Group Title: Genome biology
Title: Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants
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Title: Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants
Physical Description: Book
Language: English
Creator: Xie, Gary
Forst, Christian
Bonner, Carol
Jensen, Roy
Publisher: Genome biology
Publication Date: 2001
 Notes
Abstract: BACKGROUND:Tryptophan synthase consists of two subunits, a and ß. Two distinct subgroups of ß chain exist. The major group (TrpEb_1) includes the well-studied ß chain of Salmonella typhimurium. The minor group of ß chain (TrpEb_2) is most frequently found in the Archaea. Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies.RESULTS:Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the a chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2. In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes.CONCLUSIONS:TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional ß chain, as TrpEb_1 is absent. The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1. We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase ß chains. A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.
General Note: Periodical Abbreviation:Genome Biol.
General Note: Start page research 0004.1
General Note: Other pages research 0004.13
General Note: M3: 10.1186/gb-2001-3-1-research0004
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http://genomebiology.com/2001/3/1/research/0004

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Research

Significance of two distinct types of tryptophan synthase beta

chain in Bacteria, Archaea and higher plants

Gary Xie*t, Christian Forst*, Carol Bonner' and Roy A Jensen**

Addresses: *BioScience Division, Los Alamos National Laboratory, Los Alamos, NM 87544, USA. 'Department of Microbiology and Cell
Science, University of Florida, Gainesville, FL 32611, USA. *Department of Chemistry, City College of New York, New York, NY 10031, USA.

Correspondence: Roy A Jensen. E-mail: rjensen@ufl.edu


Published: 14 December 2001
Genome Biology 2001, 3 (I):research0004.1-0004.13
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2001/3/I/research/0004
2001 Xie et al., licensee BioMed Central Ltd
(Print ISSN 1465-6906; Online ISSN 1465-6914)



Abstract


Received: 24 September 2001
Revised: 30 October 2001
Accepted: 30 October 2001


Background: Tryptophan synthase consists of two subunits, a and p. Two distinct subgroups of
P3 chain exist. The major group (TrpEb_l) includes the well-studied P3 chain of Salmonella
typhimurium. The minor group of P3 chain (TrpEb_2) is most frequently found in the Archaea.
Most of the amino-acid residues important for catalysis are highly conserved between both TrpE
subfamilies.

Results: Conserved amino-acid residues of TrpEb_l that make allosteric contact with the TrpEa
subunit (the a chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher
plants all exist that possess both TrpEb_l and TrpEb_2. In those prokaryotes where two trpEb
genes coexist, one is usually trpEb_l and is adjacent to trpEa, whereas the second is trpEb_2 and
is usually unlinked with other tryptophan-pathway genes.

Conclusions: TrpEb_l is nearly always partnered with TrpEa in the tryptophan synthase
reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the
functional P3 chain, as TrpEb_l is absent. The six lineages show a distinctive divergence within the
overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in
TrpEa that are otherwise conserved for interfacing with TrpEb_l. We suggest that the stand-
alone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established
catalytic capability of tryptophan synthase P3 chains. A coincident finding of interest is that the
Archaea seem to use the citramalate pathway, rather than threonine deaminase (llvA), to initiate
the pathway of isoleucine biosynthesis.


Background system for many years and continues to receive attention. In
Tryptophan biosynthesis is absent in mammals but is a the current genomics era, emerging surprises reveal there is
general metabolic capability of prokaryotes, eukaryotic still much to learn. These surprises include the revelations
microorganisms and higher plants. It has been a classic that operons are being organizationally reshuffled, invaded
system for elucidation of gene-enzyme relationships and reg- by insertion of apparently unrelated genes, disrupted by
ulation, thanks largely to the lifelong efforts of Charles either partial or complete dispersal of genes to extra-operon
Yanofsky [1]. Tryptophan is biochemically the most expen- locations, or complicated by the seemingly unnecessary
sive of the amino acids to synthesize [2]. The clustered orga- presence of additional operon-gene copies located outside of
nization of the trp genes into an operon has been a model the operon.








2 Genome Biology Vol 3 No I Xie et al.


Tryptophan synthase, catalyzing the final step of tryptophan
biosynthesis, is one of the most rigorously documented
examples of an enzyme complex [3-6]. It consists of an
a subunit, which cleaves indoleglycerol phosphate to indole
and glyceraldehyde 3-phosphate, and a p subunit, which
condenses indole and L-serine to yield L-tryptophan. The
appa complex forms a tunnel into which enzymatically gen-
erated indole is released. The a monomers and P dimers
contact one another via highly sophisticated mechanisms of
allostery [7], and it is little wonder that the genes encoding
these two subunits are almost always closely linked, fre-
quently being translationally coupled.

Against this background, it seems curious that a significant
number of organisms possess more than one gene encoding
the p chain of tryptophan synthase. Usually, but not always,
the 'extra' gene is unlinked to the gene encoding the a chain,
and it also defines a distinct subgroup of the p chain. This
has been recognized in the COGs database [8] as "alternative
tryptophan synthase" (COG 1350). In this paper we present a
detailed analysis of the distribution of the two subgroups of
the p chain in prokaryotes within the context of the surpris-
ingly dynamic past and ongoing alterations of organization
for genes responsible for tryptophan biosynthesis.

Nomenclature
In recent publications involving bioinformatic analysis of aro-
matic amino-acid biosynthesis [9-14], we have implemented a
nomenclature that attempts logical and consistent naming of
genes and their gene products for different organisms. The
problem is exemplified by the contemporary naming in two
model organisms of 3-deoxy-D-arabino-heptulosonate 7-phos-
phate (DAHP) synthase, the initial enzyme step of aromatic
amino-acid biosynthesis. In Bacillus subtilis the gene, appro-
priately enough, has been named aroA, but in Escherichia coli
the equivalent function is represented by genes encoding three
differentially regulated paralogs. These E. coli genes have been
named aroF, aroG and aroH. In B. subtilis these latter three
gene designations refer to 5-enolpyruvylshikimate-3-phos-
phate synthase (step 6 of chorismate synthesis), chorismate
synthase (step 7) and chorismate mutase (initial step of pheny-
lalanine and tyrosine biosynthesis). Even in B. subtilis, where
the naming was intended to follow an orderly progression in
terms of order of reaction steps, there is the complication that
DAHP synthase is expressed as a fusion of two catalytic
domains, one being a class of chorismate mutase called AroQ.
This requires naming at the level of domain and the desig-
nation aroQ.aroA was implemented to denote such a
fusion [9-12]. Thus, a single enzymatic function in one
organism is accommodated through the cumulative expres-
sion of three paralog genes, but in another organism is only
encoded by a portion of a single gene. A universal nomen-
clature is needed that labels at the level of domain, that
labels in synchrony with order of reaction steps as much as
possible, and that labels isofunctional paralogs at the same
hierarchical level but with discriminating identifiers.


The status of nomenclature for the tryptophan pathway is
not so chaotic as the genes in almost all prokaryotes have
been named in line with E. coli, but even here distinct prob-
lems have arisen because gene fusions that exist in E. coli are
often absent elsewhere. Much of the literature up to at least
1996 used five gene designations for the seven protein
domains [15]. In B. subtilis these seven protein domains are
encoded by separate genes [16]. Thus, E. coli trpD encodes
the equivalent of B. subtilis TrpG and TrpD. The E. coli gene
fusion has been re-designated with the convention of a bullet
to represent a fusion: trpG.trpD [17]. Likewise, the E. coli
trpC encodes the equivalent of B. subtilis TrpC and TrpF,
and the E. coli gene fusion has been re-designated
trpCotrpF. The nomenclature we advocate is more easily
remembered because genes are named in the order of
pathway reaction. Subunits for a given reaction are named at
the same hierarchical level: anthranilate synthase (first reac-
tion) consists of TrpAa and TrpAb, and tryptophan synthase
(fifth reaction) consists of TrpEa and TrpEb. The implemen-
tation of a logical and consistent nomenclature, as illustrated
in Figure 1, should be most helpful in the long term.


Results and discussion
Phylogenetic tree construction
Initial amino-acid alignments were generated using
ClustalW software, version 1.4 [18]. Manual adjustments
were made through visual inspection to bring conserved
motifs and residues into register. This was implemented by
use of the BioEdit multiple alignment tool [19]. Inferences
about the evolutionary relationships within the TrpEa and
TrpEb protein families were made using the PHYLIP
package of programs [20]. The Protpars program was used
to generate a maximum parsimony tree, and the neighbor-
joining and Fitch programs were used to generate a dis-
tance-based tree. The distance matrix used in the latter
programs was produced using the program Protdist with a
Dayoff PAM matrix. The Seqboot and Consense programs
were then used to assess the statistical strength of the tree
using bootstrap resampling. Neighbor-joining (PHYLIP),
Fitch and Margolash (Fitch in PHYLIP), and maximum par-
simony methods [21] all produced trees consistent with one
another. Despite low bootstrap values at many individual
internal nodes, the clusters formed and arrangement of taxa
within them were largely identical. Ninety TrpEb and 63
TrpEa sequences were analyzed.

Distinctly different types of TrpEb
TrpEb proteins divide into two distinctly different groups, as
illustrated by the unrooted tree shown in Figure 2. The major
group, denoted TrpEb_1, includes the well-studied enzymes
from such organisms as E. coli, Salmonella typhimurium,
and B. subtilis. The minor group, denoted TrpEb_2, is repre-
sented heavily, but not exclusively, by archaeal proteins.
Among the current inventory of completed archaeal genomes,
only Methanococcus jannaschii lacks TrpEb 2. Seven








http://genomebiology.com/200 1/3/ I /research/0004.3


coo-

'COO-
OH
Chorismate


o00


NH2ranilate
Anthranilate


PRPP PR


PTrpB
[TrpD]


Ser G3P CH2 O 002 + H20
nCH2Coo- PLPC SLCH- OH- L
aL _II? H PI l U
I NH3+ TrpEb N TrpEa N OH OH TrpD
H H [TrpA] H [TrpC]


Indole


Indole 3-glycerol
phosphate


-00C
- -OH2 C 0 N H


OH OH
N-(5'-Phosphoribosyl)
-anthranilate



TrpC
[TrpF]


COO- OH OH
I I
HO C CH CH
C"NH-CH CH20-
1 -(o-Carboxyphenylamino)
-1-deoxyribulose
5-phosphate


Tryptophan synthase


Figure I
Biochemical pathway of tryptophan biosynthesis. Acronyms that are currently used for the seven monofunctional proteins of Bacillus subtilis are shown in
brackets below the acronyms used in this paper. TrpAa, large aminase subunit of anthranilate synthase; TrpAb, small glutamine-binding subunit of
anthranilate synthase; TrpB, anthranilate phosphoribosyl transferase; TrpC, phosphoribosyl-anthranilate isomerase; TrpD, indoleglycerol phosphate
synthase; TrpEa, a subunit of tryptophan synthase; TrpEb, P subunit of tryptophan synthase.


archaeal genomes possess both TrpEb_1 and TrpEb_2
(Methanosarcina barker possesses two paralogs of TrpEb_1
in addition to one species of TrpEb_2). Six archaeal genomes
possess TrpEb_2, but not TrpEb 1.

Bacterial TrpEb_2 proteins are thus far limited to Aquifex,
Thermotoga, Mycobacterium, Geobacter, Chlorobium and
Rhodopseudomonas genera. In addition, one of the multiple
TrpEb proteins present in the higher plant, Arabidopsis
thaliana, belongs to the TrpEb_2 subfamily. In view of the
distinct divergence of TrpEb_2 from TrpEb_1, one might
expect that either TrpEb_2 has lost the ability to interact
allosterically with TrpEa, or perhaps that a divergent sub-
group of TrpEa has coevolved with TrpEb_2. Multiple copies
of TrpEb are often present in genomes. Examples include


cases where two TrpEb_1 species coexist, where two
TrpEb_2 species coexist, or where TrpEb_1 and TrpEb_2
coexist in the same organism. A number of organisms
(M. barker, Rhodobacter capsulatus, Chlamydia psittaci,
and Corynebacterium diphtheriae) possess two copies of
TrpEb_1. In each case the trpEb 1 copy that is linked to
trpEa is highly conserved, whereas the remaining copy has
diverged to the extent that it may be a pseudogene (Table 2).
The TrpEb_1 products of these probable pseudogenes have
elongated branches (highlighted yellow) on the protein tree
shown in Figure 2.

It is noteworthy that cyanobacterial and higher plant amino-
acid sequences form a cohesive cluster for TrpEb_1,
as shown in phylogram form in Figure 3 (left panel). An


Figure 2 (see figure on the next page)
Unrooted phylogenetic tree (radial view) of the TrpEb protein family consisting of subfamily TrpEb_l (top) and TrpEb_2 (bottom). Phylogenetic
reconstruction of the inferred amino-acid sequence was accomplished by the neighbor-joining method using the PHYLIP program. Organismal acronyms
are defined in Table I. For economy of space, a single branch is used to represent proteins that have diverged very recently, for example, TrpEb_l
proteins from E. coli and S. typhimurium (Eco/Sty). Archaeal proteins are highlighted in magenta. The detailed order of branching for TrpEb_l proteins in
cyanobacteria and higher plants is shown in Figure 3. Probable pseudogenes are shown in yellow.


L-Tryptophan








4 Genome Biology Vol 3 No I Xie et al.


Cyanobacteria/
higher plantsJ
Cte-1/Det Sau
MIo/Ccr
Rca-1/Rpa-1 Smu/Lla
Ngo/Nme \ \ \ / /Sp Dra
Bpe/Neu \ \Pae\ \ \ /


Cdip-2
Hin

Yp,
E,


















Paer



Tac; T,,o






Sso-2




0.1


Paero-2 Ape-2 Pfu-2"
Paero-2 Pab-2


Figure 2 (see legend on the previous page)









http://genomebiology.com/2001/3/ I/research/0004.5


Key to sequence identifiers


NCBI GI number


NCBI GI number


Species name


Acronym trpEb- I


Species name

Actinobacillus
actinomycetemcomitans
Aeropyrum pernix
Aeropyrum pernix
Anabaena sp.
Anabaena sp.
Aquifex aeolicus
Aquifex aeolicus
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Archaeoglobus fulgidus
Archaeoglobus fulgidus
Bacillus halodurans
Bacillus stearothermophilus
Bacillus subtilis
Bordetella pertussis
Buchnera sp. APS
Campylobacter jejuni
Caulobacter crescentus
Chlamydia trachomatis
Chlamydia psittaci
Chlamydia psittaci
Chlorobium tepidum
Chlorobium tepidum
Clostridium acetobutylicum
Corynebacterium diphtheriae
Corynebacterium diphtheriae
Dehalococcoides ethenogenes
Deinococcus radiodurans
Escherichia coli
Ferroplasma acidarmanus
Geobacter sulfurreducens
Geobacter sulfurreducens
Haemophilus influenzae
Halobacterium sp.
Helicobacter pylori
Klebsiella pneumoniae
Lactococcus lactis
Legionella pneumophila
Mesorhizobium loti
Methanobacterium
thermoautotrophicum
Methanobacterium
thermoautotrophicum
Methanococcus jannaschii
Methanosarcina barkeri
Methanosarcina barkeri
Methanosarcina barkeri
Mycobacterium bovis
Mycobacterium leprae
Mycobacterium smegmatis
Mycobacterium smegmatis
Mycobacterium tuberculosis
Neisseria gonorrhoeae
Neisseria meningitides
Nitrosomonas europaea
Nostoc punctiforme
Nostoc punctiforme
Pasteurella multocida


Acronym trpEb- I

Aac N/A


Ape- I
Ape-2
Asp- I
Asp-2
Aae- I
Aae-2
Ath- I
Ath-2
Ath-3
Afu- I
Afu-2
Bha
Bst
Bsu
Bpe
Bsp
Cje
Ccr
Ctr
Cps-I
Cps-2
Cte- I
Cte-2
Cac
Cdip-I
Cdip-2
Det
Dra
Eco
Fac
Gsu-I
Gsu-2
Hin
Hal
Hpy
Kpn
Lla
Lpn
MIo
Mth- I

Mth-2

Mja
Mba- I
Mba-2
Mba-3
Mbo
MIe
Msm-I
Msm-2
Mtu
Ngo
Nme
Neu
Npu-I
Npu-2
Pmu


N/A
N/A
6226273

136251
1174779

3334387

10174280
226585
136270
N/A
11182450
11269304
136272
6226274
N/A
N/A
N/A

N/A
N/A
N/A
N/A
7474051
136273

N/A

1174785
14423973
7674399
N/A
267168
N/A
13474230
3334383



2501412
N/A
N/A

N/A
13093205
N/A

3024761
N/A
11269306
N/A
N/A
N/A
13432266


trpEb-2 Prochlorococcus marinus
Pseudomonas aeruginosa
Pyrobaculum aerophilum
Pyrobaculum aerophilum
7674393 Pyrococcus abyssi
7674395 Pyrococcus abyssi
Pyrococcus furious
Pyrococcus furious
Pyrococcus horikoshii
7674374 Rhodobacter capsulatus
Rhodobacter capsulatus
Rhodopseudomonas palustris
10176821 Rhodopseudomonas palustris
Salmonella typhimurium
7674372 Staphylococcus aureus
Streptococcus mutans
Streptococcus pneumoniae
Streptomyces coelicolor
Sulfolobus solfataricus
Sulfolobus solfataricus
Synechococcus sp.
Synechocystis sp.
Thermomonospora fusca
Thermoplasma acidophilum
Thermoplasma volcanium
Thermotoga maritima
N/A Thermotoga maritima
Vibrio cholera
Xylella fastidiosa
Yersinia pestis
Yersinia pseudotuberculosis
Zea mays
Zea mays


Pma
Paeru
Paero- I
Paero-2
Pab- I
Pab-2
Pfu- I
Pfu-2
Pho
Rca- I
Rca-2
Rpa- I
Rpa-2
Sty
Sau
Smu
Spn
Sco
Sso-I
Sso-2
Syn
Ssp
Tfu
Tac
Tvo
Tma- I
Tma-2
Vch
Xfa
Ype
Yps
Zma-I
Zma-2


N/A
12230946


14520675


N/A


N/A
N/A
N/A

136281
13701169
N/A
N/A
6226276


N/A
2501413
N/A


1717761

11269279
11269281
N/A
N/A
1174780
1174778


14423981

N/A
14591361



N/A






14424473
13814334



13878841
13541762

7674388


analysis of TrpEa proteins from the same organisms yields a
very similar phylogram output (Figure 3, right panel). This
higher plant/cyanobacteria relationship is pleasingly consis-
tent with the endosymbiotic hypothesis of organelle evolu-
tion. In each case Prochlorococcus marinus and
Synechococcus species are the outlying sequence group, with
the other cyanobacterial sequences (Nostoc punctiforme and
Anabaena species) being closer to the higher plant
sequences from A. thaliana and corn (Zea mays). The order
of branching shown is supported by very high bootstrap
values. Zma-3 is the TrpEa protein that has been proposed
[22] to function independently of a TrpEb partner, produc-
ing indole for entry into a pathway other than tryptophan. In
this case indole serves as a precursor for a defense metabo-
lite that is active against insects, bacteria and fungi.


TrpEa in organisms lacking TrpEb_ I
Six organisms (all Archaea) possess intact tryptophan path-
ways, but they lack TrpEb_1. TrpEb in Thermoplasma vol-
canii, T. acidophilum, and Ferroplasma acidarmanus is
represented only by a single species of TrpEb 2. Although
Sulfolobus solfataricus, Aeropyrum pernix and Pyrobaculum


Table I


Table I (continued)


trpEb-2








6 Genome Biology Vol 3 No I Xie et al.


Exceptions to residue invariance


Invariant
Protein residue*

TrpEa 57P
60D
61G
641
183T
184G
21 IG
212F/L
213G
234G
TrpEb 10IOG
21L
41F
55 R
78E
79D
80L
82H
94Q
96L
97L
102G
114Q
124A
133F/Y
141R
146V
149M
153G
156V
159V
162G
167K
173A
177W
186Y
2081
213R/K
224P
225 D
267H
269A
274G
299S
310G
3451
383D

Invariant
Protein residuet
TrpEb_2 PIO
Y14
E54
L86
E87
EI01
S108
Al 14
W138
G176


Invariant
Protein residuet Exceptions: organism (homologous residue)


Exceptions: organism (homologous residue)

Sso (A)
Hal (E)
Ctr (N)
Mth (V)
Ctr (R)
Hin (S)
Ctr (R)
Ctr (R)
Ctr (D) Paero (A) Ape (S)
Ctr (K)
Ctr (H) Cps-2 (E) Cps-1 (Y)
Cje (A)
Asp-I (Y)
Mth-1 (K)
Ccr (D)
Rca-I (E) Paeru (E)
Mth-1 (M) Dra (Q)
Mba-2 (Q) Dra (F)
Rca-2 (E)
Rpa-I (M) Spn (W) Bsp (M)
Cps-2 (I)
Bsp (K)
Rpa- I (M) Paeru (M)
Zma-2 (R) Cps- I (G)
Rca-2 (H)
Rca-2 (K)
Mba-I (A)
Cps-2 (I)
Hal (D)
Cje (I)
Bha (A)
Cdip-2 (E)
Cdip-2 (S) Sau (S)
Hal (T) Rca-2 (C)
Cps-2 (F) Rca-2 (Y)
Cps-2 (F) Rca-2 (F)
Xfa (V)
Bpe (L)
Cje (V)
Bha (T) Rca-2 (A)
Rca-2 (N)
Sau (L) Mba- I (S)
Aac (A)
Rca-2 (T)
Mba-2 (S)
Rca-2 (V)
Yps (E)



Exceptions: organism (homologous residue)
Paero-I (gap)
Aae-2 (L)
Sso- I (Q)
Msm-2 (F)
Paero-2 (D)
Paero-2 (Q)
Paero-2 (N)
Mba-3 (S)
Sso-1 (R)
Fac (N)


S184
S203
1206
A207
S209
E210
G226
N265
P272
G300
F/Y325
F37 I
P379
S410


Sso-I (T)
Fac (T)
Msm-2 (M)
Fac (G)
Ape-I (A)
Sso-2 (D)
Fac (A)
Paero-2 (S)
Mba-3 (E)
Cte-2 (A)
Mba-3 (H)
Ape-I (M)
Fac (A)
Ath-3 (C)


*Residues are numbered according to the S. typhimurium sequence.
tResidues are numbered according to the P. furious sequence.


aerophilum all possess two species of TrpEb, both are the
TrpEb_2 variety. Thus, in all six of these lineages TrpEa
either might be unable to form a tight complex with TrpEb_2,
or might have evolved different protein-protein contacts. In
the latter case, distinct TrpEa subgroupings might be
expected in parallel with the two TrpEb subgroupings. On the
contrary, all TrpEa sequences fall into a single group
(Figure 4). However, in contrast to sequences present in
those Archaea that do possess TrpEb_1 (for example,
Archaeoglobus, species of Pyrococcus, Methanococcus,
Methanobacterium, Methanosarcina and Halobacterium),
the six archaeal lineages that possess only TrpEb_2 have very
distinctive elongated branches on the TrpEa tree (Figure 4).
This suggests an elevated rate of evolutionary divergence, due
either to selection for new productive contacts of TrpEa with
TrpEb_2 or to lack of constraint to maintain TrpEa residues
previously important for contacts with TrpEb 1.

The long branch of the TrpEa sequence of Chlamydia tra-
chomatis reflects its likely status as a pseudogene. This is
consistent with the observation that C. trachomatis TrpEb 1
(Figure 2) also seems to be a pseudogene. One does not
expect positive selection for maintenance of function in
C. trachomatis as it lacks an intact tryptophan pathway.
Indeed, the alteration in C. trachomatis of many otherwise
invariant amino-acid residues is evident from the informa-
tion given in Table 2.

Overview comparison of TrpEb_l and TrpEb_2
Figure 5 shows an alignment of the amino-acid sequence of
TrpEb_l1 from S. typhimurium with TrpEb_2 from P. furio-
sus. Each sequence is shown as a template for its own sub-
family, as extracted from a refined multiple alignment.
Conserved residues deduced from a full multiple alignment
(available from the author on request) are indicated, as are
the gap positions present in the full alignment. Functional
roles in catalysis and allosteric regulation are indicated for


Table 2


Table 2 (continued)








http://genomebiology.com/2001 /3/ I/research/0004.7


Figure 3
Unrooted phylogenetic tree (phylogram view) of cyanobacterial and higher plant TrpEb_l (zoom-in expansion from Figure 2) and TrpEa (zoom-in
expansion from Figure 4) protein sequences. The higher-plant lineage is shown in green. Bootstrap values (from 1,000 replications) supporting the order
of branching shown are given at the nodes.


the S. typhimurium TrpEb_1 sequence in order to compare
similarities and differences between TrpEb_1 and TrpEb_2
proteins. Residues that are ligands of pyridoxal phosphate or
that interact with pyridoxal phosphate are scattered
throughout the sequences, including the catalytic K87, and
are highly conserved. Residue Elo9 has been shown to
render indole more nucleophilic via proton abstraction from
N1 [23]. The serine substrate-binding region is highly con-
served, as is a monovalent cation (MVC) binding region [7]
coordinating with G232, F/Y3o6, and G/A/S3o8. A number
of indels (insertions/deletions) distinguish TrpEb_1 and
TrpEb_2, and TrpEb_2 is about 50 residues longer overall
than TrpEb_1. In addition to other residues conserved
between both TrpEb_1 and TrpEb_2, each subgroup has its
own repertoire of uniquely conserved residues. The COMM
domain [24], a rigid but mobile domain as originally defined
with S. typhimurium TrpEb_1 [25], differs from the corre-
sponding region of the TrpEb_2 subfamily by the presence
of an indel. Key TrpEb_1 regulatory residues (R141, K167)
within this region as well as one residue near the MVC site
(D305) are not conserved in the TrpEb_2 subgroup.

Loss of intersubunit contacts in TrpEa-TrpEb_2
systems
The tryptophan synthase of S. typhimurium is a rigorously
documented example of substrate channeling in which
indole generated as an intermediate is passed through an
internalized tunnel ([7] and references therein). Ligand
binding at the a-site and covalent transformations at the
P-site accomplish mutually reinforcing overall allostery. The
movable COMM domain is comprised of residues G102 to
G189 in S. typhimurium TrpEb_2. COMM interacts with
both the p-active site and with a-subunit loops 2 and 6 in
response to allosteric signals. Within the COMM domain of
TrpEb_1, S178 participates in intersubunit signaling with


G181 of TrpEa. Competing allosteric conformations are
mediated by alternative salt bridges between K167 of
TrpEb_1 and D305 of TrpEb_1 on the one hand, or between
K167 of TrpEb_1 and D56 of TrpEa, on the other. When
D305 of TrpEb_1 is not occupied with K167, it forms an
alternative salt bridge with R141, as shown in Figure 5.

As intersubunit signaling between TrpEb_2 and TrpEa is
either lacking or involves different contacts, one might
expect the important catalytic residues, but not the allosteric
residues, to be conserved in comparison of TrpEb_2 with
TrpEb_1. This comparison is shown in Figure 5. Likewise, in
those TrpEa proteins that lack a TrpEb_1 partner, instead
being forced to function in concert with TrpEb_2, one might
expect retention of catalytic residues but loss of allosteric
residues. This comparison is shown in Figure 6. Of the inter-
subunit signaling pair TrpEb_1 S178/TrpEa G181, 8178 is
not conserved within its own subfamily and equivalent
residues seem to exist [26]. It is, however, striking that G181
is invariant in all TrpEa proteins, except for those belonging
to the six archaeal lineages lacking TrpEb_1. It is even more
striking that K167, which participates in the alternative P3-
or p-a salt bridges is invariant in TrpEb_1, but absent in
TrpEb_2. Likewise, the salt-bridge partner D305 is invariant
in TrpEb_1, but absent in TrpEb_2. The salt-bridge partner
D56 in TrpEa is invariant except for the six archaeal lineages
that lack TrpEb_1. Figure 5 shows that TrpEb_2 sequences
carry an insertion of 16 residues in the general region corre-
sponding to the COMM domain of TrpEb_1 proteins.

Tryptophan gene organization in TrpEb_2-containing
genomes
The organization of tryptophan-pathway genes in the ten
archaeal genomes and six bacterial genomes that are thus far
known to possess TrpEb_2 are displayed on a 16S rRNA tree


'- Asp-1 999 Ath-1
1000 Ath-1 L At h-2
Ath-2 Zma-1
200 786 Zma-2
00 1000 Zma-2 786 1000 Zma-3
Zma-1

TrpEb_1 TrpEa
0.1








8 Genome Biology Vol 3 No I Xie et al.


Figure 4
Unrooted phylogenetic tree (radial view) of the TrpEa protein family. Organismal acronyms are defined in Table I. Phylogenetic reconstruction of the
inferred amino-acid sequence was accomplished by the neighbor-joining method using the PHYLIP program. The detailed order of branching for
cyanobacterial and higher-plant sequences is shown in Figure 3. Archaeal proteins are visualized in magenta, and the yellow line indicates the TrpEa
protein from C trachomatis, which is probably encoded by a pseudogene.


in Figure 7. Pyrococcus horikoshii has lost the entire
pathway, and only trpEb 2 is present. In Aquifex and
Chlorobium all trp genes are dispersed, and trpEa is not
linked to either trpEb 1 or trpEb 2. In Bacteria and
Archaea the gene order trpEbL -> trpEa is one of the most
highly conserved of genomic gene arrangements. Often
translational coupling exists, as seen in Figure 7 for P. furio-
sus, P. abyssi, M. thermoautotrophicum, T. volcanium,
Archaeoglobus fulgidus and T. maritima. In each of the
latter genomes trpEb 2 is outside the trp operon. Both


species of Thermoplasma possess an otherwise intact trp
operon that lacks trpEb 1 (also not present elsewhere in the
genome). Hence, by default it appears that the unlinked
trpEb 2 must function for tryptophan biosynthesis in these
organisms. In A. pernix and S. solfataricus all of the trp
genes are adjacent, but trpEb_2 flanks trpEa, and trpEb_ is
absent from these genomes. In each case, a second unlinked
copy of trpEb_2 is present. In Geobacter sulfurreducens,
both trpEa and trpEb_ (presumably partnered based on the
results shown in Figure 4) are unlinked to one another, and


Tvo


Paero
Ctr
Tac





Cte Cps

Dra Msm MIe
Fac Mtu/Mbo
Det Sco

Tfu


Gsu Xfa
Neu
Sso Nme/Ngo
a ad Paeru
Tma Rpa

Smu Aae
Spn Mba
Sau
Hal
Cje Bsu Ba Mth
0.1
Hpy
[cyanobacteria and
higher plants / / c dip
PfuI \ Yp E Bsp
Pab Afu \ Eco/Sty








http://genomebiology.com/200 1/3/ I /research/0004.9


F/Y Y/F
I I
Sty 1 MTTLLNPYFG- - EFGGMYVPQILMPA - - -LNQLEEAFVRAQKDPE ----- -FQAQFADLLKNYA TALTKCQNITAGTR ------- 70
Pfu-2 1 MKVVLPDGRIPRRWYNILPDLPEPLAPPLDPET- NEPVDPKKLERIFAKELVKQEMSTKRYIKIPEEVRKMYSKI G- TPLFRATNLEKYLNTP ------ 93


A/S Comm domain
\86 87 Q/N G/A 109 110 114 Y/F
_ nl-4 / / :: -_n ,< 141
Sty 71 ---TTLY LLH VLGQALLAKRMGKSEII SALASALLGLKCRIYMGAKDVERQSPNVFt EVIPVHS----- GS :163
Pfu-2 94 - -ARIY ATV LAQAYYAKKEGIERLVT ALSLAGALMGIKVRVYMARASYEQKPYRKV EVFPSPSENTEIGK 191

D-56 G-181 S/T-190F
167 178 -
Sty 164 A----------- K N RDWSGSYETAY GPHPYPTIVREFQRMIG ET9LDKEG-R AVIAC GGS IGMPADFINDTS-- 249
Pfu-2 192 R-FLSENPNHPG Lj S4AEDVLKDE-KAY LN---H---VLMHQTVIG K EEFEE--- DVIIGC GGS AGLA--YPFVKEVL 278
Salt bridge %
305 F/Y-306 G/A/S-308
G/A 303
Sty 250 --------------VGLIGVEPGGHGIETGE ------HGAPLKHGR--VGIYFGMKAPMMQTADGQIEESYSISAD GSPHAYLNSIGRADYVSIT 328
Pfu-2 279 DG -------- DNEYEFIAVEPKAAPSMTRG ------ VYT-YDFGDS-GELTPKLKMHTLGHR---YHVPPIHAGJ APH LSVLVNNGIVKPIAYH 359



E-350 377 378
Sty 329 DDEALEAFKTLCR 1EG[ I SSHAHALKMMREQPEK--EQLLVVNLS FTVHDILKARGEI 397
Pfu-2 360 QTEVFEAAALFA KIEG VP SA KATIDKAIEAKREGKEIVILFNL S3Lt HGYEEYLEGRLQDYEPKDLPISNPLNPKP 446
E/Qndole PLP Serine Metal



Figure 5
Alignment of TrpEb_l from S. typhimurium (Sty) and of TrpEb_2 from P. furious (Pfu-2). The sequences are shown as they appear in a comprehensive
alignment, including gaps. Invariant residues in each subfamily are highlighted and near-invariant residues (delineated in Table 2) are shaded. Invariant or
near-invariant residues common to both subfamilies are boxed. Residues shown in Sty to be relevant to metal coordination or to the binding of indole,
serine or PLP are color-coded as indicated at the bottom.


both are outside an otherwise intact trp operon. In this case it
seems curious indeed that the operon contains trpEb_2.

A snapshot of the incredibly dynamic alteration of trypto-
phan gene organization in prokaryotes is apparent from
Figure 7. Of the organisms shown in Figure 7, the most con-
sistent linkage is that of trpAa and trpAb. In A. fulgidus
trpD and trpB are fused, whereas in Rhodopseudomonas
palustris trpAa and trpAb are fused. Operons are sometimes
incomplete as with P. aerophilum and G. sulfurreducens, or
fragmented, as with R. palustris. In A. pernix an inverted
arrangement yields a divergent transcription of trpEa ->
trpEb 2 -4 trpC -> trpD and trpB -> trpAa -> trpAb. This is
also one of the very few cases where the order is trpEa -*
trpEb instead of the usual trpEb -> trpEa.

The Ferroplasma genome illustrates a case where a non-
tryptophan pathway gene, aroA encoding DAHP synthase, is a
member of the operon (translationally coupled). The implied
transcriptional control of DAHP synthase by L-tryptophan
potentially could produce growth inhibition by L-tryptophan


because of limitation of precursors needed for L-phenylala-
nine and L-tyrosine biosynthesis. This is because no other
genes encoding DAHP synthase appear to be present in the
genome. It would be interesting to know whether the Ferro-
plasma DAHP synthase is sensitive to allosteric control or
not. The phenomenon of growth inhibition triggered by
exogenous amino acids is exemplified by the effect of exoge-
nous L-phenylalanine upon DAHP synthase in Neisseria
gonorrhoeae [27] and in other organisms ([27] and refer-
ences therein).

What is the function of TrpEb_2? As previously discussed, it
seems clear that TrpEb_2 has sometimes been pressed into
service with TrpEa to function as tryptophan synthase. What
might be its function in those situations where trpEb 2 is
isolated away from a typical tryptophan operon, which pos-
sesses closely linked or translationally coupled genes speci-
fying trpEa and trpEb_ ?

TrpEb_1 from S. typhimurium is the prototype member
of a superfamily of pyridoxal phosphate (PLP)-dependent









10 Genome Biology Vol 3 No I Xie et al.


D56Salt bridge with l K167


Sty 1 MERYENLFAQLNDRREGAFVP'i TLGDPG--IEQSLKIIDTLIDAGADALi GVPFSELAD;PTIQNANLRAFAAGVTPAQCFEMLALIRE----KHPTIPIGLLMYAN 104

Tac 1 ------------------ MKPYFTLGY ---RYDAIAEFRNSDGVYAFGFPTSKPVYIRIRKTHDPELNRYSEAENSRIFKIAD --DIGSKKYALMYYEV 83
Tvo 1 -------- ---------- MKPYFTLGY --- KYNEVKKFIKNSDGVYAF 3GFPTENPLY DKKIKATHSFAVNK--FSHDDN AEIFKLAE -- SMGIKKYALLYYSV 83
Fac 1 ---------------KNLIFTLGYPN--NETLSHFLDLIPVDKINYIGFPSNRY VIRKTHNVGNINFSDDFYKKYFDHFHK ----NHVKYSLSYYS 86
Sso 1 ----------- MEMGKML TLGYPN--VQSFKDFIIGAVENGADIL GIPPKYAKYPVIR--KSYDKVKGLDIWPLIEDIRKDVG--------VPIIALTYLE 86
Paero 1 ------------LSKPGLGVLVASWPS KDTYEKAIRGLEGIADFFGLPSKNPKYDFIRKAHREAGEP ----------- LWLRP -----QAPTYIMTYWE 77
Ape 1 -----------AIARPGFSVTIAWPS--PDTFLEIASTLKGCVDYL GIPTPKPLY TIRLTHLKAVESGYSGPKTLSLAEEASQ --EAGVPYIVMAYAT 91

G181 Intersubunit
signaling
with 0 S178

Sty 105 LVFNNGIDA----FYARCEQVGVDSVLVADVPV ---- EESAPFRQAALRHNIAPIFICPPNADDDLLRQVASYGRGYLLSRSGGAENRGAL---P-LHHLIEKLKE 202

Tac 84 LSANPGILD------- YLNRNGFAGAILPDLMIDH-RDAFFDAVERLRQYSLDYVPFVTPITPVKVMEEQIAAGGDWI GM-PATGVQLPYS------IDAIYNHIRP 178
Tvo 84 FSGKKGILD------- YLSSSRFDGVMVPDVGIDY-PDRIEQVAKAIADHGMDYVPFVTPVTPFKIM ERQKVSGEIV G -PATGIQLPYD ------IGTIYGHIRD 178
Fac 7 D-IKARFDE----FIDYLQKRNFSGIIIPDLIIDYYSEGKEIINKLNDRGFEYIPFFSPSTPDSIIKDVSSTNSWYGLQPSTGIDIPYE -----LDYTTERINE 183
Sso 97 D-WVDQLEN---- FLNMIKDVKLDGILFPDLLIDY-IDDLDKIDGIIKNKGLKNVIFTSPSVPDLLIHKVSKISDLFLYGVR-PTTGVPIPVS------VKQLINRVRN 183
Paero 78 E-HRGNLDG---- LFTLAAEIRARGVLAPDLLIDFP-EELELYLKYAREYALAPAFFVPGKFPHWLVKRLTSAEPDFILGLY-AATGVELPVY -----VERNVKIIRQ 174
Ape 92 E-QPWSFSE---- VLREASRKGALSVLPPDLPFELPGHVEWYVEESRRLGEPSLFASPKFPHRWLDRYRRLDPLLILGLQPATGVKLPLA----FLRNVKTARK 188




Sty 203 YHAA---PALQG?3SSSPEQVSAAVRAGAAGAIS IVKIIEKNLA----------SPKQMLAE ----LRSFVSAMKAASRA---------- 269
Sty 203HA IAQAVKIKL


Tac 179 YAGGK--RIVYG? RDAGTMKMLASY EAFGIA 3VVEMMENL----------------DTAS ----YRRLIETIMEA------------- 236
Tvo 179 FTSGK--RIVYG ? RNKCTIAKIAKYGGFGIA3VVEMLDDG----------------NVKA----YRQLINDILGAS------------ 237
Fac 14 LLPGR--EITYGIKTDDD IRDLKNGGSGVAIGYLIKMTEAG----------------DEKG----FIDYINKMRGVLDE---------- 244
Sso 14 LVEN ---KLIVG ? LSSESDLRDALSAGADGIAI31 VFIEEIERN ----------------GVKS----AINLVKKFRAILDEYK-------- 245
Paero 175 LAGD--VYIVAG DSPSKAARLIEAGADGV FMRRLQNS ------------ --VDS ----ALSFLKSIKEALK----------- 233
Ape 19 IVGQ VYMLAG 3IKSPEDALRVLEAGADAV EVARLVSSG----------------RLGE----ARHLACSIRAAISERGG------- 252


Figure 6
Alignment of TrpEa from S. typhimurium with TrpEa proteins restricted to co-function with a TrpEb_2 type of P subunit. Catalytic residues Sty E49 and
D60, as well as other conserved residues shown to be important for indoleglycerol phosphate binding are shown in blue. Other invariant (highlighted) or
near-invariant (shaded) residues are marked in yellow. Conserved residues that important for intersubunit signaling with TrpEb_l are shown in green.


enzymes that are of remote relationship and that catalyze
P-replacement and p-elimination reactions [28-30]. These
include O-acetylserine sulfhydrylase, threonine deaminase,
threonine synthase, cystathionine p-synthase, 1-aminocyclo-
propane-i-carboxylate deaminase, L-serine dehydratase, and
D-serine dehydratase. Isolated TrpEb_1 does catalyze the
reaction of L-serine dehydratase (deaminase) in vitro, but
does not support significant levels of the other activities. It
does not seem likely that TrpEb_2 catalyzes the latter reac-
tions either, as Psi-Blast of TrpEb_2 sequences did not
return hits for them any more avidly than was the case for
TrpEb_1 queries.


Perhaps the most likely 'alternative' function of TrpEb_2
proteins is a catalytic activity already established as an in
vitro activity of isolated TrpEb_1, of which there are two.
The L-serine + indole -> H20 + L-tryptophan reaction might
function alone for tryptophan biosynthesis in cells that
acquire indole from the environment. Little information
about the availability and utilization of indole in nature


seems to exist. Model organisms such as E. coli and B. sub-
tilis transport indole poorly and it tends to be toxic, but
these organisms lack TrpEb_2.


As stated above, the second established in vitro activity of
isolated TrpEb_1 dimers is L-serine dehydratase (deami-
nase): L-serine -> pyruvate + ammonia. It is indeed sugges-
tive that the presence of TrpEb_2 correlates almost perfectly
with the absence of the primary L-serine deaminase
(COG176o) used in nature (Table 3). This SdaA class of
serine deaminase is an iron-sulfur protein, not a pyridoxal
5'-phosphate protein [31]. It is absent from the Archaea.
Although widely distributed in the Bacteria, it is not present
in Thermotoga, Aquifex, Chlorobium, Geobacter and
Rhodopseudomonas. Mycobacterium smegmatis is the only
TrpEb_2-containing organism to possess an SdaA homolog.


There are other candidates for carrying out the serine deami-
nase reaction. A serine deaminase that is PLP-dependent
exists in eukaryotes, but seems to be absent or rare in








http://genomebiology.com/2001/3/11/research/0004.1 I


Pyrococcus horikoshii

Pyrococcus furious

Pyrococcus abyssi

Methanobacterium thermoautotrophicum

Thermoplasma acidophilum

Thermoplasma volcanium

Ferroplasma acidarmanus

Methanosarcina barker

-Archaeoglobus fulgidus

Pyrobaculum aerophilum

CAeropyrum pernix
Sulfolobus solfataricus

-Aquifex aeolicus [co

-/ Thermotoga maritima

- Mycobacterium smegmatis

Chlorobium tepidum [co

-- Geobacter sulfurreducens

0.1 Rhodopseudomonas palustris


[Pathway has been completely lost]

2 -14 4 2 -11 -5

-4 -16 -1 2 -11 -5

-1 4 -17 -4 38 -4

4 34 -29 -4 -4

-23 -29 -23 -4 -4
B C Ab D [aroA>
-4 -8 -10 -13 -23 -4

272 1 119 4 -14 167

-19 -1 -8 -11 -1

253 -1 -7 4

2 5 5 151 339 -4

-8 14 -8 -23 0 3
mplete dispersal of all pathway genes] i:


-4 40 -13 2 -1

-4 76 35 -4
mplete dispersal of all pathway genes]

1 61 4 153 4

65 0 14


Figure 7
Organization of tryptophan-pathway genes in those archaea and bacteria that possess trpEb_2. The 16S rRNA tree of the organisms that possess trpEb_2
is shown at the left. The tree was obtained from the SUBTREE program of the Ribosomal Database [41,42]. Each of the eight genes is color-coded
differently. For economy of space trpD is D, trpC is C, etc. aroA in F. acidarmanus belongs to the AroAi, grouping [12]. Flanking genes are shown as boxes
touching one another with intergenic spacing indicated. Minus values indicate translational coupling. The open gray box is a conserved hypothetical
protein. Pointed box ends indicate the direction of transcription. Space between boxes indicates a lack of genetic linkage.


prokaryotes. Threonine dehydratase has a lesser capability
to carry out the serine dehydratase reaction in vitro. 'Biosyn-
thetic' (IlvA) and 'catabolic' (TdcA) threonine deaminase are
homologs, except that IlvA has a unique carboxy-terminal
extension that provides an allosteric module. Most archaea
lack ilvA, and only a limited number possess catabolic threo-
nine dehydratase.

If archaea lack ilvA, what is the source of a-ketobutyrate for
isoleucine biosynthesis? It appears likely that a-ketobutyrate
is generated instead by the 'pyruvate' pathway in which cit-
ramalate is generated as the initial intermediate. A few
tracer studies in the older literature have indicated deriva-
tion of a-ketobutyrate from pyruvate rather than from threo-
nine [32,33], and (R)-citramalate synthase activity has been
shown recently in M. jannaschii (MJ1392) [34]. This
pathway was initially shown to exist in Leptospira [35], and
it seems likely that more examples will surface. Indeed, it is


interesting that an enteric bacterium has a latent potential to
replace the threonine deaminase step with a pyruvate-
derived pathway under appropriate selective conditions
[36]. The citramalate pathway for conversion of pyruvate to
2-ketobutyrate involves a carbon-chain elongation mecha-
nism that uses an initial step of condensation with acetyl-
CoA, followed by rearrangement, oxidation and elimination
of a carbon to produce a keto acid differing from the original
substrate by the presence of an additional carbon. This
mechanism is familiar in nature, analogous steps being used
in the TCA cycle, the ketoadipate pathway of lysine biosyn-
thesis, and in leucine biosynthesis. These analogous steps in
different pathways were originally used in formulating the
recruitment hypothesis for evolutionary acquisition of new
function [37], and it is interesting that the citramalate syn-
thase gene shown in M. jannaschii had previously been
annotated as a-isopropylmalate synthase (which catalyzes
the initial step of leucine biosynthesis).


ow



ow
S^


w^

w^









12 Genome Biology Vol 3 No I Xie et al.


Table 3

Possible sources of serine deaminase in organisms containing
TrpEb_2

Serine deaminases Threonine deaminases


Organisms


Fe-S* PLP*


Pyrococcus horikoshii
Pyrococcus furious
Pyrococcus abyssi
Methanobacterium
thermoautotrophicum
Thermoplasma acidophilum
Thermoplasma volcanium
Ferroplasma acidarmanus
Methanosarcina barkeri
Archaeoglobus fulgidus
Pyrobaculum aerophilum
Aeropyrum pernix
Sulfolobus solfataricus
Aquifex aeolicus
Thermotoga maritima
Mycobacterium smegmatis
Chlorobium tepidum
Geobacter sulfurreducens
Rhodopseudomonas palustris


TdcA* IlvA*


+
+-
+-


+
-I-

+-
+

+


*Sequences that were used to query the databases at NCBI [38], atJGI
[39] for Fac and Mba, and at ERGO [40] for Paer and Rpa were gi
2501150 (iron-sulfur (Fe-S) serine deaminases), gi 134387 (PLP-dependent
serine deaminases), gi 135723 (catabolic threonine deaminase), and gi
135720 (biosynthetic threonine deaminase).




Overall, the cumulative evidence indicates that established
sources of L-serine dehydratase are low or absent in organ-
isms that possess TrpEb_2, and therefore points to a plausi-
ble role for TrpEb_2 as L-serine dehydratase.



Acknowledgements
The graduate research studies of G.X. were partially supported by funding
from the National Institutes of Health at Los Alamos National Laboratories.




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