCG computer software package (26) was used to
analyze nucleotide and deduced amino acid sequences of
cloned cDNAs.
Biochemicals
Arogenate (90% pure) was isolated from a multiple
auxotroph of Neurospora crassa ATCC 36373 (42) or isolated
from a tyrosine auxotroph of Salmonella typhimurium (4).
Prephenate (85% pure) was prepared as the barium salt from
culture supernatants of the tyrosine auxotroph of S.
typhimurium and was converted to the potassium salt with
excess K2S04 prior to use. Dehydroquinate (90% pure) was
chemically synthesized by following the method of Haslam et
al. (37) using a platinum-catalyzed dehydrogenation of
quinic acid (about 90% pure). 2'5' ADP Sepharose-4B and AH-
Sepharose 4B were purchased from Pharmacia, Piscataway, NJ.
Bradford reagent was purchased from Bio-Rad, Rockville

44
Center, NY. DTT and hydroxylapatite were purchased from
Research Organics, Inc. Cleveland, OH. Shikimate, NADP,
NADPH, benzamidine, leupeptin, and PMSF were purchased from
Sigma Chemical Co., St. Louis, MO. All other chemicals were
purchased through Fisher Scientific Co., Orlando, FL.

45
CHAPTER III
PHYSIOLOGICAL PROFILE OF AROMATIC PATHWAY ENZYMES IN PLANTS
The activities of dehydroquinase and shikimate
dehydrogenase (of the common portion of aromatic
biosynthesis), and prephenate aminotransferase, arogenate
dehydratase and arogenate dehydrogenase (of the post-
prephenate portion of the pathway) were examined under
selected physiological conditions in the higher plant N.
silvestris and in the green alga Chlorella sorokiniana.
Prephenate aminotransferase from Chlorella exhibited several
of the striking characteristics of higher plant enzyme.
Arogenate dehydratase activities were followed in a
growth culture cycle of N. silvestris suspension cells
similarly as was followed for PAT and ADH (12).
Results
Specific activities from crude extracts
Specific activities have been compared for several
aromatic amino acid pathway enzymes of N. silvestris
cultured cells and from the green alga C. sorokiniana (Table
3-1). A constant ratio of about 5 for SDH and DQT activity
was found in N. silvestris, compared to a ratio of 1 for
crude extracts of C. sorokiniana. The activities of DQT and

46
SDH from Chlorella extract were unaffected when incubated
with SP-I N. silvestris specific antibody. Activity was not
found when QDH was assayed in N. silvestris and C.
sorokiniana crude extracts.
Table 3-1. Specific activities from N. silvestris and C.
sorokiniana aromatic pathway enzymes.
Specific Activity
(nmol min'1 mg'1 protein)
Enzyme
N. silvestris
C. sorokiniana
QDT
12.00
6.20
SDH
60.00
6.00
PAT
16.00
3.75
ADH/NADP
1.00
NF
ADH/NAD
NF
NF
PDH/NADP
NF
NF
PDH/NAD
NF
NF
ADT
0.6
NF
PDT
NF
NF
NF, not found; Crude extracts were used for assay.
Separation of activities
Column chromatography was used to separate the enzymes
of interest for this study from N. silvestris and C.
sorokiniana. DQT and SDH of the common pathway were not
separable in N. silvestris or in C. sorokiniana after two
steps of column chromatography (DE-52 and HA). Figure 3-1
shows a DEAE-cellulose chromatographic profile of
N. silvestris suspension cell extract with overlapping

Fig. 3-1. DEAE-cellulose chromatography of aromatic pathway enzymes from N.
silvestris. Extract of five day suspension cells was applied to a DE-52 column as
described in Materials and Methods. PAT (a) , ADH (O) , ADT (a) , and SDH (•) eluted
in the KCl gradient (—) and protein was monitored (...) .

FRACTION NUMBER
2.4
2.0
1.6
0.8
0.4
oo
M KCI

49
enzyme activities of PAT, ADH, ADT and SDH (DQT activity was
coincident with SDH activity; not shown). Although ADT and
ADH activities were not found in crude extracts of C.
sorokiniana, they were located in DEAE-cellulose fractions
at very low activities as shown in Fig. 3-2A. ADH was
specific for cofactor NADP+. Prephenate dehydratase and
prephenate dehydrogenase activities were not found in these
column fractions. The ADH and ADT activities overlapped
those of PAT, DQT, and SDH in the KCl gradient fractions.
The coeluting DQT and SDH activity peak fractions, as shown
in Fig. 3-2B, were pooled, dialyzed and applied to an HA
column where they once again coeluted, this time in the wash
fraction, data not shown.
PAT in C. sorokiniana
Several of the unique properties earlier shown for
prephenate aminotransferase in N. silvestris suspension
cells (5) were studied in C. sorokiniana extracts. A high
temperature optimum of about 70°C for PAT was also found in
the alga extract as shown in Fig. 3-3. In Table 3-2, it is
shown that PAT from Chlorella has a preference for GLU as
the amino acid donor in combination with the keto acid
substrate, PPA. As found in higher plants, ASP was utilized
at about 50% of the activity with GLU. Slight activity was
seen with TYR as the amino acid donor in C. sorokiniana but
not in N. silvestris.

Fig. 3-2. DEAE-cellulose chromatography of aromatic
pathway enzymes from C. sorokiniana. Alga extract was
applied to a DE-52 column as described in Materials and
Methods. A) PAT (•), ADH (■), and ADT (a) eluted in the
KCl gradient (—) and protein was monitored (...) . B) SDH
(O) and DQT (•) eluted in the gradient.

51

52
¡H
H
H
>
H
H
U
«<
<
CU
TEMPERATURE (°C)
Fig. 3-3. Temperature optimum for activity
prephenate aminotransferase in C. sorokiniana.
temperatures range used was from 20°C to 80°C for 5 min.
of
The

Table 3-2. Prephenate aminotransferase amino acid donor
specificity in C. sorokiniana.
53
L-Amino Acid Donor (10 mM)
Specific Activity
GLU
16.0
ASP
8.0
TYR
2.0
LEU
0
ALA
0
aA concentration of 1 mM PPA was used in combination with
the amino acids. S. A. is nmol min' mg protein'
When C sorokiniana extract was incubated at 65°C for 10
min and then assayed at 37°C, activity with PPY as keto acid
substrate was lost, but activity with PPA remained constant.
At 70°C for 20 min, however, about 60% of the PAT activity was
lost (Table 3-3).
Table 3-3. Prephenate aminotransferase activity after
thermal treatment in C. sorokiniana11.
Aminotransferase
No
65°C
70°C
70UC
Couple
treatment
10 min
10 min
20 min
PPA/GLU
0.61
0.60
0.61
0.25
PPY/GLU
0.29
0
a—rz—
0
0
aReaction mixtures were incubated at 37°C following thermal
treatment. Assays contained 1 mM PPA or 10 mM PPY, 10 mM GLU
and 0.05 mg protein.

54
APT activities in cultured N. silvestris cells.
When N. silvestris stationary phase cells were diluted
into fresh medium, a substantial elevation of soluble
protein occurred which increased until day 2, leveled off
between days 2 and 3, and began a steady decline between
days 3 and 7 as shown in Fig. 3-4B. This rise ceased before
mid-exponential growth was reached. That soluble protein
synthesis was terminated before reaching a maximum was
indicated by the finding that EE cells achieved a two-fold
higher content of soluble protein. EE cells exhibited a
consistently higher soluble protein content of about 360 mg
g dry wt._1.
During the transition of cell populations in lag phase
to exponential-phase growth, new synthesis of macromolecular
constituents must have occurred. Figure 3-5A shows that the
rise in ADT activity expressed as units of activity per g
dry wt., paralleled the rise in soluble protein between
subculture and day 2 (Fig. 3-4B). The rise in ADT activity
levels continued until day 3, whereas overall soluble
protein content plateaued between days 2 and 3. Thus,
specific activity or activity expressed on a cell basis
peaked at day 3. Since the specific activity of E cells and
EE cells are similar and since the ratio of soluble protein
to dry weight is greater in EE cells than in E cells, the
activity of ADT expressed as units per g dry wt was greater
in EE cells than in E cells. The decline in specific

Fig. 3-4. Growth curve of N. silvestris in
suspension cultures. A) Cell growth monitored as cell
count or as dry weight. The physiological stages of
growth: lag (L), exponential (E) and stationary (S) are
indicated in each panel. B) Soluble protein content was
determined in samples harvested daily and was related to
the dry wt. and cell number. Comparable protein values
obtained with EE cells (average of ten determinations)
are indicated with stippled bars, the widths of which
indicate the range of variation.

56
O
33
-<
5
m
a
i
H
3
(Q
0
DAYS AFTER SUBCULTURE
T
7

Fig. 3-5. Arogenate dehydratase activities followed
throughout a growth curve of N. silvestris. Activity was
followed in samples harvested daily throughout the growth
curve shown in Fig. 2-4. A) Activities expressed as
units/g dry wt. , B) as units/cell, C) as specific
activity. The stippled bars indicate the corresponding
activities found in EE cells (average of 10
determinations), the width of the bars indicating the
range of variation. D) total cumulative units of
activity per culture. See Fig. 2-4 for meaning of L, E
and S.

AROGENATE DEHYDRATASE ACTIVITY
in
oo

59
activity (Fig. 3-5C) and units of activity per cell (Fig. 3-
5B) between days 3 and 5 (mid-to late-exponential growth
reflected a rate of enzyme synthesis that was slower than
the growth rate since total units of activity (Fig. 3-5D)
continued to increase during that time. From days 5 to 7
the decline in specific activity reflected a net loss of
enzyme activity.
Discussion
C. sorokiniana aromatic-pathway enzyme activities
appear to be somewhat lower but consistent with those found
in higher plants. The S-protein from N. silvestris must be
antigenically different from that of C. sorokiniana, since
the S-protein specific antibody did not precipitate the
protein from Chlorella. Specific antibodies made to PAT and
ADH are now available (see Chapter V) to test cross
reactions with these proteins from Chlorella.
Although the coincident DQT and SDH activity peak from
the DEAE column which eluted protein from C. sorokiniana
extract was only purified through two steps, it is
conceivable that further purification would yield a
bifunctional S-protein like that found in higher plants.
Activities from column fractions indicated that DQT activity
was about half that seen for SDH, whereas crude extracts
indicated that activities were about equal. Possible
explanations would be that (i) more than one protein is

60
present in Chlorella for DQT and/or SDH, (ii) some component
in crude extract inhibits the SDH activity or (iii) DHS, the
product of the reaction is being utilized by another
protein. The second possibility may also apply to ADT and
ADH since activity was not found in crude extracts but was
located in column fractions.
Most interesting, is the similarity of PAT in Chlorella
to that of higher plants, each having the high optimal
temperature for activity. At 65°C, the aminotransferase(s)
using the PPY/GLU couple was inactivated. The high
specificity of Chlorella for the substrate couple of PPA and
GLU was also consistent with higher plant PAT (5, 6). From
these data, it is suggested that C. sorokiniana has an
aromatic biosynthetic pathway similar to that established
for higher plant chloroplasts. Supportive evidence would
include the isolation of Chlorella chloroplasts to determine
the location of the aromatic pathway enzymes. The isolation
and sequencing of cDNA clones coding for S-protein, PAT and
ADH (see Chapter VI) may be useful as probes to clone the
appropriate genes in Chlorella.
Arogenate dehydratase activities paralleled a similar
experiment with respect to the chloroplast isoenzyme of DAHP
synthase (Ds-Mn), where growth of cells and enzyme
activities where followed (32). Both enzymes rise in
exponential growth and decline in stationary phase of
growth. ADT exhibited an opposite pattern of activity to

61
three other aromatic pathway enzymes, SDH, PAT, and ADH.
The three enzymes are known to exist in the chloroplast of
higher plants, but the extent to which a fraction of each of
the total activities measured might be derived from the
cytosol is unknown. All three enzymes exhibited elevated
levels of activity in stationary phase and minimal levels of
activity in exponential-phase growth (11). Aromatic-pathway
enzymes may be sorting out into two different patterns of
expression for reasons yet to be elucidated. Alternatively,
since enrichment of Ds-Co (the cytosolic isoenzyme of DAHP
synthase), occurred in stationary phase, rising levels of
SDH, PAT and ADH in stationary phase may reflect the
expression of cytosolic isoenzymes. If so, the rise in
activity of cytosolic isoenzymes exceeds the decrease of the
plastidial isoenzyme counterparts. These data suggest that
while ADT is present in the chloroplast pathway, it may not
function in the cytosol.If so, perhaps prephenate
dehydratase of the alternative route (activity, not as yet
determined in plants, see Fig. 1-1) , functions in the
cytosol to synthesize PHE that may be used extensively for
secondary metabolism.

62
CHAPTER IV
PURIFICATION OF PROTEINS
FROM THE AROMATIC AMINO ACID PATHWAY
This chapter includes the separation and purification
of the common pathway bifunctional DQT/SDH proteins from
Nicotiana silvestris suspension cells. Six to eight steps
of column chromatography, including a specific NADP+
affinity column have been used. Prephenate aminotransferase
and arogenate dehydrogenase were also purified by a series
of chromatographic steps.
Results
DOT/SDH purification
In crude extracts, activities for SDH and DQT (specific
activity of 61 and 13, respectively) were detected by
fluorometric or spectrophotometric assays. The enzyme(s)
are not stable at 4°C after several days of storage, unless
glycerol and DTT are present. Under these conditions
activities remained stable to freeze/thaw, at either -20 or
-70°C. Two peaks of activity for the bifunctional S-protein
were eluted from a Celite 545 column with an ammonium
sulfate gradient (Fig. 4-1). Elution of the major peak
began at about 50% of ammonium sulfate saturation and was

Fig. 4-1. Elution profile of SP-I and SP-II from Celite 545 column
chromatography. Two activities for SDH (•) were separated by a decreasing gradient
of ammonium sulfate (solid line) on a Celite 545 column at about 50% and 25%
saturation of ammonium sulfate as labeled. Dehydroquinate dehydratase (O) activity
peaks were coincident with the SDH activity peaks. Protein was monitored at
A2so nm (•••) • See Materials and Methods for details of chromatography.

200
0
20
40 60 80
FRACTION NUMBER
100
90
80 h
¡2
70 z>
CO
100
120
=3
Z
o
60
50
40
LU
30 O
0C
LU
20 CL
10
3.20
2.80
2.40
o
oo
CM
2.00 <
LU
1.60 H-
O
CC
1.20 CL
0.80
0.40
0

65
almost completely removed from the column by the end of a
45% of ammonium sulfate saturation step. The major activity
peak fractions were completely separated from fractions
containing the minor peak of activity and eluted at about
25% of ammonium sulfate saturation. Fractions with DQT
activity were located coincidently with the two peak of SDH
activity. There were no column fractions containing only
one of these two activities. Fractions from each activity
peak were pooled and labeled SP-I for the major peak of
activity and SP-II for the minor peak of activity. Proteins
(SP-I and SP-II) were further purified similarly by
chromatography steps. It was calculated from this
chromatographic step that the major peak, SP-I, accounts for
90% or greater of the total activity in the crude extract.
The fold purifications calculated for the SP-I and SP-II
were based on this assumption. The proteins eluted in the
wash fractions of the CM columns in the second
chromatographic step of purification. Figure 4-2 shows
profiles of SP-I and SP-II elutions from the third step of
purification on DEAE-cellulose columns in Epps buffer at pH
8.6. Each protein peak eluted at about 0.3M KCl in the
gradient and had coincident DQT and SDH activities. The
fourth chromatographic step was an HA column which eluted
each protein in the wash fractions. This step was followed
by another DEAE column, equilibrated at a lower pH of 7.2.
Each bifunctional protein eluted in the gradient of an NADP+

Fig. 4-2. Elution profiles of SP-I and SP-II from
DEAE- cellulose chromatography. The third step of
purification of the two S-proteins was a DEAE-cellulose
chromatographic column equilibrated in EPPS buffer (see
Materials and Methods for details). SDH (•) activity
remained coincident with DQT (O) activity for both SP-II
(panel A) and SP-I (panel B). The proteins began to elute
at about 0.25MKC1 of the gradient (solid line) . Protein
profiles were followed at A280 nm (...) .

FU min-1 FU min'
67
FRACTION NUMBER

68
specific 2'5'-Sepharose-4B affinity column at 1 ¡jlM NADP+ and
18 mM NaCl (Fig. 4-3). Activities for DQT and SDH remained
coincident for each protein. The fold purification at this
step was 195 for SP-I and 918 for SP-II. Only SP-I was
purified further by repeating the DEAE-cellulose
chromatography step (Epps-KOH buffer), followed by a second
application on the affinity column. The final fold
purification was 1077 for the SP-I. Purification yields,
purities, specific activities and ratios of SDH/DQT
activities are shown in Table 4-1 and in Table 4-2.
Throughout the purification of SP-I and SP-II, the
ratio of shikimate dehydrogenase to dehydroquinase activity
was calculated to be within the range of 4.7 to 6.2 for SP-I
(at an average of about 5.1) and within a range of 4.4 to
5.4 for SP-II (at an average of about 4.8). Each protein was
concentrated on a PM-10 membrane to about 1 or 2 ml before
storage at -70° C.
Purification of ADH and PAT
When ADH was purified along with the SP-II, it was realized
that they eluted with overlapping activity peaks on the
Celite 545 column using the decreasing ammonium sulfate
gradient at about 25 to 20% of saturation as shown in Fig.
4-4. A critical step of separation occurred when SDH did
not bind to HA and ADH bound to HA (Fig. 4-5). Without the
inclusion of this step, the two enzymes co-purified
throughout the entire purification procedure, both eluting

Fig. 4-3. Elution profiles of S-proteins from an
NADP+ specific affinity column. A 2'5'-Sepharose-4B
column with a combined NADP+ (0-10 uAf) and NaCl (0-0.16M)
gradient was used as final steps of purification of the
S-proteins, each eluting at about 0.018M NaCl and luAf
NADP. Both SDH (•) and DQT (O) activities coeluted. A)
step six of purification of SP-II protein. B) step eight
of purification of SP-I protein.

FU min'1 FU min
70
I
0.12
0.06
0.18
0.12
0.06
0
0
10 20 30 40 50 60 70 80
FRACTION NUMBER
M NaCI M NaCI

71
Table 4-1. Purification of SI protein from N.
silvestris suspension cultured cells.
Step
Vol
ml
Total
Protein
mg
SDH
DQT
Ratio
S/D
SA
Purity
fold
Yield
%
SA
Purity
fold
Yield
%
Crude
540
605
56
1
100
12
1
100
4.7
Celite
643
206
126
2
70
25
2
65
5.0
CM-52
720
49
412
7
55
94
8
59
4.4
DEAE-1
279
35
561
10
53
113
9
50
5.0
HA
618
8.7
1,515
27
36
257
22
28
5.9
DEAE-2
106
1.95
4,609
82
24
941
78
23
4.9
Aff-1
88
0.42
14,500
259
17
2,342
195
13
6.2
DEAE-3
83
0.16
33,878
605
15
7,365
614
15
4.6
Aff-2
54
0.07
65,877
1176
9
12,923
1077
12
5.1
S/D is
shikimate dehydrogenase/dehydroquinase.
Table
4-2 .
Purification of S2 protein from N.
silvestris suspension cultured cells.
SDH
DQT
Total
Vol
Protein
Purity
Yield
Purity
Yield
Ratio
Step
ml
mg
SA
fold
%
SA
fold
%
S/D
Crude
540
605
5
1
100
1
1
100
4.6
Celite
348
97
26
5
68
5
5
64
5.0
CM-52
345
69
31
6
59
8
8
73
4.4
DEAE-1
77
31
66
13
55
14
14
53
5.1
HA
104
5
295
59
43
75
75
53
5.4
DEAE-2
31
1
757
151
34
187
187
34
4.1
Aff-1
15
0.006 4,590
918
7
880
880
7
5.2
S/D is shikimate dehydrogenase/dehydroquinate.

72
2.0
1.0
0
<
FRACTION NUMBER
Fig. 4-4. Celite 545 column chromatography overlap of
arogenate dehydrogenase and SP-II. Both SP-II (•) and ADH
(O) elute in the 45 to 0% ammonium sulfate gradient (solid
line) with overlapping activity peaks. Protein was
monitored at A280 „„ (...) .
280

73
500
400
300
200
100
0
FRACTION NUMBER
Fig. 4-5. Hydroxylapatite column chromatography
separation of arogenate dehydrogenase and SP-II. The SDH
activity (■) was found in the wash fractions and ADH (•)
eluted at about 120 mM KP04.
mM KPO

74
in similar fractions from the NADP+ affinity column.
Therefore, when ADH was purified by the earlier mentioned
procedure an HA column step was included to completely
remove SDH activity. Purification of PAT followed the
previously demonstrated procedure with a purification of
about 250-fold.
Discussion
The SP-I and SP-II proteins, well separated from each
other on the Celite 545 column, were each purified further
to about 1100- and 900-fold, respectively, by a series of
chromatographic steps. This complete separation was always
repeatable under similar conditions of the Celite 545 column
step (at least 12 times). SDH and DQT activities coeluted
at an activity ratio of about 5 throughout the entire
purification regimen for both bifunctional proteins. These
proteins may be isoenzymes from different compartments
(chloroplast and cytosol) or they may be isoenzymes residing
in different locations within the chloroplast. This second
possibility was reported for two DAHP synthase isoenzymes
(DS-1 and DS-2) presumably located in chloroplasts of
Arabidopsis (2). Further studies which may elucidate
differential properties of the two proteins and localization
studies should provide information with respect to possible
isoenzyme species and their location within the plant cell.

75
The purified SP-I and SP-II proteins were studied further to
characterize both the DQT and the SDH activities in (Chapter
V). The purified SP-I was used to produce specific antibody
against the protein (see Chapter VI) and for N-terminal
sequencing (see Chapter VII). Purification of PAT and ADH
was completed and then used to produce specific antibodies
to each protein for use as probes in cDNA cloning.

76
CHAPTER V
PROPERTIES OF THE S-PROTEINS
The main focus in my studies of the properties of the
two bifunctional proteins was to detect possible
differential properties which might suggest that these
proteins are isoenzymes. Molecular mass, pH and temperature
optima, thermal inactivation, and Km values for both
functional domains of the bifunctional proteins, SP-I and
SP-II, have been determined. Effects on activity of each
protein by various compounds, including PCMB, were also
studied.
RESULTS
Activity peaks on a Celite column
A Celite 545 column resolved about 6 peaks of SDH
activity when a more shallow gradient of ammonium sulfate
was applied than that used during the separation of the S-
proteins, Chapter IV (Fig. 5-1). Dehydroquinase activity
was coincident with SDH activities. Fractions of the six
peaks containing the enzyme activities were pooled
separately, avoiding overlapping fractions as much as
possible, and were labeled as shown in Fig. 5-1. The six

Fig. 5-1. Elution profile of SDH activities from a Celite 545 column run with
a shallow gradient. Five possible peaks of SDH activity eluted on a Celite 545
column with an ammonium sulfate gradient from 55% to 45% of saturation for about
600ml (labeled I to V) . A sixth peak (VI) of SDH activity eluted in a second
gradient from 45% to 0% ammonium sulfate.

70
60
50
40
30
20
10
0
SDH (•) FU min"
i n m iv v vi
n i i i i i 1 i—i i
FRACTION NUMBER
-j
oo

79
pooled samples containing DQT/SDH activities were
concentrated and examined for possible differential
properties, substrate requirements for saturation with SHK
and NADP*, alternative substrate specificities (i.e., NAD*
and QA), temperature optimum, thermal inactivation, SDS PAGE
and activity-stained native PAGE. No significant
differences were found between the overlapping peak samples
of I to V (data is not shown). All six protein samples were
specific for NADP* and SHK when assayed for SDH activity.
Pool sample IV (or perhaps I through V) corresponds to SP-I,
and Peak VI corresponded to SP-II. SP-I and SP-II exhibited
similar properties except for Mr, Km and activity stained
gels.
Molecular Mass
A difference was seen in the molecular mass of SP-I and
SP-II as shown in Fig. 5-2. SP-I migrated slightly ahead of
SP-II on SDS gels (A) and on a silver stained SDS gels (B).
Both gels showed bands (or set of bands) at about 59 kD and
40 kD for purified SP-I, and bands at about 61 kD for
purified SP-II. A band at 42 kD can be detected when higher
concentration of SP-II was used in these experiments. The
silver stained gel offered higher resolution and indicated
that the affinity purified SP-I also had a set of bands at
about 29 kD. Native Mr was determined on a Sephadex G-200
column,( Fig. 5-3, panels A and B), to be about 59 kD and 62
kD for SP-I and SP-II, respectively. In crude extract

Fig. 5-2. Molecular weight determinations by SDS
PAGE and by silver stain of the two S-proteins. A) The
SDS gel shows 2 bands for SP-I (584 ng) at molecular
weights of about 60,000 and 40,000 in lane 3. Lane 5 has
a single detectable band at a molecular weight of about
62,000 for the SP-II (155 ng) . Lanes 1 and 7 are
molecular weight markers with MW as indicated. B) The
silver staining technique was also used to determine
molecular weights of the S-proteins. Lanes 1, 9, and 16
show molecular weight markers. Lanes 2 through 7
represent SP-I and lanes 11 through 14 represent SP-II at
various stages of purification as follows:
Lane
Protein
Durification steD
Molecular weiaht(s)
2
SI
25ul
Affinity pure
60,000
40,000
3
SI
lOul
Affinity pure
60,000
40,000
4
SI
25ul
DEAE-cellulose
60,000
40,000
29,000
5
SI
lOul
DEAE-cellulose
60,000
40,000
29,000
6
SI
lOul
Affinity pure
(AB)
60,000
40,000
29,000
7
SI
25ul
Affinity pure
(AB)
60,000
40,000
29,000
11
S2
25ul
Affinity pure
(1)
62,000
12
S2
lOul
Affinity pure
(1)
62,000
13
S2
25ul
Affinity pure
(2)
62,000
14
S2
lOul
Affinity pure
(2)
62,000
(AB) is purified enzyme from several runs that were
combined, passed through the affinity column a third time
and concentrated. For SP-II the (1) represents the
purified protein after the first pass through the
affinity column and (2) represents combined column runs
of SP-II passed a second time through the affinity column
and concentrated.

81
94,000
67,000
43,000
30,000
20,100
14.400
B
12345678 910111213141516
94,000
67,000
43,000
30,000
20,100
14,400

Fig. 5-3. Gel filtration chromatography of S-
proteins. A) Concentrated crude extract (â– ) , pool IV
(SP-I) (•) and pool VI (SP-II) (a) (from the shallow
gradient Celite 545 column) were passed through a
Sephadex G-200 column. The dotted profile represents the
three marker enzymes (alcohol dehydrogenase, BSA and
carbonic anhydrase). B) A molecular weight curve based
on the column profile from A suggests the molecular
weights for the SP-I to be about 59,000 and about 62,000
for SP-II. Crude extract S-protein is suggested to be
about 63,000.

MOLECULAR WEIGHT
83
FRACTIONS
Fu min’

84
(containing all fractions of the S-protein), the
bifunctional protein was determined to have only one native
molecular weight of about 63 kD.
Activity stained gels
In order to optimize conditions for a native PAGE
activity stain gel, various combinations of substrate and
dyes were studied with concentrated crude extract, data not
shown. All activity stained gels were assayed at optimal
conditions as described in Methods (Chapter II). Activity
stained bands required both substrate, SHK and NADP+ for
visibility. Crude extract revealed 3 bands on native gels
after application of the activity stain (Fig. 5-4). In Fig.
5-5, purified SP-I and SP-II had 2 activity stained bands
each. SP-II migrated at a faster rate than did SP-I.
pH and temperature optima of S-proteins
Both functional domains of the two bifunctional
proteins were studied with respect to pH optima (Fig. 5-6).
SDH activity was followed in a pH range of 6.5 to 10 in 200
mtf Bis Trispropane buffer and showed optimal activity at pH
9.0 for both proteins. DQT, assayed by the coupled assay
method had very low activities when the same buffer was used
over the same pH range for both SP-I and SP-II. A pH range
from 6.1 to 9.5 in potassium phosphate, Epps or glycine
buffers, each at 100 mM resulted in higher activities and
showed optimal activity at pH 7.25 for both proteins. When
the three buffers above were used for the SDH assay there

Fig. 5-4. Optimal PAGE activity staining for SDH
in crude extract. An activity stain of a native
polyacrylimide gel was done at optimal conditions when
concentrated crude extract of 360 ug was applied to the
gel. Three bands are shown after incubation in
substrates at concentrations for optimal activity.


Fig. 5-5. PAGE activity staining of purified SP-I
and SP-II. SP-I was added to lanes 2 and 3, and SP-II
was added to lanes 1 and 4. Activity staining was done
under optimal conditions. A) After incubation for about
15 min, SP-I showed two bands of activity in both lanes.
SP-II, at lower concentrations, showed two bands in lane
4, each migrated faster than the SP-I bands. B) The gel
was incubated further for about 15 min. The SP-II in lane
1 was then visible. C) After incubation for 1 h, 2 bands
were more pronounced for all four lanes.

88

Fig. 5-6. S-proteins pH optima for activity. SP-I
and SP-II pH optima were determined with purified enzymes
(using 4ng and 8ng protein, respectively) in a range from
pH 6 to pH 10. Shikimate dehydrogenase activity was
determined in the presence of 200 mM Bis Trispropane
buffer at the indicated pH values. Dehydroquinate
dehydratase activity was determined with 100 mM potassium
phosphate, Epps, or glycine buffer at appropriate pH
values for each.

9.5 10.0
ACTIVITY (nmol min 1 )
opQ Q ^ *-**-* ^ ’-* ro ro ro ro ro co
ro £ a> oo oro^cncoo KS-^ooooro

91
was no difference in activity levels from that seen with Bis
Trispropane buffer.
A temperature curve was followed for each activity of
the two bifunctional proteins (Fig. 5-7) using optimal assay
conditions for activity. Optimal activity occurred at about
40°C for the SDH functional domain and at about 32°C for the
DQT functional domain of either protein. Extract of each
bifunctional protein was heated to 30°C, 40°C or 50°C for 5
to 25 min, then assayed at room temperature (24°C) as shown
in Fig. 5-8. Activity decreased rapidly at all three
temperatures, by 10 min of incubation at 50°C no activity
was detected for either protein. Even at 30°C the enzymes
were subject to inactivation. Substrate was shown to
protect the functional SDH domain of both enzymes from
thermal inactivation (Fig. 5-9). With the addition of NADP+
prior to incubation at 50°C for 5 min, SP-I retained 67% of
its SDH activity and SP-II retained about 61% of the SDH
activity compared to 8% and 15% SDH activity retained,
respectively, when NADP+ was not added. SHK was
considerably less protective against thermal inactivation
with only 18% and 21% retention of SDH activity for SP-I and
SP-II, respectively. Without substrate present, SP-I was
thermally inactivated to 50% and SP-II to 60% of activity
after 2 min at 50°C.

Fig. 5-7. Temperature optima for S-proteins.
Optimal activity was determined for both SP-I and SP-II
purified proteins over a range of temperatures from 24°C
to 66°C. A) Dehydroquinate dehydratase activity was
determined for the temperature range given. B) Shikimate
dehydrogenase activity was determined over the given
temperature range.

ACTIVITY (nmol min'
93

Fig. 5-8. Thermal inactivation of S-proteins.
Purified extracts containing the S-proteins were heated
at 30°C (•),40°C (a), or 50°C (■) from 5 to 25 min, then
assayed at 25°C at saturating substrates. A) SP-I (4 ng
protein) was assayed for SDH activities at various
temperatures. B) SP-II (6 ng protein) was assayed for
SDH activity at various temperatures.

95
5 1 0 1 5 20 25
TIME (min)

Fig. 5-9. Substrate protection against thermal
inactivation of S-proteins. Purified enzyme was
incubated at 50°C for 2 or 5 min without substrate added
or 5 min with NADP+ or SHK added, assayed at 25°C and
compared to unheated extract activity. Conditions for
assay were the same as described in Fig. 5-8. SP-I
results are shown in panel A and SP-II results are shown
in panel B.

ACTIVITY (nmol min
97
INACTIVATION AT 50°C (min)

98
Kinetic constants
Affinity purified SP-I and SP-II were used to
characterize the properties of the two proteins. Both
proteins were specific for their substrates and neither
enzyme would utilize NAD* as cofactor or quinate as
substrate. Saturation of the SDH activity domain of the
bifunctional enzyme with substrates, NADP* (at 0.14 mM) and
SHK (at 5.0 mM) , is shown in Fig. 5-10 (A and C) for SP-I
and in Fig. 5-11 (A and C) for SP-II (saturating at 0.2 mM
NADP* and 4.0 mM SHK. From the double-reciprocal plots in
Figs. 5-10 and 5-11 (panels B and D), the Km values for the
SDH domain of both SP-1 and SP-II were found to be 0.02 mM
for NADP+, However, different values for shikimate of 0.8 mM
for SP-I and 0.36 mM for SP-II were obtained. Saturation
curves and double-reciprocal plots were determined for both
proteins (data not shown) when assayed in the forward
direction with DHS and NADPH. SP-I had a Km of 0.36 mM with
DHS and SP-II had a difference of 0.26 mM for DHS. The Km
value for NADPH was the same for SP-I and SP-II at 0.01 mM.
SP-I and SP-II had different Km values for DQT activity with
substrate DHQ (0.07 and 0.04 mM DHQ, respectively, data not
shown). Table 5-1 relates the Km and Vmax values and the
ratio of Vmax to Km for each functional domain activity of
the two proteins.

Fig. 5-10. Saturation curves and double reciprocal
plots for SP-I. A) Saturation of shikimate dehydrogenase
when substrate, NADP+, was held constant at 0.5 mM and
substrate, shikimate, was varied over a range of 0.25 mM
to 5 mM. B) A double reciprocal plot of the data obtain¬
ed in A when SHK was the variable. C) Saturation of SDH
when SHK was held constant at 5 mM and NADP+ was varied
over a range of 0.01 mM to 0.5 mM. D) A double recipro¬
cal plot of the data obtained from B when NADP+ was the
variable.

100
mw SHIKJMATE mM NADP*
[SHK]
[NADP 1

Fig. 5-11. Saturation curves and double reciprocal plots for SP-II. A)
Saturation of shikimate dehydrogen-ase when substrate, NADP+, was held constant at
0.5 mM and substrate, shikimate, was varied over a range of 0.25 mM to 5 mM. B)
A double reciprocal plot of the data obtained from A when SHK was the variable.
C) Saturation of SDH when SHK was held constant at 5 mM and NADP+ was varied over
a range of 0.01 mM to 0.5 mM. D) A double reciprocal plot of the data obtained
from B when NADP+ was the variable.

ISHK] [NADP
REACTION VELOCITY
301

103
Table 5-1. Kinetic parameters for SP-I and SP-II.
Enzyme
Substrate
Km (mM)
Vmax (nmol/min)
(Vmax/Km)
SP-I SDH
SHK
0.80
0.60
0.75
NADP
0.02
0.59
29.50
DHS
0.36
0.25
0.69
NAD PH
0.01
0.26
26.00
QDT
DQH
0.07
1.96
28.00
SP-II SDH
SHK
0.36
0.60
1.66
NADP
0.02
0.59
29.50
DHS
0.26
0.27
1.04
NADPH
0.01
0.26
26.00
QDT
DQH
0.04
1.47
36.00
Inhibitor effects on enzyme activities
Protocatechuic acid (0.5 mM) inhibited SDH activity of
both SP-I and SP-II at about 15% at saturating substrate
concentrations (5.0 mM SHK and 1.0 mM NADP*) and about 57%
for each at 0.8 mM SHK (SP-I), 0.36 mM SHK (SP-II) and 0.2
mM NADP+. PCA did not inhibit the DQT activities of the two
proteins. PCMB was inhibitory to SDH activities for both
functional domains and activated the DQT functional domains
from both bifunctional proteins as shown in Table 5-2.
Neither SP-I or SP-II were effected by additions of 0.5
mM PHE, TYR, TRP, quinate, and cinnamic acid when added to
saturated substrate reaction mixes and assayed for DQT or
SDH activities. Metals at 0.2 mM concentrations (calcium,
magnesium, manganese, cobalt, zinc, and iron) had no effect
on activities of either protein. Incubation of DTT had no
effect on any of the activities. EDTA at 10 and 125 mM had

104
no effect on activity when tested at pH 7.0, 8.6, or 9.5.
Table 5-2. Effect of PCMB on Enzyme Activities
Protein
Enzyme
mM
PCMB
Inhibition
Percent 0.
Prevention
. 5 mM DTT
Reversal
Percent
Activation
SP-1
SDH
0.000
0.0
NA
NA
NA
SDH
0.125
100
100
0.0
NA
SDH
0.250
100
100
0.0
NA
SDH
0.500
100
95
0.0
NA
SDH
1.000
100
81
0.0
NA
DQT
0.050
0.0
NA
0.0
50
DQT
0.100
0.0
NA
NA
50
SP-II
SDH
0.000
0.0
NA
NA
NA
SDH
0.500
100
96
0.0
NA
DQT
0.050
0.0
NA
NA
55
DISCUSSION
A shallow ammonium sulfate gradient on a Celite 545
column revealed about 6 peaks of SDH activities. All SDH
activity peaks had DQT activity as well. No differential
properties were found between the five overlapping peaks
containing SDH and DQT activities which eluted in the
gradient from 55% to 45% of (NH4)2S04 saturation. These
could be isoforms of SP-I or artifacts of the column
chromatography. The sixth peak (also referred to as SP-II)
had several differential properties when compared to SP-I
which contained all of or some of the five activity peaks
from the Celite 545 shallow gradient column profile. A
difference of molecular mass of about 3,000 was determined

105
between SP-I (Mr at 59,000 to 60,000) and SP-II (Mr at about
62,000) from gel filtration, SDS and silver stained PAGE.
Bands were also present at about 40,000 (SP-I) and at about
42,000 (SP-II). It was not clear what the lower molecular
mass bands represent. These bands could be contaminating
protein, isoforms of each protein, or proteolytically
damaged protein. Activity stained gels showed three bands
(each of which may contain two bands for a total of six
bands) of activity for SDH from crude extracts, while,
purified SP-I had two bands that migrated slower than the
two bands of SP-II. The difference in Mr, may suggest these
proteins are isoenzymes. One possibility, is that SP-I, the
smaller Mr protein (the major fraction of activity that was
located on the Celite column) may be the mature chloroplast
isoenzyme with transit peptide removed and that SP-I, the
larger Mr protein (the minor fraction of activity from the
column) might be the cytosolic preprotein and isoenzyme that
functions in aromatic biosynthesis. A second differential
property, that of Km values was observed for SP-I and SP-II.
SP-II has greater affinity for SHK, DHS and DHQ, substrates
for both functional domain of the bifunctional protein.
Both proteins have about the same affinity for cofactor,
NADP+ or NADPH. These Km values are in the range of Km
values found in the literature (listed in Table 1-1). The
difference in affinity between SP-I and SP-II for substrates
is also suggestive of differentially located isoenzyme.

106
The Vmax/Km ratio reflects catalytic efficiency of enzymes
that catalyze the same reaction in cells. SP-I and SP-II
have the same Vmax, but different Kms, therefore, the
Vmax/Km ratio suggests that SP-II with the lower Km , is
catalytically more efficient than SP-I.
PCMB, a potent inhibitor of SDH activity in plant
species (48, 53) inhibited both bifunctional proteins,
implying that the presence of sulfhydryl groups are critical
for catalytic activity of the proteins. This inhibition can
be prevented by the addition of thiol reagents, such as DTT,
which did, in fact, protect the enzyme from inhibition by
PCMB. Most interesting is that PCMB activated activity of
the DQT functional domain. My interpretation is that this
may reflect physical overlapping of catalytic sites on the
protein. Protocatechuic acid, also a known inhibitor of
SDH activity (53) inhibited SDH activity of both proteins,
but had no effect on the DQT functional domain. PCA is the
product of the quinate catabolic system, and its
physiological significance as an inhibitor of SDH is
unclear.
The pH and temperature optima did not indicate any
differences between the two S-protein, but were different
for the functional domains of the two proteins. The SDH
functional domain of the bifunctional proteins had the
higher pH and temperature optima. Thermal inactivation
studies of the two bifunctional protein showed that the SDH

107
functional domain of each protein rapidly lost activity over
time even at 30°C and was protected from inactivation by
cofactor much better than by substrate. From the data
obtained, it appears that SP-II was stabilized to a slightly
greater degree than the SP-I (about 6% greater when NADP+
was present at incubation and about 4% greater for SHK).
Substrate protection of the DQT functional domain of the two
proteins with DHQ was not performed at this time.

108
CHAPTER VI
SPECIFIC ANTIBODY TO THE BIFUNCTIONAL S-PROTEIN
AND TO TWO POST-PREPHENATE PROTEINS
Specific antibody was made to the purified bifunctional
SP-I, to purified arogenate dehydratase and to purified
prephenate aminotransferase. SP-I specific antibody was
characterized with respect to both SP-I and SP-II.
Results
Antibodies made to each protein (SP-I, ADH and PAT)
precipitated crude protein preparations or purified antigens
on "Ouchterlony" plates. Each enzyme incubated with the
appropriate antibody was precipitated and activity was lost.
SP-I specific antibody precipitated both SP-I and SP-II
antigen on "Ouchterlony" plates at dilutions of antibody up
to 32-fold as seen in Fig. 6-1. The precipitant band was
more pronounced for the SP-I plate because more highly
concentrated protein was available. A saturation curve of
inhibition produced by antibody was similar for both SP-I
and SP-II when equal activities were established using
0.0084 //g of SP-I and 0.031 /¿g of SP-II (between 0.230 and
0.267 nmol min", respectively). Increasing amounts of

Fig. 6-1. Precipitation of S-proteins with SP-I specific antibody on
Ouchterlony plates. SP-I specific antibody was added to the outer wells of two
Ouchterlony plates, at dilutions of 1, 2, 4, 8, 16, and 32-fold. SP-I was added
to the middle well of the plate marked SI on the right and SP-II was added to the
middle well of the plate marked S2 on the left.

110

Ill
antibody showed maximal inhibition of both proteins with
about 100/il antibody (Fig. 6-2) .
Crude extract was applied to several wells of a native
gel. After electrophoretic migration, the gel was divided.
One half of the gel was used for Western blotting with Sl-
antibody and the other half of the gel was used with SDH
activity stain. Figure 6-3 shows the visible bands for
each.
When SP-I specific antibody was incubated with
E. coli crude extract and N. silvestris crude extract and
then assayed for SDH activity, (Table 5-1) the E. coli SDH
activity was retained, while the plant SDH was precipitated
and activity was lost.

Fig. 6-2. Effect of SP-I specific antibody on SP-I
and SP-II activities. SP-I antibody was added to equal
activity levels of SP-1 (•) and SP-II (O), and was then
incubated at 37°C for 10 min, microfuged and assayed for
SDH activity.

(]*) AGOailNV NBlOHd-S
o
§?
ACTIVITY (nmol min*1) SDH.
(H SDH2
o
o
b
ro
p
k
o
p
A
p
00
o o
ro to
KS cn
113

Fig. 6-3. Comparison of Western blot and activity-
stained gels. The number of visible bands from a
Western Blot is compared to the number of visible bands
from PAGE SDH specific activity stain. The native gel
was loaded with crude extract from N. silvestris
suspension cells, divided and treated (A) for a western
blot using SP-I specific antibody and (B) for SDH
specific activity stain.

115

116
Table 6-1. S-Protein Antibody Effect on N. silvestris
and E. colia extracts.
Antibody (¿il)
Crude
Extracts (nmol
min'1)
Antigen (/¿l)
E. coli
N. silvestris
0
25
2.73
0.364
25
25
2.72
0.031
50
25
2.73
0.016
aAntibody (0.16mg ml"1) , crude extracts (. 019mg ml-1)
and buffer were added at a total volume of 100/xl,
incubated at 37°C for 10 min and microfuged before
assay at 24°C.
Discussion
The two bifunctional S-proteins of N.silvestris, were
each precipitated by antibody made to purified SP-I as seen
by precipitant bands on" Ouchterlony" plates, and by loss of
activity following incubation of antibody and antigen.
There was no loss of activity for SDH and DQT from E. coli
crude extracts. These results suggested that the antibody
would be a suitable probe for selection of cDNA encoding for
the DQT/SDH bifunctional protein of higher plants after
treatment with an E. coli lysate to remove background
contaminants. Results from a Western blot indicate the
possibility of multiple SDH activities in crude extracts of
N. silvestris. From six to seven bands may be visible on the
Western blot. There are from three to six bands that are
visible when an activity stain specific for SDH is applied
to PAGE containing crude extract from the same protein
sample. The possibility of a pentafunctional arom protein

117
like that found in yeast and fungi was considered, but not
evident since no high molecular-weight band (would have to
be in the range of about 150 kD) was found on these gels
when crude extract was applied.

118
CHAPTER VII
CLONING cDNAS ENCODING THE BIFUNCTIONAL
S-PROTEIN, ADH AND PAT
A cDNA library from N. tabacum was available for use in
cloning cDNA encoding proteins from the aromatic amino acid
biosynthetic pathway. Thus far, genes have not been cloned
for the bifunctional S-protein, arogenate dehydrogenase or
prephenate aminotransferase of higher plants.
RESULTS
Cloning aromatic pathway genes
S-protein cDNA. Greater than 106 PFU carrying inserts
from a N. tabacum library were plated with E. coli XLl-Blue.
Probing the library with S-protein antibody, five plaques
that gave blue reactions on IPTG saturated nitrocellulose
filters were designated SP3, SP5, SP6, SP10, SP33 and
studied further. The size of the cDNA coding for the
bifunctional protein was expected to be about 2 kB, based on
molecular-weight estimations (Chapter V) and based on known
sequences of yeast and E. coli (Table 1-1). Gel
electrophoresis of purified pBluescript containing the
inserted cDNAs had different mobilities on the gel (plasmid
DNA was a mixture of circular and supercoiled DNA). The

119
expected size of the plasmid was 5 kB (2 kB insert plus
about 3 kB for pBluescript). Although the size cannot be
quantitated from this gel, it suggested that the DNA of the
various samples were of different sizes and that the three
DNA samples (SP3, SP5 and SP6) were identical in size (Fig.
7-1) .
PAT and ADH cDNA. The tobacco cDNA library was
prepared similarly as above to clone cDNA encoding PAT or
ADH by using the specific antibody probe for each. About
twelve possible clones for each were removed from plates and
plaque purified. Several of the plasmids containing cDNAs
appear to be of an acceptable size (Fig. 7-1, although not
quantitatively determined). The expected size of cDNA
encoding PAT is about 3 kB, based on a previous molecular-
weight estimation of 88,000 D (7). A preliminary Mr
estimation of about 130,000 for ADH by gel chromatography in
N. silvestris (unpublished data, Bonner and Jensen, 1986)
suggests that cDNA encoding ADH might be about 2.1 kB if the
plant protein is a dimer (dimers from 57,600 up to 158,000 D
have been described in bacteria (46, 56). Figure 7-1 shows
the plasmid DNA containing possible clones.
Functional complementation
Both aroD (DQT~) and aroE (SDH') mutants transformed
with SP3, SP5 or SP6 yielded transformants which grew on M9
medium without addition of aromatic amino acids. This
result was a strong indication that all three cDNA clones

Fig. 7-1. Agarose gels of cDNA encoding aromatic
pathway proteins. A) The cDNA inserts in pBluescript
are shown. B and C) Restriction enzymes (all found in
the multiple cloning site of pUC 18, pGEM or pBluescript
plasmids) were individually used to determine the number
of RE sites (or fragments) of the cloned cDNA insert in
pBluescript encoding the S-protein:
Lanes
A
(left)
B
(right)
cDNA
Clones
Restriction
Enzyme Sites
Restriction
Enzyme Sites
1
PAT 5
HindiII
3
Sail
1
2
SP10
SacI
0
BamHI
0
3
SP33
Kpnl
0
Ndel
1
4
SP3
SP3
-
Spel
0
5
PAT 7
STD
-
Bañil
3
6
SP5
Xbal
0
SP3
-
7
STD
Xhol
0
STD
-
8
pBluescript
Not I
0
EcoRV
3
9
SP6
EcoRI
1
10
ADH1
Smal
1
11
ADH4
PstI
0
12
ADH8
Bbul(SphI)
1

121
A
Jase Pairs
23,130
9,416
6,557
4,361
2,322
2,027
500
125
B

122
must encode an intact bifunctional S-protein. On the
other hand, SP10 and SP33 transformants would not grow on M9
medium unless aromatic amino acids were supplied. Table 7-
1 shows results obtained by direct enzyme assays of
transformants carrying an aroD*aroE insert, in comparison to
appropriate controls. These data show that expression
levels of both DQT and SDH are about 15-fold higher than the
wild type E. coli levels which have the corresponding
activities.
Table 7-1. SDH/DQT activities in transformed aroD
and aroE mutant strains of E. coli.
Enzyme
Preparation
Specific Activities
DQT SDH
aroD
AT1360
0
1.3
aroD
AT1360
(+SP3)
15.0
18.7
aroE
Sk4 94
0.7
0
aroE
SK4 94
(+SP3)
11.4
17.9
Initial sequence analysis of cDNA clones
Analysis of the sequenced 5' and 3' ends of the 5
possible SP-clones, by the GCG Blast computer program,
revealed that one clone, SP10 was NADP+-specific malate
dehydrogenase and that SP33 was NADP+-specific cinnamyl-
alcohol dehydrogenase (about 100% identity). The three
remaining clones, SP3, SP5, and SP6 were identical. The 5'

123
end (444 bases sequenced from the T3 promoter) was
translated to yield a sequence having about 23 to 25%
identity with the DQT functional domain of the
pentafunctional amino acid sequence from S. cerevisiae or A.
nidulans, respectively, and about 22% identity with the
monofunctional AroD sequence from E. coli, Fig. 7-2. The
sequence determination at the 3' end (sequenced from the T7
promoter) was not informative at this stage because the
deduced amino acid sequence showed very little conservation
between the known genes of E. coli, S. cerevisiae or A.
nidulans at the C-terminal region.
Partial sequencing of 5' and 3' ends of several of the
possible ADH and PAT cDNA clones showed no significant
identity to known genes.
Subcloninq cDNA encoding the S-protein
Since the partial sequence at the 5'end of the cloned
insert, the positive functional complementation results and
the direct assay of activities were all mutually reinforcing
that the cDNA encoding the S-protein had been cloned, the
SP3 clone was used further for subcloning analysis in order
to obtain a complete nucleotide sequence.
Based on the multiple cloning sites of the vectors to
be used for subcloning, pUC18, pGEM5Zf(+) and pBluescript+,
restriction enzymes were chosen and tested in order to
locate and map the restriction sites present in the SP3 cDNA
clone. Analysis of agarose gels (Fig. 7-1B, right and

124
* *
*
* ** * **
Ec AroD
41
En AroM
1075
Sc Arol
1099
clone SP3,
SP5, SP6
57
dfDilEwRvDhya 53
GsDaVElRvDlLK 1087
GceaVEVRvDhLa lili
GaDlVEVRlDsLK 69
75 kPILFTfRsakEGGe 89
1113 LPiiFTiRtqsqGGr 1127
1131 iPiiFTvRtmkqGGn 1145
85 LPtLFTyRptwEGGg 99
Fig. 7-2. Preliminary alignment of translated sequences
of clone SP3 with established DQT sequences. The deduced N-
terminal amino acid sequence of three cDNA clones, SP3, SP5,
and SP6 displayed two highly conserved regions with known
amino acid sequences from E. coli (AroD), E. nidulans (AroM)
and S. cerevisiae (Arol). All amino acids showing identity
with the SP3 clone in each group are in caps. Amino acids
conserved in all four proteins are noted with an asterisk.

125
left) indicated that HindIII, Bañil and EcoRV had at least
several restriction sites within the cDNA insert, and that
SphI and Ndel, each had one restriction site located within
the insert. The remaining restriction enzymes shown in Fig. 7-
1B, only had one site within the multiple cloning region of
the plasmid. From this information and from the known RE
sites of the 3' and 5' sequenced ends of the cDNA clone
(determined by GCG Map analysis) a strategy for subcloning was
begun and continued as information about RE sites was obtained
through subcloning (Fig. 7-3).
After appropriate RE were used to obtain fragments of
suitable size for sequencing, the bands were excised from
agarose gels (Fig. 7-4A) and prepared for transformation.
To confirm that the transformed cells carried plasmids
containing the appropriate fragments, a cracking procedure
(Promega protocol, Madison, WI), followed by an agarose gel
was performed (Fig. 7-4B). This gel showed that the plasmids
contained inserts of the correct size and were ready to be
purified for sequencing. Agarose gels containing the purified
plasmid DNA carrying the various fragment sizes are shown in
Fig. 7-4C.
After sequencing the cDNA fragments on an available Li-
Cor sequencer, overlapping areas were placed together to yield
a complete cDNA sequence. For correction of sequencing
errors, multiple sequencing of all fragments was done in both
directions. The complete nucleotide sequence and the deduced

126
5'end 3'end
E pBluescript 2958 bp E
c c
o o
R R
<-MCR I insert about 2000bp I MCR->
HE S
ic a
no 1
dR I
IV
I
I
444
423 <-- 1
E
c
o
R
I
*
227 --> 844 603 <-- 67
H BE H
i a c i
n no n
d I R d
I IV I
I I
I I
320
S
a
1
I
Plasmid <-- 487 -->
H N E
id c
n e o
d I R
I V
I
I
-> 1536
B S N
a p d
n h e
II I
I
1230
B B
a a
n n
I I
I I
558 1376
E
c
o
R
V
S
P
h
I
Fig. 7-3. Sequencing strategy for cDNA encoding the bifunctional
protein. Overlapping fragments were obtained when the SP3 cDNA clone
was cut with restriction enzymes in the sizes denoted in the Figure.
The cDNA clone was first cut with Hind III only, and Hind III plus
EcoRV. From this four of six possible fragments were obtained for
sequencing, two are shown in the second step of the sequencing strategy.
Next the cDNA clone was cut with Ban II and two of three fragment were
obtained. Two combinations of RE were used, NdeI plus Sal I, and Sphl
plus EcoRV. Sequencing was completed for multiple samples of these
fragments in both directions.

Fig. 7-4. Agarose gels of SP3 cDNA fragments during
stages of subcloning. SP3 fragments were visualized on
agarose gels after incubation with restriction enzyme (s) .
A) The first three lanes contained cDNA fragments cut
with Hindlll (from 500 to about 800 bp), the second set
of three lanes contained cDNA fragments cut with SphI and
Sail (about 300 and 1100 bp) and the last set of three
lanes contained fragments cut with Bañil (about 500,700
and 900 bp) . B) The first three lanes contained cDNA
fragments cut with NdeI and EcoRV (about 300, 500 and 900
bp). C) All lanes show that plasmids carrying the cDNA
fragments were transformed into competent cells. The
lowest bands represent RNA, the mid bands are the plasmid
DNA bands and the top bands are genomic DNA. D) Lanes
contain purified plasmid DNA containing cDNA fragments.
Bands are of different sizes depending on the size of the
ligated fragment.

128
HI •••

129
amino acid sequence is shown in Fig. 7-5. The sequence has
an untranslated 3' end of 277 bases following a TAA stop
codon and has three to four probable polyadenylation sites
prior to the beginning of a nine base poly A-tail.
N-terminal amino acid sequence
The first 7 N-terminal amino acid residues of purified
SP-I were found tobeGEAMTRN. These amino acids
corresponded to the deduced amino acid sequence starting at
position number 24 (underlined in Fig. 7-5).
Analysis of cDNA coding for the S-protein
GCG sequence analysis. The GCG sequence analysis
program (26) was used for analysis of the presumed mature S-
protein cDNA sequence starting at amino acid residue number
24. The overall isoelectric point was 5.96 for the
bifunctional protein (isoelectric points of 5.04 for the
AroD functional domain and 6.99 for the AroE functional
domain). A Mr of 60,388 (556 amino acids) was determined
for the proposed mature protein (Mr of 28,515, 260 amino
acids was determined for the AroD functional domain and Mr
of 34,651, 320 amino acids for the AroE functional domain).
There were no remarkable differences in amino acid
composition when the AroD and AroE domains were compared.
The bias of codon usage was similar to that of other higher
plant genes, with A and T being favored in the third
position of most alternative codons. Rare codons were TGC
(one), CGC (zero), CCG (zero), and ACG (two).

Fig. 7-5. Nucleotide and deduced amino acid sequence
of cDNA coding for the bifunctional S-protein. The
nucleotide sequence of the cDNA encoding the S-protein,
containing 2041 bases, is numbered on the right. The
deduced 679 amino acid sequence is numbered on the left.
The N-terminal sequenced amino acids are in bold and
underlined. The stop codon is designated by the asterisk
and four possible polyadenylation sites are underlined with
hashed marks.

60
131
CCATTTTTGTGCTCTACAAGTTGGTTGCTAATGGAGTTGGTAGTGGATTCAGGGGTGAGG
IP PLCSTSWLLMBLVVDSGVR
21 K M B <3 B A M T E W BTLZCAPIMA
GACACAGTGGATCAAATCTTGAATCTAATGCAAAAGGCTAAAATTAGTGGTGCTGATCTT 180
41DTVDQMLNLMQKAK I SGADL
GTGGAAGTTCGATTGGATAGCTTGAAAAGCTTTAATCCTCAATCAGATATCGATACTATT 240
61VBVRLDSLKSPNPQSDZDTZ
ATCAAACAGTCCCCTTTGCCTACCCnTTCACTTACAGGCCCACTTGGGAAGGGGGTCAG 300
81ZKQSPLPTLPTYRPTWBGGQ
TATGCTGGTGATGAAGTGAGTCGACTGGATGCACTTCGAGTAGCAATGGAGTTGGGAGCT 360
101 YAGDBVSRLDALRVAMBLGA
GATTACATTGATGTTGAGCTAAAGGCTATTGACGAGTTCAATACTGCTCTACATGGAAAT 420
121 DYZOVBLKAZDBPNTALHGN
141 K S A
CKVZVSSHNY
H T P S S
GAGGAGCTCGGCAATCTAGTAGCAAGAATACAGGCATCTGGAGCrGACATTGTGAAGTTT 540
161 BBLGNLVARZQASGADZVKP
GCAACAACTGCACTGGATATCATGGATGTTGCACGTGTATTCCAAATTACTGTACATTCT 600
181 ATTALDZMDVARVPQ ZTVHS
CAAGTACCAATAATAGCCATGGTCATGGGAGAGAAGGG'rrTGATGTCTCGAATACmO 1' 660
201 QVPZ ZAMVMGBKGLMSRZLC
CCAAAArTrGGTGGATACCTCACAnTGGTACTCTTGAAGTGGGAAAGGTTTCGGCTCCT 720
221 PKPGGYLTPGTLBVGKVSAP
GGGCAACCAACAATTAAAGATCTTTTGAATATATACAATTTCAGACAGTTGGGACCAGAT 780
241 GQPTZKDLLNZYNPRQLGPD
ACCAGAATATTTGGCATTATCGGGAAGCCTGTTAGCCATAGCAAATCACCTTTATTGTAT 840
261 TRZPGZZGKPVSHSKSPLLY
AATGAAGCTlTCAGATCAGTrGGGTTTAATGGTGTl'TATATGCCTTTGCTGGTTGATGAT 900
281 NBAPRSVGPNGVYMPLLVDD
GTTGCAAAri"rCITrCGGACTTACTCATCTTTAGA'lTriG<-‘mGCTCAGCTGTAACAATT 960
301 VANPPRTYSSLDPAGSAVTZ
CCTCACAAGGAAGCCATTGTTGACTGCTGTGATGAGTTGAATCCTACCGCTAAAGTAATA 1020
321 PHKBAZVDCCDBLNPTAKVZ
GGGGCTGTCAATTGTGTCGTAAGCCGACTCGATGGGAAG’rrGT’llGG'ri'GCAATACAGAC 1080
341 GAVNCVVSRLDGKLPGCNTD
TATGTGGGTGCAATCTCCGCCATTGAAGAAGCGTTGCAAGGCTCACAGCCTAGTATGTCT 1140
361 YVGAZSAZBBALQGSQPSMS
GGGTCTCCCTTAGCTGGTAAATTA'ITIG'mG'lXJATTGGTGCTGGTGGCGCTGGCAAGGCA 1200
381 GSPLAGKLPVVZGAGGAGKA
CTTGCTTATGGTGCAAAGGAAAAGGGGGCTCGGGTGGTGATTGCTAACCGTACCTATGAA 1260
401 LAYGAKBKGARVV Z ANRTYB
CGAGCGAGAGAACTTGCTGATGTAGTTGGAGGTCAGGCTriXrid’ClTUACGAGCTTAGC 1320
421 RARBLADVVGGQALSLDBLS
AATTTCCATCCAGAAAATGACATGATTCTTGCAAATACCACCTCCATTGGCATGCAACCA 1380
441 MPHPBNDMZ LANTTS ZOMQP
AAGGTTGATGATACACCAATCriTAAGGAAGCTriTjAGGTACTACTCACrTGTATTTGAT 1440
461KVDDTPZ PKBALRYYSLVPD
GCTGTTTATACGCCCAAAATCACTAGACTCTTGCGGGAAGCTCACGAGAGTGGAGTAAAA 1500
4B1 AVYTPKZTRLLRBAHB8GVK
ATTGTAACAGGAGTTGAAATGTTTATCGGCCAGGCATATGAACAATATGAGAGATTTACA 1560
501ZVTOVBMPZO0AYBQYBRPT
GGGCTTGCCAGCTCCAAAGGAACTTTTCAAGAAAATTATGGCTGGATATTGAGAGCAAGG 1620
521 GLASSKGTPQBNYGMZLRAR
TGTGTGATAGCAATOGTCITAGATTCCTCTGCCCTACCArnGTGCITCGGAGGAATTAA 1740
561 CVZAMVLDSSALPPVLRRN*
TTCGTTCCAGGTAAATCGTGA rrrrCACCAAAACAAA'riX7ITGAGGATCTTCAGGAAGGC 1800
AGTCAGACAT ACCAGTGGACAATCGCCGTCATTCTGGCTTATATT AGACTCTTG T AGCAC 1860
TTCA TTCnTGACAACTATGGTATCTCTAA TIG 1X3CT1TCATTAAACACAGATGTATCAG 1920
TGTTTCTCATTGTGACCCCATACTTGGAATTCCTCTTGATTATCATTATTATTAAATCTT 1980
GTCACATTATTGTATGATTTGTATCAAAAAAAAA
2041

132
Discussion
S-protein cDNA cloning
From five possible cDNA clones isolated by antibody
screening of a Lambda Zap II cDNA library from N. tabacum,
three identical clones encoding the bifunctional S-protein,
were obtained. Subcloning analysis and sequencing of the
entire cDNA of clone SP3 was performed.
The other two cDNA clones, probably code for two other
NADP+-dependent plant proteins, malate dehydrogenase and
cinnamyl-alcohol dehydrogenase. Antibody selection of these
two cDNA clones could be due to the presence of these
proteins as contaminants in the purified SDH/DQT
bifunctional protein. Consistent with this, both MDH and
CDH have very similar calculated isoelectric points (5.78
and 6.07, respectively) compared to the mature S-protein
isoelectric point of 5.96. Neither CDH nor MDH activity
could be detected in the purified S-protein preparation,
even at high protein concentration. Alternatively, MDH and
CDH might have common epitopes recognized by the S-protein
specific antibody. As a final possibility, the rabbit may
have developed an immune response separate from that
developed to the injected protein, perhaps by ingestion of
some plant similar to tobacco. Using the GAP analysis in
the GCG program, the SDH functional domain of the
bifunctional protein had 21% and 23% identities with CDH and

133
MDH, respectively. MDH had about 51% similarity with the
SDH functional domain. These proteins did not show any
striking conservation of residues aligned with the SDH
functional domain and its homologues, except for a region
thought to be important for NADP+ binding.
Possible cDNAs coding for PAT or ADH
Of the number of possible cDNA clones isolated with
specific antibodies to either PAT or ADH, no determinations
have been made as to whether the cDNAs of interest have been
cloned. The terminal regions (5' and 3') of the cDNAs that
were sequenced did not offer enough information. Neither
PAT nor ADH have been sequenced in any organism, so that
comparisons of homologues are not possible at present.
Although many aminotransferases have been cloned from plant,
animal and microbial species, and some very highly conserved
regions have been determined, they all seem to be located
toward the center of the genes, with very little
conservation at either terminus. There are no auxotrophic
mutants available to attempt functional complimentation for
these putative cDNA clones. Extract preparation and enzyme
assay may help to identify the correct clones if the cDNAs
are intact and functional.
Homology relationships of the DOT domain
A multiple alignment is shown in Fig. 7-6 of the AroD
functional domain of the S-protein (denoted Nta-AroD»E) with
its homologues. The corresponding dendrogram is shown in

Fig. 7-6. Multiple amino acid alignment of the AroD
domain of AroD»E with its homologues. Nine proteins have
been aligned and conserved regions are boxed. Three
conserved residues that have been shown as active site
residues in E. coli have asterisks (H (25) , L (20) , M
(47) } . The amino acid residue numbers for each sequence
are on the right of the figure:
Designation
Organism
Protein
Accession
Number
Eco-AroD Escherichia coli
Sty-AroD Salmonella typhimurium
Bsu-AroD Bacillus subtilus
Efa-AroD Enterococcus faecalis
Nta-AroD«E Nicotiana tabacum
Sce-Arol Saccharomyces cerivisiae
Eni-AroM Emericella nidulans
Ncr-Qa-lS Neurospora crassa
Eni-QutR Emericella nidulans
dehydroquinase
dehydroquinase
dehydroquinase
dehydroquinase
bifunctional S-protein
arom pentafunctional protein
arom pentafunctional protein
quinate repressor protein
quinate repressor protein
P085S6
P07547
P11637
M59935
S14750
P24670
L09228
L23802

135
Eco-AroD
[Ml
K
T
V
T
V
K
D
L
V
I
G
T
fol
A
P
K
[xl
I
V
S
L
M
A
K
D
I
A
S
V
K
s
E
A
34
Sty-AroD
M
K
T
V
T
V
K
N
L
I
I
G
E
G
M
P
K
I
I
V
s
L
N
G
R
D
I
N
s
V
K
A
E
A
34
Bsu-AroD
M
N
V
L
T
I
K
G
V
s
I
G
E
G
M
P
K
I
I
I
p
L
M
G
K
T
E
K
Q
I
L
N
E
A
34
Efa-AroD
M
K
P
V
I
V
K
N
V
R
I
G
E
G
N
P
K
I
V
V
p
I
V
A
P
T
A
E
D
I
L
A
E
A
34
Nta-AroD*E
P F L C S
T
S
W L
L
[mJ
B
L
V
V
D
S
G
V
R
K
M
E
[Gj
E
A
M
T
R
N
E
T
L
I_
c
A
p
Z
M
A
D
T
V
D
Q
M
L
N
L
M
50
Sce-Arol
. . . . Y
I
A
T .
rri
T
G
V
R
E
I
E
I
P
S
G
R
S
A
F
V
c
L
T
F
D
D
[l]
T
E
Q
T
E
N
1092
Eni-AroM
V
A
T G Q
i
D
S
L
S
I
I
K
E
G
E
H
S
F
F
A
s
L
T
L
P
D
L
R
E
A
G
D
I
1068
Ncr-Qa-lS
L
P
K G
T
i
P
F
V
E
s
A
F
P
L
A
S
V
P
V
E
Q
R
R
F
T
Y
A
L
A
L
P
V
S
A
L
L
D
K
G
V
D
328
Eni-QutR
L
A
T G
N
|_XJ
P
B
L
R
N Q
L
s
P
F
P
L
H
M
Q
P
I
E
S
R
K
F
T
Y
A
A
T
V
P
I
s
H
L
L
E
N
D
V
D
328
Eco-AroD
Sty-AroD
Bsu-AroD
Efa-AroD
Nta-AroD*E
Sce-Arol
Eni-AroM
Ncr-Qa-lS
Eni-QutR
LAYREADP
LAYREATP
EAVKLLNP
TASQTLDC
QKAKISGA
LTPICYOCE
LEEVCVGSD
IQELDVGVD
IEELESTAD
L
B
w
R
V
D
H
Y
A
D
L
s
N
V
E
S
V
M
A
A
A
K
I
L
R
E
T
M
72
L
E
w
R
V
D
H
F
M
D
I
A
S
T
Q
S
V
L
T
A
A
R
V
I
R
D
A
M
72
V
E
w
R
V
D
V
F
B
K
A
N
D
R
E
A
V
T
K
L
Z
S
K
L
R
K
S
L
72
V
E
w
R
L
D
Y
Y
E
N
V
A
D
F
s
D
V
C
N
L
s
Q
Q
V
M
E
R
L
72
V
E
V
[rJ
L
D
S
L
K
S
F
N
P
Q
S
D
z
D
T
Z
z
K
Q
83
V
E
V
[*1
V
D
H
L
A
N
Y
S
A
D
F
V
S
K
Q
L
S
I
L
[rI
K
A
T
1129
V
E
L
*
V
D
L
L
K
D
P
A
S
N
N
N
I
P
s
V
D
Y
V
V
E
Q
L
s
F
L
R
S
R
1111
I
E
I
I
V
D
D
L
A
T
S
E
S
G
P
T
s
P
L
G
L
A
P
H
R
A
S
B
z
s
R
V
V
G
E
I
R
R
D
T
378
F
[bJ
L
K
I
IdJ
V
S
A
A
P
S
A
R
L
G
T
E
S
N
L
A
D
S
z
s
H
T
V
A
T
V
[rJ
R
N
I
374
Eco-AroD
P
E
K
P
L
L
F
T
F .
R
S
A
K
E
G
G
E
Q
A
I
S
T
E
A
Y
I
A
L
N
R
A
A
I
D
S
G
L
V
D
M
X
D
L
E
L
F
T
119
Sty-AroD
P
D
I
P
L
L
F
T
F .
R
s
A
K
E
G
G
E
Q
T
I
T
T
Q
H
Y
L
T
L
N
R
A
A
I
D
S
a
L
V
D
M
Z
D
L
E
L
F
T
119
Bsu-AroD
E
D
K
L
F
L
F
T
F .
R
T
H
K
E
G
G
S
M
E
M
D
E
S
S
Y
L
A
L
L
E
S
A
I
Q
T
K
D
I
D
L
z
D
I
E
L
F
S
119
Efa-AroD
G
Q
K
P
L
L
L
T
F .
R
T
Q
K
E
G
G
E
M
A
F
S
E
E
N
Y
F
A
L
Y
H
E
L
V
K
K
a
A
L
D
L
L
D
I
E
L
F
A
119
Nta-AroD*E
S
p
L
P
T
L
F
T
Y .
R
P
T
W
E
G
G
Q Y
A
G
D
E
V
S
R
L
D
A
L
R
V
A
M
E
L
a
A
D
Y
z
D
V
E
L
K
128
Sce-Arol
D
S
I
P
I
I
*
T
V . .
*
T
M
K Q
G
G
N
F
P
D
E
E
F
K
T
L
R
E
L
Y
D
I
A
L
K
N
G
V
E
F
L
D
L
E
L
T
L
1176
Eni-AroM
V
T
L
P
I
I
r
T
I . .
*
T
Q
S
Q
G
G
R
F
P
D
N
A
H
D
A
A
L
E
L
Y
R
L
A
F
R
S
G
C
E
F
V
D
L
D
I
A
F
1158
Ncr-Qa-lS
V
I
P
I
I
L
H
V .
V
F
P
E
R
A
L
Y
E
E
A
L
L
A
L
Y
M
T
Y
L
N
H
A
L
R
L
A
P
D
Y
L
T
V
D
L
G
L
423
Eni-QutR
I
V
P
M
Lil
Y
H
V B S
S
V
F
P
D
S
A
P
L
R
R
S
D
A
S
Y
L
E
L
V
L
H
G
L
R
L
G
P
E
F
V
T
V
D
L
S
F
421
V K V V
V Y V V
V Y V V
I K I V
C X V X
MSN
MSN
MSN
L C N
V S S
D F H K
D P H Q
D P E K
D F Q K
N Y D N
T P
T P
T P
T P
T P
Eco-AroD
G
D
D
Q v
K
B
T
V
A
Y
A
H
A
H
D
Sty-AroD
G
D
A
D
V
K
A
T
V
D
Y
A
H
A
H
N
Bsu-AroD
G
D
A
N
V
K
A
L
V
S
L
A
E
B
N
N
Efa-AroD
N
P
L
A
A
D
T
L
X
H
E
A
X
K
A
G
Nta-AroD»E
A
Z
D
E
F
N
T
A
L
H
G
If
X
S
A
X
Sce-Arol
P
T
D
I
Q
Y
B
V
I
N
X
R
G
N
Eni-AroM
P
E
D
M
L
R
A
V
T
E
M
K
G
F
Ncr-Qa-lS
D
S
G
L
L
G Q
L
T
T
V
Q
G
T
Eni-QutR
E
D
S
I
L
S
Q
I
I
G
T
K
G
S
E
A
E
E
I
I
A
R
L
R
K
M
Q
S
F
D
A
166
S
A
B
B
M
V
S
R
L
R
K
M
Q
A
L
G
A
166
V
K
D
B
I
I
S
R
L
R
K
M
Q
D
L
G
A
166
s
Q
X
X
I
V
A
R
L
R
Q
M
Q
M
R
Q
A
166
s
s
B
E
L
G
N
L
V
A
R
Z
LaJ
A
S
G
A
175
D
D
A
B
W
B
N
R
F
N
Q
A
L
T
L
D
V
1223
A
N
M
S
w
X
K
F
Y
N
K
A
L
E
Y
G
1204
G
D
P
S
w
L Q
A
Y
E
K
A
Q
N
T
G
c
471
S
D
P
X
Y
B
A
I
Y
B
R
A
K
K
L
G
c
469
ECo-AroD
D
I
P
K
I
A
L
M
P
Q
S
T
s
D
V
L
T
L
L
A
A
T
L
E
M
Q
E
Q
Y
A
D
R
P
Z
Z
T
M
S
M
A
K
T
G
V
I
Sty-AroD
D
I
P
X
I
A
V
M
P
Q
S
X
H
D
V
L
T
L
L
T
A
T
L
E
M
Q
Q
H
Y
A
D
R
P
V
z
T
M
S
M
A
K
E
G
V
I
Bsu-AroD
H
I
P
X
M
A
V
M
P
N
D
T
G
D
V
L
T
L
L
D
A
T
Y
T
M
K
T
I
Y
A
D
R
P
I
z
T
M
s
M
A
A
T
G
L
I
Efa-AroD
D
I
C
X
I
A
V
M
P 0
D
A
T
D
V
L
T
L
L
S
A
T
N
E
M
Y
T
H
Y
A
S
V
P
z
V
T
M
s
M
G
Q
L
G
M
I
Nta-AroD*E
D
_X
V
X
F
ihl
T
T
A
L
D
Z
M
D
V
r
A
R
V
F
Q
Z
T
V
H
S
Q V
P
z
z
A
M
V
M
G
B
X
G
L
M
Sce-Arol
D
V
V
*
r
V
G
T
A1
V
N
F
E
D
N
L
R
L
E
H
F
R
D
T
H
K
N
K
P
L
I
A
V
N
M
T
S
X
G
S
I
Eni-AroM
D
I
I
K
L
V
G
V
A
R
N
Z
D
D
N
T
A
L
R
K
F
K
N
W
A
A
E
A
H
D
V
P
L
z
A
I
N
M
G
D
Q
G
Q
L
Ncr-Qa-lS
D
L
V
R
L
T
R
P
A
S
N
P
R
D
N
T
D
I
R Q
F
H
V
A
V
E
A
V
G
G
P
R
L
P
F
z
A
Y
N
T
G
R
L
G
R
T
Eni-QutR
D
M
V
R
L
T Q
P
A
T
T
z
D
D
N
F
A
V
E
R
F
R
H
Q
I
K
T
L
P
G
P
Q
L
P
V
z
A
Y
N
S
G
P
L
G
R
Q
215
215
215
215
219
1268
1252
521
519
Eco-AroD
G
E
V
F
G
S
A
A
T
F
G
A .
. V
K
X A
S
A
P
G Q
I
S
V
N
D
L
Sty-AroD
G
E
V
F
G
S
A
A
T
F
G
A .
. V
K
Q A
S
A
P
G Q
I
A
V
N
D
L
Bsu-AroD
G
E
V
F
G
S
A
C
T
F
F
A .
. G
E
B A
S
A
P
G Q
I
P
V
s
E
L
Efa-AroD
G
Q
L
F
G
S
A
L
T
F
G
S .
. A
0
Q A
S
A
P
G Q
L
S
V
Q
V
L
Nta-AroD*E
C
P
X
F
G
G
Y
L
T
F
G
T L
E V
G
X V
S
A
P
GO
P
T
z
X
D
L
TVLTILHQA .
SVLMILHNA .
SVLDILHKNT
MYLKTFBQNK
NXYNPRQLGP
R G
H .
252
252
256
253
206
Sce-Arol
Eni-AroM
Ncr-Qa-lS
Eni-QutR
N
N V L
T P
V
N
G F M
T P
V
N
E I L
T P
V
N
P V L
T P
V
TSDLLPNS
SHPSLPFK
TP. . V P T K
IPRSLISQ
a[a1 IPOQL t
aLaJ ip q QL s
E D A I G L R
S G T K G[L .
V A Q I N K
A T E I R K
N P A H R Y
MYTSMGGIB
GLSLMGEIK
LQPPLTALE
. . P S I T I Q B
1305
1289
A T Q 559
AQEALYSSPV 557

Fig. 7-7. Dendrogram of dehydroquinase homologues. The
dendrogram is based upon the multiple alignment which includes
the nine designated proteins explained in the legend of Fig.
7-6.

137
E. coli AroD
S. typhimurium AroD
B. subtilus AroD
E. faecalis AroD
N. tabacum AroD«E
S. cerivisiae Arol
E. nidulans AroM
N. crassa Qa-lS
E. nidulans QutR

138
Fig. 7-7. The latter shows two clusters, with Nta-AroD«E
grouping with the monofunctional proteins of the
prokaryotes: E. coli, Salmonella typhimurium, Bacillus
subtilis, and Enterococcus faecalis. Thus, the
bifunctional plant enzyme is evolutionarily closer to the
prokaryote monofunctional proteins than to the AroD domains
of the pentafunctional proteins present in the eukaryotes,
yeast (Arol) and fungi (AroM).
The proteins in the multiple alignment shown in Fig. 7-
6 are separated between the two clusters that are shown in
Fig. 7-7 for clarity. This alignment shows the conserved
residues within each cluster, as well as those conserved
throughout both clusters. Three amino acids shown to be
important catalytic residues for the E. coli AroD protein,
HIS (25), MET (47), and LYS (20) are marked with asterisks,
and these are conserved in all proteins having AroD
catalytic activity. It has been suggested that the
repressor proteins from Neurospora crassa (Qa-lS) and from
Emericella nidulans (QutR) evolved from three of the five
domains of the pentafunctional protein corresponding to
dehydroquinase, shikimate dehydrogenase, and shikimate
kinase (38). These retained the ability to bind what
previously were substrate molecules to function in a new
role as regulatory agents. Thus, critical residues such as
those marked by asterisks were altered to retain binding but
to lose catalysis. Note that nine other residue positions

139
are completely conserved in the catalytic proteins, but not
in the two regulatory proteins. This implies that some or
all of these residues may be important for catalysis. Six
residues are absolutely conserved for all nine proteins.
Homology relationships of the SDH domain
A multiple alignment is shown in Fig. 7-8 of the AroE
domain of the S-protein with its homologues. The
corresponding dendrogram is shown in Fig. 7-9. Again two
evolutionary clusters are seen in the dendrogram, and the
AroE domain clusters with the monofunctional AroE protein of
E. coli, rather than with the AroE domains of the
pentafunctional proteins (Arol and AroM) or with the
repressor proteins (Qa-lS and QutR). The Nicotiana AroD
domain is also homologous with the catabolic quinate
dehydrogenase of yeast and fungi, which are monofunctional
and specific for NAD+. However, the latter (QutB and Qa-3)
are evolutionarily closer to the repressor proteins (Qa-lS
and QutR) than to the shikimate dehydrogenase domain of the
plant bifunctional S-protein. It is surprising that the
catabolic QDH exhibits homology with biosynthetic SDH while
the catabolic dehydroquinases display no obvious homology
with biosynthetic dehydroquinases (32, 35). This is all the
more striking in that the former differ in substrate
specificity, whereas the latter do not. One might also have
expected the catabolic dehydroshikimate dehydratase to be
homologous with the biosynthetic dehydroquinases or

Fig. 7-8. Multiple amino acid alignment of the shikimate
dehydrogenase domain of AroD»E and its homologues. Nine pro¬
teins have been aligned and conserved regions are boxed. The
probable domain for the NADPH binding site is underlined. The
amino acid residue numbers for each sequence are on the right
of the figure:
Designation
Organism
Protein
Accession
Number
Eni-QutB Emericella nidulans
quinate dehydrogenase
quinate dehydrogenase
quinate repressor protein
quinate repressor protein
arom pentafunctional protein
arom pentafunctional protein
dehydroquinase
bifunctional S-protein
unidentified reading frame
P25415
P11635
P11637
M59935
P08566
P07547
P15770
Ncr-Qa-3 Neurospora crassa
Ncr-Qa-lS Neurospora crassa
Eni-QutR Emericella nidulans
Sce-Arol Saccharomyces cerivisiae
Eni-AroM Emericella nidulans
Eco-AroE Escherichia coli
Nta-AroD»E Nicotians tabacum
Eco-URF Escherichia coli
P28244

141
Eni-QutB
M
E
P
I
T
I
P
T
D
R
D
G
V
A
Y
L
Y
G
H
P
L
R
N
S
L
S
P
P
L
H
Q
T
V
Y
N
A
L
G
L
N
w
T
Q
I
P
L
s
T
A
T
50
Ncr-Qa-3
L
T
S
T
P
D
I
T
P
Y
T
R
H
G
Y
L
F
G
0
K
L
A
A
s
M
S
P
L
L
H
s
I
V
Y
S
H
L
S
L
N
w
A
Q
L
R
L
D
73
Ncr-Qa-lS
D
P
M
K
L
Y
V
F
G
A
N
V
G
Y
s
L
S
P
A
M
H
N
A
A
L
K
A
C
G
I
P
H
H
Y
K
P
L
S
T
A
N
608
Eni-QutR
D
P
M
0
F
F
V
F
G
A
N
T
T
Y
s
L
S
P
A
M
H
N
A
A
F
K
V
R
G
M
P
H
I
Y
R
I
H
Q
S
P
T
599
Sce-Arol
P
K
E
L
F
V
V
G
K
P
I
G
H
s
R
S
P
I
L
H
N
T
G
Y
E
I
L
G
L
P
H
K
P
D
K
F
E
T
E
S
1345
Eni-AroM
P
K
K
F
A
I
F
G
S
P
I
S
Q
s
R
S
P
A
L
[hJ
N
T
L
F
A
Q
V
G
L
P
H
N
Y
T
R
L
B
T
T
N
1329
Eco-AroB
M
B
T
Y
A
V
F
G
N
I
A
[H
s
K
s
P
F
I
H
0
Q
F
A
Q
Q
L
N
I
E
H
P
Y
G
R
V
Íl"
A
P
I
40
Nta-AroD»E
D
T
R
I
F
G
I
I
G
*
ÍJ
V
s
H
s
K
s
P
L
L
Y
N
E
A
F
R
s
V
G
F
N
G
V
Y
M
P
L
L
.
V
298
Eco-URP
Eni-QutB
G
T
S
F
T
R
S
P
B
I
S
T
F
L
S
s
V
R
s
N
P
K
7
V
[gT
s
s
V
T
M
P
W
T
V
A
i
M
P
H
L
*
D
D
L
T
E
D
A
R
Q
A
100
Ncr-Qa-3
S
P
S
I
P
L
F
L
Q
L
A
0
H
P
D
F
Y
G
A
s
V
T
M
P
H
K
V
A
i
I
P
H
L
D
H
L
T
P
E
C
R
D
V
116
Ncr-Qa-lS
I
G
T
L
R
E
V
I
s
D
P
Q
F
A
G
A
s
V
G
L
P
F
K
V
E
i
I
S
L
T
H
S
L
S
R
H
A
K
A
I
648
Eni-QutR
L
R
G
I
N
Y
L
V
E
N
P
N
F
G
G
T
s
V
S
L
P
Y
K
T
E
V
I
P
L
L
H
S
M
S
P
H
A
R
A
I
639
Sce-Arol
A
Q
L
V
K
E
K
L
L
D
G
N
K
N
F
G
G
A
A
V
T
I
P
L
K
L
D
i
M
Q
Y
M
D
E
L
T
D
A
A
K
V
I
1387
Eni-AroM
A
Q D
V
Q
E
F
I
R
S
P
D
LfJ
G
G
A
F
R
N
N
S
L
K
L
D
i
M
P
L
L
D
E
V
A
A
B
A
E
I
I
1369
EcoAroE
N
D
F
I
N
T
L
N
A
F
F
â– 
-si
A
G
G
K
G
A
N
V
T
V
fp
F
K
E
E
A
F
A
R
A
D
E
L
T
E
R
A
A
L
A
82
Nta-AroD»E
D
D
V
A
N
F
F
R
T
Y
S
â– 
Ü
L
D
F
A
G
S
A
V
T
I
P
H
K
E
A
I
V
D
C
C
D
E
L
N
P
T
A
K
V
I
338
ECO-URF
S
M
\k
N
K
Q
L
A
C
E
Y
V
D
E
L
T
P
A
*
*
L
V
22
Eni-QutB
G A
C
N
T
I
Y
L
R
K
E
D
D
G
K
T
Q
Y
Ncr-Qa-3
G
A
C
N
T
L
F
L
K
T
D
. P
A
T
G
R
R
L
Y
Ncr-Qa-lSc
G
A
V
N
T
L
I
P
V
R
H
L T A
D
G
G
I
P
D
E
V
S
M
F N
N I
S Q
A
G
A
V
R
A
L
Eni-QutR
G
A
V
N
T
L
I
P
I
R
N
L . .
E
G
S
T
D
N
A
L
D
L
E K
N .
. R
A
G
P
I
K
G
L
Sce-Arol
G
A
V
N
T
V
I
P
L
G
N
. K
K
F
Eni-AroM
G
A
V
N
T
I
I
P
V
S
T
. G
K
N
T
P
S
R
L
Eco-AroE
G
A
V
N
T
L
K
R
I
E
[Di
. . .
[O'
R
[l|
Nta-AroD*E
G
A
V
N
C
V
V
S
R
L
D
G
K
L
ECO-URF
G
A
I
N
T
I
V
N
D
D
G
Y
L
mi
O
O
o
o
o
R B A
V R B S
I R A C
I G I C
I R N A
MILS
L L S D
A I S A
H I R A
131
148
698
685
1414
1401
109
365
48
Eni-QutB
L
L
Q G
S
P
N
G
A
E
H
P
K
G
K
P
A
L
I
V
G
G
G
G
T
A
R
T
A
I
Y
V
L
R
K
W
L
G
V
S
K
I
Y
I
V
N
177
Ncr-Qa-3
F
V Q
N
V
S
D
P
A
R
V
Y
E
S
R
P
A
L
V
I
G
G
G
G
A
A
R
S
A
V
Y
A
L
H
K
W
L
G
A
T
D
I
Y
L
V
N
194
Ncr-Qa-lS
L
R
R
G
L
S
P
A
N
A
V
R
S
T
S
T
G
L
V
I
G
A
G
G
M
A
R
A
A
V
Y
A
M
L
Q
L
G
V
K
K
I
L
I
F
N
743
Eni-QutR
I
R
R
G
L
S
P
A
N
A
I
R
P
S
T
T
G
L
I
I
G
A
G
G
M
A
R
A
G
I
Y
A
M
I
H
L
G
V
Q
N
I
F
I
W
N
730
Sce-Arol
L
I
N
N
G
V
P
E
Y
V
G
H
T
A
G
L
V
I
G
A
G
G
T
S
R
A
A
L
Y
A
L
H
S
L
G
c
K
K
I
F
I
I
N
1456
Eni-AroM
L
R
K
A
G
V
Y
G
P
K
R
K
D
Q
B
Q
S
A
[Lj
V
V
[gJ
G
G
G
T
A
[R
A
A
I
Y
A
L
H
N
M
[g|
Y
S
P
I
Y
I
V
G
1447
Eco-AroB
L
E
R
L
s
F
I
R
P
G
L
R
I
L
L
i
G
A
G
G
A
S
R
G
V
L
L
P
L
L
S
L
D
c
A
V
T
I
T
»l
149
Nta-AroD*K
I
E
E
A
L
Q
G
S
Q
P
3
M
S
G
S
p
L
A
G
K
L
F
V
V
i
G
A
G
G
A
G
K
A
L
A
Y
G
A
K
E
K
G
A
R
V
V
I
A
414
ECO-URF
I
K
B
S
G
F
D
I
K
G
K
T
M
V
L
L
G
A
G
G
A
S
T
A
I
G
A
Q
G
A
I
E
G
L
K
E
I
K
L
F
iü
89
Eni-QutB
[r]
D
A
K
B
V
E
A
I
L
A
E
D
K Q
R
N
P
S
P
Q
V
A
L
V
P
V
S
D
P
S
A
A
A
T
212
Ncr-Qa-3
R
D
K
S
B
V
D
A
V
I
A
E
C
T
E
R
G
Y
G
D
R
L
V
H
V
A
S
V
E
Q
A
E
G
227
Ncr-Qa-lS
R
T
F
A
N
A
E
K
L
V
L
H
F
B
N
L
L
V
R
D
A
L
P
L
L
S
T
G
P
R
S
H
D
N
T
C
F
H
I
I
R
S
R
D
D
P
L
P
E
N
793
Eni-QutR
R
T
V
A
N
A
E
K
L
A
Q
H
Y
M
R
L
N
L
C
T
L
G
G
S
G
S
A
S
Y
T
I
H
V
L
K
S
L
Q
E
S
W
P
A
N
774
Sce-Arol
R
T
T
S
K
L
K
P
L
I
E
S
L
P
S
E
F
N
I
I
G
I
E
S
T
K
S
I
E
E
I
1488
Eni-AroM
R
T
P
S
K
L
E
N
M
V
S
S
F
P
S
S
Y
N
I
R
I
V
E
S
P
S
s
F
B
S
V
1478
Eco-AroE
R
T
V
S
R
'Al
E
E
L
A
K
L
F
A
H
T
G
S
I
Q A
I*
TI
M
D
*
fxH
B
G
H
E
F
D
L
I
184
Nta-AroD*B
R
T
Y
B
*
A
*
E
L
A
D
V
V
G
G
Q
A
L
S
L
D
*
L
S
N
F
H
P
E
N
D
M
I
449
ECO-URF
_r|
R
D
E
F
F
D
K
¿Ü
A
F
A
Q
A
L
M
K
T
P
I
V
S
S
R
S
P
I
S
P
i
U
K
P
U
K
P
W
I
P
P
T
F
133
Eni-QutB
L
E
A
P
V
A
V
V
s
G
I
[71
N
Y
P
P
Q
T
E
E
B
I
R
A
R
B
T
L
R
L
F
L
N
R
Q
T
H
E
K
D
Q
G
V
I
256
Ncr-Qa-3
L
E
G
P
G
A
I
V
A
C
I
P
D
F
P
P
K
T
E
K
B
M
L
V
R
R
I
V
E
T
F
L
M
K
E
E
K
G
A
M
267
Ncr-Qa-lS
F
K
N
P
T
M
I
V
s
C
I
P
T
H
T
V
D
N
T
P
D
P
E
F
T
V
P
L
H
W
L
D
N
P
T
G
G
I
V
831
Eni-QutR
Y
K
Q
P
T
I
V
V
s
G
I
P
A
H
R
I
G
D
Q
P
A
P
N
F
Q
L
P
P
Q
W
I
E
S
P
T
G
G
V
V
813
Sce-Arol
K
E
H
V
G
V
A
V
s
C
V
P
A
D
K
P
L
D
D
E
L
L
S
K
L
E
R
F
L
V
K
G
A
H
A
A
F
V
P
T
L
1529
Eni-AroM
P
H
V
A
I
G
T
I
LlI
A
D
Q
P
I
D
P
T
M
R
B
T
L
C
H
M
F
B
R
A
Q
B
A
D
A
B
A
V
K
A
I
B
H
A
P
R
I
L
1525
Bco-AroE
I
'Ñl
A
ffi
S
S
"ol
.
I
S
G
D
I
P
A
I
P
S
S
L
I
H
P
G
I
Y
C
210
Nta-AroD*E
L
A
N
T
T
S
I
0
.
M
Q
P
K
V
D
D
T
P
I
F
K
E
A
L
R
Y
474
ECO-URF
L
T
N
G
T
K
V
0
.
M
K
P
L
R
M
N
H
W
L
M
I
S
I
C
156
Eni-QutB
L
E
M
C
Y
H
P
L
P
w
T
D
I
A
Q
I
A
Q
D
A
R
W
K
V
I
L
G
S
E
A
L
I
w
Q G
L
E
Q
A
R
V
W
T
G
K
D
V
303
Ncr-Qa-3
L
E
M
C
Y
N
P
S
P
F
T
E
L
G
A
L
A
E
H
E
G
w
Q
V
I
L
G
T
E
A
L
I
w
Q G
I
E
Q
V
C
I
R
P
E
L
T
L
314
Ncr-Qa-lS
L
E
L
D
Y
K
C
L
T
S
P
L
L
E
Q
T
R
R
E
A
H
R
G
w
V
A
M
D
G
L
D
L
L
P
E
Q G
F
A
Q
F
E
L
F
T
G
R
R
A
881
Eni-QutR
V
D
L
A
Y
K
P
L
N
T
P
L
M
R
0
I
R
S
L
S
H
R
G
w
A
A
L
D
G
L
D
V
L
P
E
Q G
F
A
Q
F
E
L
F
T
G
C
R
A
862
Sce-Arol
L
E
A
A
Y
K
P
S
V
T
P
V
M
T
I
S
Q
D
K
Y
0
w
H
V
V
P
G
S
Q
M
L
V
H
Q G
V
A
Q
F
E
K
W
T
G
F
K
G
1576
Eni-AroM
L
E
M
A
Y
K
P
Q
V
T
A
L
M
R
L
A
s
D
S
G
w
K
T
I
P
G
L
E
V
[lJ
V
G
q5]
W
Y
LaJ
V
C
F
L
A
1567
Eco-AroE
r*l
D
M
F
Y
Q
K
G
K
T
P
F
L
A
W
C
E
Q
R
G
S
K
R
N
A
D
G
L
G
[SI
L
V
A
qÍ
A
H
A
F
L
L
w
H
'ol
V
L
P
257
Nta-AroD*E
Y
s
L
V
F
D
A
V
Y
T
P
E
I
T
R
L
L
R
B
A
H
E
S
G
V
K
I
V
T
G
V
*
«
X
I
G
0 *
E
Q
Y
E
R
F
T
G
«■
A
S
524
ECO-URF
1¿I
I
R
D
F
H
S
L
K
L
A
C
I
T
R
I
172
Eni-QutB
V
S
E P G
L
V
B
K
V
Q
A
P
V
A
Q T
I A
B
R
S K S
N
L . .
Ncr-Qa-3
Y
c
G G Q
L
N
Ncr-Qa-lS
P
R
R L M
R
R
B
V
L
R
A
Y
P
D
D Q
A K
S
H
T A Q
• L Q
P R
L N G
I A T
Q I S
918
Eni-QutR
P
R
R L M
R
T
V
I
L
Q
E
Y
K
E
E B
Q G
B
B
Y D Q
S
AMR
T R
L E N
L D G
Q P M
901
Sce-Arol
P
F
K A .
I
F
D
A
V
T
K
E .

Fig. 7-9. Dendrogram of shikimate dehyrogenase
homologous. The dendrogram is based on the multiple amino
acid alignment and includes the nine proteins defined in the
legend of Fig.7-8.

143
E. nidulans QutB
N. crassa Qa-3
N. crassa Qa-lS
E. nidulans QutR
S. cerivisiae Arol
E. nidulans AroM
E. coli AroE
N. Cabacum AroD^E
E. coli URF

144
catabolic dehydroquinases. Table 7-2 shows each pairwise
comparison of N. tabacum AroD*E with its homologues (Gap
program).
Table 7-2. Pairwise comparisons of S-protein with its
homologues.
Organism
and Gene
Percent
DQT
Identity Similarity
SDH
Identity Similarity
Sce-Arol
29
53
28
56
Eni-AroM
24
52
27
51
Eco-AroD
30
52
-
-
Eco-AroE
-
-
31
55
Sty-AroD
29
51
-
-
Efa-AroD
34
54
-
-
Bsu-AroD
32
53
-
-
Ncr-Qa-lS
22
44
30
53
Eni-QutR
21
46
26
47
Eni-QutB
-
-
28
53
Ncr-Qa-3
-
-
26
50
Eco-URF
-
-
36
61
StU-MDH
-
-
23
51
Nta-CDH
—
“
21
47
NAD(P)H-binding proteins often share a motif (Rossman
fold) dominated by glycine or other residues with small
sidechains such as alanine (72). Table 7-3 shows a
comparison of this region in the organisms compared in this
dissertation with a number of others recovered by the GCG
Blast program. The S-protein fits the NADPH binding motif
perfectly with GxGxxAxxxA (72).
A survey of some chloroplast transit peptides

145
Table 7-3.
NAD(P)H Binding Motif of Enzymes3.
Conserved Region
Organism cDNA or Gene Product
GxGxxGG
GxGxxVxxxA Accession
GxGxxAxxxA Number
E. nidulans
N. crassa
E. nidulans
N. crassa
E. nidulans
S. cerevisiae
E. coli
E. coli
N. tabacum
Quinate repressor (QutR)
Quinate repressor (Qa-lS)
Quinate dehydrogenase (QutB)
Quinate dehydrogenase (Qa-3)
Shikimate dehydrogenase (AroM)
Shikimate dehydrogenase (Arol)
Shikimate dehydrogenase (AroE)
unidentified reading frame
S-protein (AroD»E)
S TTGL11GAGGMARAGIYAM M59935
TSTGLVIGAGGMARAAVYAM P11637
GKPALIVGGGGTARTAIYVL P25415
SRPALVIGGGGAARSAVYAL P11635
EQSALWGGGGTARAALYAL P07547
HTAGLVIGAGGTSRAALYAL P08566
GLRILLIGAGGASRGVLLPL P15770
GKTMVLLGAGGAS TAIGAQG P28244
GKLFWIGAGGAGKALAYGA
S. tuberosum
N. tabacum
N. tabacum
H. vulgare
T. repens
Malate dehydrogenase KMKIWAGAGSAGIGVLNAA
Cinnamyl-Alcohol dehydrogenase1 GFRGGILGLGGVGHMGVKIA
Cinnamyl-Alcohol dehydrogenase2 GLRGGILGLGGVGHMGVKIA
Alcohol dehydrogenasel GSTVAIFGLGAVGLAAAEGA
Alcohol dehydrogenasel GSSVAIFGLGAVGLAAAEGA
Z23023
P30360
P30359
P05336
P13603
C. roseus S-Adenosylhomocysteinase
T. aestivum S-Adenosylhomocysteinase
N. silvestris S-Adenosylhomocysteinase
GKVAWAGYGDVGKGC AAAL P3 5 0 0 7
GKVAWCGYGDVGKGCAAAL P32112
GKVALVAGYGDVGKGCAAAL D16138
H. sapiens
H. sapiens
M. musculus
Alcohol dehydrogenase gamma
Glutathione reductase
Lactate dehydrogenase
GSTCAVFGLGGVGLSWMGC P00326
SYDYLVIGGGSGGLASARRA S08979
NNKITWGVGQVGMACAISI P16125
P. stipitis
S. cerevisiae
K. marxianus
C. synechocystis
D-Xylulose reductase
Succinate dehydrogenase
Alcohol dehydrogenasel
Ketolate reductoisomerase
GDYVAVFGAGPVGLLAAAVA P22144
WIGAGGAGLRAAFGL Q00711
TKLPLVGGHEGAGVWAMGE P20369
GKTVAIIGYGSQGHAHALNL A47037
E. coli
E. coli
T. brucei
T. brockii
P. putida
Threonine dehydrogenase
Glutamate synthase
Phosphogluconate DH
NADH oxidase
Formaldehyde DH
GEDVLVSGAGPIGIMAAAVA P07913
GKKVAIIGAGPAGLACADVL B29617
WGLGVMGANLALNI P31072
KKWWGGGPAGMQAAITA P32382
GSTVYVAGAGPVGLAAAASA D21201
aMotifs were taken from Scrutton et al., 1990. The first nine set of
organisms are those used in the SDH multiple allignment of Fig. 7-8.

146
established in the literature is shown in Table 7-4. The
transit peptides vary from 34 to 88 amino acid residues.
The sequence context around the translation start (AUG) is
proposed to most often be AACAAUGGC in plant genes (54) .
Included in this survey are the seven known sequences for
aromatic pathway proteins. From this selection in Table 7-
4, only one of sixteen, chorismate synthase in barley
(aromatic pathway protein) has this proposed conserved
region. The cleavage site often exhibits the pattern (V/I)-
X-(A/C)¿A, with R frequently at position -2 or between -6 to
-10 (33). Of the seven aromatic pathway proteins, shikimate
kinase in tomato conforms to the motif before the cleavage
point and five of the remaining nine sequences conform to
the cleavage point (two of these also have the proposed ALA
after the cleavage point). The S-protein does not fit this
cleavage site motif. The overall features of transit
peptides, show that they are rich in hydroxylated amino
acids, SER and THR (20-35%), small hydrophobic amino acids
such as VAL and ALA, not especially rich in basic amino
acids (but do have a net positive charge), and usually have
only one or two acidic groups (45). The aromatic pathway
protein transit peptides follow these features, being
especially rich in SER. The S-protein truncated transit
peptide (23 residues) has 17% SER (3) and THR (1), 35%
hydrophobic residues, 0.09% basic residues {R (ARG) and K
(LYS) near the cleavage point} and 13% acidic residues.

147
Table 7-4. Transit Peptides of Aromatic Pathway proteins
and of other Plant Proteins.
Amino
Enzyme Start Acid Mature
Plant Transit Peptide > Number Protein
DAHPsyn
agcaatggc
potato
MALSSTSTTNSLLPNRSLVQNQPLLPSPLKNAFFSNNSTKTVRFVQPISAVHSSDSNKIPIVSDKPSKSSPPAA
74
TATTAP
EPSPsyn
agaaatggc
tomato
MAQISSMAQGIQTLSLNSSNLSKTQKGPLVSNSLFFGSKKLTQISAKSLGVFKKDSVLRWRKSSFRISASVATAE
76
KPHEIV
EPSPsyn
aagaatggc
petunia
MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLLNSANSMLVLKKDSIFMQKFCSFRISASVATAQ
72
KPSEIV
EPSPsyn
tccgatggc
cruc. weed MAQVSRICNGVQNPSLISNKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIGSELRPLKVMSSVSTAE
74
KASEIV
CHAsyn
aacaatggc
barley
MASSLSTKPFLSGSRRRSTTDGSGWSYFQTSDLRQLSNQSVQISVRRQTAPLKLWQASG
57
ASGSSF
SHKkin
gactatgga
tomato
MEARVSQSLQLSSWINSDKWRKPSGLLRFSEKWNEKPRHRVWSCHLQPRKAAHSDRRVQLKVSC
65
SPQNVQ
TRPsyn
agtaatggc
cruc. weed MAASGTSATFRASVSSAPSSSSQLTHLKSPFKAVKYTPLPSSRSKSSSFSVSCTIAKDPPVLMAAGSDPALW
72
QRPDSF.
AS Pat
aaacatggc
soybean
MASSFLSAASHAVSPSCSLSTTHKGKPMLGGNTLRFHKGPWSFSSSRSRGRISMAVAVNV
60
SRFEGI.
GSAat
catcatggc
barley
MAGAAAAVASGISIRPVAAPKISRAPRSRSWRA
34
AVSIDE.
L12ribp
tacaatggc
tobacco
MASTLSTITLRSPSPSTASSTHASIPFPKKALEFPIRTPKLHHRRAT
53
FLRPLA.
IPMdh
tgaaatggc
rape
MAAALQTNIRPVKFPATLRALTKQSSPAPFRVRC
33
AAASPG.
PCPoxre
agtaatggc
pea
MALQTASMLPASFSIPKEGKIGASLKDSTLFGVSSLSDSLKGDFTSSALRCKRELRQKVGAVRA
64
ETAAPA.
FEDred
cgccatggc
spinach
MTTAVTAAVSFPSTKTTSLSARSSSVISPALISYKKVPLYYRNVSATGKMGPIRA
55
QIASDV.
FBPald
taagatggc
spinach
MASASLLKTSPVLDNPGFLKGQTLRIPSVAGVRFTPSGSSSLTVRA
46
SSYADG.
CARanhy
accaatgtc
pea
MSTSSINGFSLSSLSPAKTSTKRTTLRPFVFASLNTSSSSSSSSTFPSLIQDKPVFASSS
60
PIITPV.
G3Pacyt
gggatgac
pea
MTDSFAHCASHINYRHKMKTMFIFSTPCCSPSTAFFSPFRASNSKPLRSTLSLRSSISSSSITSTSHCSLAFNI
IVKHKE KNWSANMT
88
SSVSSR.
S-PROT
not known
tobacco
PFLCSTSWLLMELWDSGVRKME
XX
GEAMRR.

148
SUMMARY
In conclusion, with the cloning of the aroD»aroE cDNA
encoding the S-protein (the first to be cloned and sequenced
in higher plants), other studies may now be undertaken.
First of all, a complete sequence is desirable, since it is
proposed that the 5' region has been truncated. This may be
accomplished by obtaining mRNA from Nicotiana tabacum plants
for primer extension. Other studies may include cloning of
the gene from a genomic library, determining the number of
gene copies in Silvestris or in other plant species by use
of Southern blots, performing site directed mutagenesis or
chemical treatment to study the active site residues, use of
the cloned cDNA coding for the S-protein as a probe to
obtain the cDNA from other plants and to determine what the
cytosolic pathway has with respect to dehydroquinase and
shikimate dehydrogenase. The probable location of the
bifunctional gene product in the chloroplast remains to be
proven. Clarification of the relationship of SP-I and SP-II
is needed. The possibilities are: (i) that one is an
artifact of limited proteolysis, (ii) that one is an
active, uncleaved preprotein in the cytosol, (iii) or that
they are separately subcompartmented in the chloroplast.

149
REFERENCES
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157
BIOGRAPHICAL SKETCH
Carol A. Bonner graduated with a New York State High
School Equivalency Diploma in 1967. A B.S. in biochemistry
was undertaken in the Department of Biology at State
University of New York at Binghamton and was completed in
May of 1982. Education was continued from 1982 to 1985,
whereupon an M.S. degree was completed in biological
sciences in the Department of Biology at the State
University of New York at Binghamton under the supervision
of Professor Roy A. Jensen.
At this time, the author accepted a position as Bio
Science Manager at the University of Florida in the
Department of Microbiology and Cell Science. A
continuation of education to receive a Ph.D. degree in the
Department of Microbiology and Cell Science under the
supervision of Professor Roy A. Jensen was begun in the fall
semester of 1989 and completed in April of 1994.

I certify that I have read this study and in my opinion
it conforms to acceptable standards of scholarly presenta¬
tion and is fully adequate, in scope and quality, as a dis¬
sertation for the degree of Doctor of Philosophy.
^
Roy A.<|Jensán, Chair
Professor of Microbiology and Cell Science
I certify that I have read this study and in my opinion
it conforms to acceptable standards of scholarly presenta¬
tion and is fully adequate, in scope and quality, as a dis¬
sertation for the degree of Doctor of Philosophy.
Lonnie 0. Ingram (j
Professor of Microbiology and Cell Science
I certify that I have read this study and in my opinion
it conforms to acceptable standards of scholarly presenta¬
tion and is fully adequate, in scope and quality, as a dis¬
sertation for the degree of Doctor of Philosophy.
Robfert R.’ Schmidt
Graduate Research Professor of
Microbiology and Cell Science

I certify that I have read this study and in my opinion
it conforms to acceptable standards of scholarly presenta¬
tion and is fully adequate, in scope and quality, as a dis¬
sertation for the degree of Doctor of Philosophy.
-oZtWxx • T.
%-
Keelnatham T. Shanmugam
Professor of Microbiology and Cell Science
I certify that I have read this study and in my opinion
it conforms to acceptable standards of scholarly presenta¬
tion and is fully adequate, in scope and quality, as a dis¬
sertation for the degree of Doctor ,-a£ Philosophy.
Richard P. Boyce
Professor of Biochemistry and Molecular
Biology
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
Dean, Graduate School

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UNIVERSITY OF FLORIDA
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