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J Bacteriol, January 1998, p. 119-127, Vol. 180, No. 1
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Substrate Ambiguity of
3-Deoxy-D-manno-Octulosonate 8-Phosphate
Synthase from Neisseria gonorrhoeae in the Context of
Its Membership in a Protein Family Containing a Subset of
3-Deoxy-D-arabino-Heptulosonate
7-Phosphate Synthases
Prem S.
Subramaniam,
Gang
Xie,
Tianhui
Xia, and
Roy A.
Jensen*
Department of Microbiology and Cell Science,
University of Florida, Gainesville, Florida 32611
Received 6 August 1997/Accepted 23 October 1997
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ABSTRACT |
3-Deoxy-D-manno-octulosonate 8-phosphate
(KDOP) synthase and
3-deoxy-D-arabino-heptulosonate 7-phosphate
(DAHP) synthase catalyze similar phosphoenolpyruvate-utilizing
reactions. The genome of Neisseria gonorrhoeae contains one
gene encoding KDOP synthase and one gene encoding DAHP synthase. Of the
two nonhomologous DAHP synthase families known, the N. gonorrhoeae protein belongs to the family I assemblage. KDOP
synthase exhibited an ability to replace arabinose-5-P with either
erythrose-4-P or ribose-5-P as alternative substrates. The results of
periodate oxidation studies suggested that the product formed by KDOP
synthase with erythrose-4-P as the substrate was
3-deoxy-D-ribo-heptulosonate 7-P, an isomer of
DAHP. As expected, this product was not utilized as a substrate by
dehydroquinate synthase. The significance of the ability of KDOP
synthase to substitute erythrose-4-P for arabinose-5-P is (i)
recognition of the possibility that the KDOP synthase might otherwise
be mistaken for a species of DAHP synthase and (ii) the possibility
that the broad-specificity type of KDOP synthase might be a relatively
vulnerable target for antimicrobial agents which mimic the normal
substrates. An analysis of sequences in the database indicates that the
family I group of DAHP synthase has a previously unrecognized
membership which includes the KDOP synthases. The KDOP synthases fall
into a subfamily grouping which includes a small group of DAHP
synthases. Thus, family I DAHP synthases separate into two subfamilies,
one of which includes the KDOP synthases. The two subfamilies appear to
have diverged prior to the acquisition of allosteric-control mechanisms
for DAHP synthases. These allosteric control specificities are highly diverse and correlate with the presence of N-terminal extensions which
lack homology with one another.
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INTRODUCTION |
3-Deoxy-D-arabino-heptulosonate
7-phosphate (DAHP) and
3-deoxy-D-manno-octulosonate 8-phosphate (KDOP)
are analogous seven- and eight-carbon 2-keto-3-deoxy sugars that are
synthesized by enzymes which belong to functionally unrelated pathways.
DAHP synthase forms DAHP as the acyclic precursor of the aromatic amino acids in bacteria, lower eukaryotes, and plants (3); KDOP
synthase is best known for its role in the formation of KDOP as a
critical component of the lipopolysaccharide of gram-negative bacteria (37), but its distribution in nature has recently been
recognized to be broader (13). Both enzymes catalyze an
overall condensation of phosphoenolpyruvate (PEP) with an aldose, i.e.,
erythrose-4-phosphate (E4P) in the case of DAHP synthase and
arabinose-5-phosphate (A5P) in the case of KDOP synthase. The reactions
are irreversible and are not aldol-type condensations, which
unfortunately has been implied by the Enzyme Commission naming that has
been recommended for DAHP synthase.
As might be expected from the close structural relationship of A5P and
E4P, the reactions are strikingly similar. This similarity is reflected
at the level of mechanistic detail (see reference 16
and references therein). DAHP synthase and KDOP synthase, along with
enolpyruvoylshikimate 3-phosphate synthase and
UDP-N-acetylglucosamine enolpyruvoyl transferase, comprise a
small class of PEP-utilizing enzymes that catalyze C---O bond cleavage
with respect to the release of Pi from PEP (1,
27). This contrasts with the more familiar nucleophilic attack at
the phosphorous atom of PEP that results in P---O bond cleavage by the
action of enzymes such as pyruvate kinase (25), PEP
carboxylase (34), and PEP carboxykinase (8).
In classical studies with Escherichia coli, DAHP synthase
(44, 45) and KDOP synthase (41) are specific for
E4P and A5P, respectively. In contrast, we found that the KDOP synthase
of Neisseria gonorrhoeae possessed the ability to utilize
E4P in place of A5P. We addressed the question of whether KDOP synthase of N. gonorrhoeae in the presence of E4P and PEP was able to
form DAHP, in which case it would also have the potential to function as a DAHP synthase. The time-dependent cleavage of the product was
investigated by the periodate-oxidation-thiobarbituric acid (TBA)
assay, and these results allow some speculation on the stereospecific course of the reaction in comparison with the reaction of DAHP synthase.
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MATERIALS AND METHODS |
Terminology.
It is becoming increasingly awkward, especially
when multiple molecular-genetic comparisons are under way, to use
acronyms whose meanings are different for different organisms.
Therefore, we employ the following definitions. aroA encodes
monofunctional DAHP synthase, appropriately designated because this
catalyzes the first reaction of aromatic biosynthesis. Unique feedback
inhibition specificities are designated by subscripts corresponding to
the appropriate amino acid symbols. Thus, the three E. coli
paralogs currently known as AroF, AroG, and AroH are denoted
AroAY, AroAF, and AroAW,
respectively. The species inhibited by both L-phenylalanine and L-tyrosine is denoted AroAFY.
aroA encodes the DAHP synthase domain of the bifunctional
chorismate mutase: DAHP synthase (AroGAroA). The convention of
using a bullet to separate the potentially independent domains of a
fusion protein (or their coding regions in the gene) is according to
the precedent of Crawford with tryptophan pathway fusions
(10).
Data analysis.
An updated version of the sequence analysis
software offered by the Genetics Computer Group (GCG) (21)
was used. The multiple alignment presented in Fig. 7 required some
manual alignment. The GCG PILEUP program was used to align members of
subfamily I
and class I
D (see Fig. 6). Class
IK members were then manually added to the alignment,
which was guided by use of a multiple alignment generated for subfamily
I alone by the PILEUP program.
Chemicals and biochemicals.
E4P was prepared according to
procedure B as described by Ballou (2), except that the
monosodium salt of glucose-6-phosphate was used and the sulfuric acid
treatment of the salt was omitted. The concentration of E4P was
estimated by using a partially purified preparation of DAHP synthase
from N. gonorrhoeae ATCC 27630. Dehydroquinate was
synthesized according to the procedure of Haslam et al.
(26). The product was obtained as an oil that could be dried
to an extremely hygroscopic solid but could not be crystallized. The
free acid was dissolved in water (to a concentration of 175 mM), the pH was adjusted to 7.0, and the solution was stored at 
80°C. It was
found to contain ~8% of dehydroshikimic acid by its
A234.
DAHP was isolated from the culture supernatant of an overproducing
mutant of E. coli according to the procedure of Mehdi et al.
(36). The mutant strain E. coli JB5 was a
gift from Jeremy Knowles (Harvard University).
The trisodium salt of PEP was purchased from Sigma Chemical Co. (St.
Louis, Mo.). Dithiothreitol (DTT) was purchased from
Research Organics,
Inc. (Cleveland, Ohio). Hydroxylapatite (Bio-gel
HTP) was purchased
from Bio-Rad (Rockville Centre, N.Y.). Bio-gel
A (0.5 m, 100 to 200 mesh; exclusion limit, 500,000 Da) was purchased
from Bio-Rad as a
fully hydrated suspension in 10 mM Tris-EDTA
buffer. This buffer was
replaced with appropriate buffers after
packing of the column.
Bacterial strains, media, and growth conditions.
N.
gonorrhoeae 2013 (ATCC 27630) is a clinical isolate which is
auxotrophic for proline and resistant to growth inhibition by
L-phenylalanine (4). Strain 2011 (ATCC 27628) is
a clinical isolate which is prototrophic and sensitive to growth
inhibition by L-phenylalanine (4). These strains
were grown in the minimal medium formulated by Hendry (28).
Growth at 525 nm was monitored by measuring the optical density of a
5-ml sample of the culture at regular intervals. Supplementation of
media to appropriate concentrations with required components was done
through a sterilizing membrane after the complete medium was made.
Cells in the late-exponential phase of growth were harvested by
centrifugation at 4°C, washed once with buffer A (20 mM K-phosphate
at pH 7.0 containing 1.0 mM DTT), and stored at 89°C until used.
The source and cultivation of
Erwinia herbicola
(
53) and
Pseudomonas acidovorans (
48)
were as referenced.
Preparation of crude extracts.
Cells were resuspended by
thawing the frozen cells in buffer B (100 mM K-phosphate at pH 7.0 containing 1.0 mM DTT) at room temperature. A buffer-to-cell ratio of
2:1 (vol/wt) was used. The cells were broken in a French pressure cell
at 16,000 lb/in2. The suspension was centrifuged at
150,000 × g for 35 min, and the supernatant was
collected as the crude extract. Extracts were desalted by passage
through an Econo Pac 10DG (Bio-Rad) desalting column. All operations
were conducted at 4°C.
Hydroxylapatite chromatography.
A 75-mg amount of the
desalted crude extract in buffer A was loaded on a column (1.5 by 20 cm) of hydroxylapatite equilibrated in buffer A. After the unbound
protein was washed with 2 bed volumes of buffer A, adsorbed protein was
eluted with a linear gradient between buffer A and 300 mM K-phosphate
at pH 7.0 (containing 1.0 mM DTT) in a total volume of 340 ml.
Fractions of 2.2 ml were collected. For KDOP synthase, the band within
the two peak tubes exhibited a specific activity of 174.5 nmol/min/mg,
compared to a specific activity of 3.1 nmol/min/mg in crude extract.
This represents a purification factor of 563.
Enzyme assays.
DAHP synthase was assayed as described by
Jensen and Nester (30). Standard reaction mixtures contained
1.5 mM PEP, 1.0 mM of E4P, and 0.5 mM MnSO4, and
appropriate aliquots of enzyme, which were incubated at 37°C for 20 min. Activity was expressed as A549, where a
value of 1.0 corresponds to 0.3 mM DAHP formed. An 
value of
5.0 × 104 M
1 cm1 was used
(36). KDOP synthase was assayed and activities were quantitated as specified by Ray (41). In some experiments,
we found it convenient to assay KDOP synthase in crude extracts. In
such preparations, DAHP synthase activity could be reduced to less than
3% by omission of MnSO4 and inclusion of 5 mM
L-phenylalanine.
Dehydroquinate synthase was assayed by monitoring the disappearance of
substrate DAHP by the TBA assay method for DAHP synthase.
The reaction
mixtures contained 0.3 mM DAHP, 50 µM NAD
+, and 0.5 mM
Co
2+ and were incubated at 37°C for 20 min. In some
experiments, partially
purified DAHP synthase from
N. gonorrhoeae was used to generate
DAHP. A DAHP synthase reaction
mixture containing 0.8 mM PEP,
1.0 mM E4P, 0.5 mM MnSO
4,
and an aliquot of DAHP synthase was
prepared. Catalysis was conducted
at 37°C until a DAHP yield equivalent
to an
A549 value of 1.0 to 1.5 was obtained. The DAHP
synthase
was destroyed by heating to 60°C. The formation of the
product,
dehydroquinate, was verified with a highly purified
preparation
of a bifunctional dehydroquinate dehydratase
shikimate
dehydrogenase
from
Nicotiana silvestris (
6).
Cloning of aroAW from E. herbicola.
The gene aroAW from E. herbicola was cloned by selection for the ability of amplified
AroAW to suppress the nutritional requirement of a leaky
pheA phenylalanine auxotroph of E. coli by the
general methodology described by Xia et al. (53).
Nucleotide sequence accession number.
The sequence for
E. herbicola aroAW has been assigned GenBank
accession no. U93355.
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RESULTS |
Relaxed substrate specificity of the KDOP synthase reaction.
Figure 1 shows the activity profiles for
DAHP synthase and KDOP synthase after fractionation of crude extracts
of N. gonorrhoeae VHC 3102 on hydroxyapatite. KDOP
synthase eluted ahead of the major protein eluate, thus yielding an
excellent partial purification. When fractions under the KDOP synthase
peak were assayed under conditions used to detect the DAHP synthase
band (1 mM E4P), an activity just marginally above the background was
detected. The substrate ambiguity of KDOP synthase was verified by more
detailed work with the peak fractions. Accuracy was maximized by use of a longer assay duration (30 min) and use of saturating concentrations of E4P (3.1 mM). The inset of Fig. 1 shows a comparison of the activities of the peak fraction when assayed with 1.25 mM A5P, 1.25 mM
E4P, and 3.1 mM E4P. The amount of the product formed with E4P was
maximal at 3.1 mM E4P and equaled as much as 8% of the product formed
with saturating concentrations of A5P (at a fixed concentration of 2 mM
PEP). A standard periodate oxidation time of 30 min was used. No
activity was seen with D- or
L-glyceraldehyde-3-phosphate (1.5 mM). However,
D-ribose-5-phosphate (3 mM), an isomer of A5P, was utilized
to the same extent as E4P. Additional confirmation of substrate
ambiguity was obtained by demonstrating that E4P inhibited the KDOP
synthase reaction with A5P in a competitive fashion.
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FIG. 1.
Separation of DAHP synthase and KDOP synthase by
chromatography of crude extracts of N. gonorrhoeae VHC 3102 on hydroxylapatite. A 100-mg amount of crude extract prepared as
described in Materials and Methods was loaded on a column (1.5 by 23 cm) equilibrated with 20 mM K-phosphate (pH 7.0) containing 1.0 mM DTT
(buffer A). After washing with 2 bed volumes of buffer A, absorbed
proteins were eluted by application of a linear gradient between buffer
A and 300 mM K-phosphate (pH 7.0, 1.0 mM DTT). DAHP synthase and KDOP
synthase were assayed as described in Materials and Methods. The
reaction mixtures contained 1.0 mM E4P/A5P, 1.0 mM PEP, and 0.5 mM
Mn2+. Enzyme activities are reported at
A549. Protein was monitored as
A280. Dashed line, progress of the gradient. The
relative abilities of fraction 104 to use E4P and A5P at 2 mM PEP are
shown in the inset. The amounts of product plotted on the ordinate
scale were calculated by using an value of 1.03 × 104 M1 cm1 for the KDOP-derived
chromophore and assuming an value equal to that for DAHP (5.0 × 104 M1 cm1) for the
E4P-derived product.
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The stereochemistry of the resultant C7 product.
Periodate oxidation provides a simple method to examine the relative
configuration of the hydroxyl groups at C-4 and C-5 in 2-keto, 3-deoxy
sugars such as DAHP and KDOP. The rate of chromophore formation
measured by the TBA assay is directly proportionate to the ease of
oxidative cleavage at C-4-C-5 (23); upon C-4-C-5 cleavage,
2-keto-3-deoxy-sugars yield the TBA-reactive product -formylpyruvate. KDOP is rapidly oxidized by periodate as a
consequence of the cis orientation of the C-4 and C-5
hydroxyl groups (23), in contrast to the much slower
oxidation encountered when the trans configuration of these
hydroxyls occurs, as seen in DAHP. The orientation of the C-4 hydroxyl
in KDOP and DAHP is dictated by the stereochemistry of the addition
across the face of the carbonyl group in the substrate sugar. In the
case of both KDOP synthase and DAHP synthase, the orientation of the
hydroxyl generated by the addition of PEP is such that C-4 has an (R)
configuration in the products KDOP and DAHP.
In order to accumulate sufficient amounts of the C
7 product
derived from the use of E4P by KDOP synthase (denoted
C
7-X), the
reaction was scaled up as follows. A 1-ml
reaction mixture containing
3.6 mM E4P, 2 mM PEP, 1 mM DTT, 0.5 mM
Mn
2+, and 375 µg of partially purified KDOP synthase in
75 mM potassium
phosphate buffer (pH 7.0) was incubated at 37°C for
5 h. The reaction
mixture was then transferred to 4°C and stored
overnight. A 40-µl
amount of 100% (wt/vol) trichloroacetic acid was
added, mixed,
and retained on ice for 5 min. The precipitated protein
was removed
by centrifugation, and the supernatant was diluted twofold
with
buffer and used for oxidation studies.
DAHP was accumulated in a similar 1-ml reaction mixture, except that it
contained 1 mM E4P, 1 mM PEP, and 940 µg of crude
extract from
Pseudomonas testosteroni (ATCC 17409) incubated at
37°C
for 20 min; the supernatant was diluted twofold with buffer
before use.
A 100-µl sample of each supernatant was used. A concentration
corresponding to 0.35 mM DAHP was formed.
Figure
2 shows a comparison of the
kinetics of periodate oxidation of the C
7-X product and
authentic DAHP. It is evident that
the C
7-X product is
rapidly oxidized in comparison to DAHP, with
the oxidation being
complete within 4 min. Almost 65% of chromophore
from C
7-X
was released within 1 min of the oxidation (cf. 17%
for DAHP). These
results exactly parallel those of Doong et al.
(
13), who
compared the rates of periodate oxidation of KDOP
and DAHP under
identical conditions. The C
7-X product behaves
like
authentic KDOP or KDO, i.e., maximal oxidization occurs within
5 min of
incubation. Thus, the relative configuration of the hydroxyls
at
C-4-C-5 in the predominant C
7-X product must be
cis. It follows
that C
7-X is the
4(
S)-isomer, unlike 4(
R)-KDOP and
4(
R)-DAHP.
The product is accordingly concluded to be the
D-
ribo-analog of
DAHP, i.e.,
3-deoxy-
D-
ribo-heptulosonate 7-phosphate. Since
DAHP
is not formed, these results eliminate the possibility that the
E4P-utilizing activity might have been due to the presence of
a minor
DAHP synthase species having a coincident elution profile
with KDOP
synthase.
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FIG. 2.
Kinetics of periodate oxidation of the enzymatic
products of KDOP synthase and DAHP synthase reactions with E4P and PEP.
Products were accumulated as described in the text, and a 100-µl
sample was used in the periodate-oxidation-TBA assay procedure. The
oxidation was terminated at the specified time intervals. Maximal
A549 values in each case were assigned a
relative value of 100% and corresponded to 0.721 for DAHP and 1.482 for the E4P/KDOP synthase-derived product (C7-X).
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The C
7-X product was tested as a substrate for
dehydroquinate synthase, the enzyme that uses DAHP as a substrate to
form dehydroquinate.
Figure
3 shows the
elution profile of
N. gonorrhoeae dehydroquinate
synthase
from hydroxylapatite. The enzyme was assayed by monitoring
the
disappearance of DAHP by the TBA assay. The C
7-X product
accumulated
as described above was incubated with a partially purified
preparation
of dehydroquinate synthase for 20 min in a reaction mixture
containing
0.5 mM Co
2+ and 50 µM NAD
+. The
C
7 product was not utilized as substrate (Fig.
3, insert).
As a positive control, the enzyme was incubated with authentic
DAHP
(Fig.
3, insert). Under the same conditions used for C
7-X,
almost 90% of the DAHP was utilized. Thus, the C
7-X
product is
not a substrate for dehydroquinate synthase, consistent with
its
likely identity as
3-deoxy-
D-
ribo-heptulosonic acid 7-phosphate.
Absolute proof of product identity requires nuclear magnetic resonance
and mass spectral analysis.
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FIG. 3.
The reaction of C7-X with N. gonorrhoeae dehydroquinate synthase. Dehydroquinate synthase
activities are profiled as eluted off the column described in the
legend to Fig. 1. Enzyme activities are reported for simplicity as
A549. Note that the values actually represent
 A549 where a value of 1.0 corresponds to 48.0 nmol of DAHP consumed. The insert shows the fraction of product
remaining after incubation of C7-X and DAHP with
dehydroquinate synthase as described in the text. A value of 100%
corresponds to 0.24 mM DAHP and 0.1 mM DAHP equivalents of
C7-X.
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DISCUSSION |
Mechanism of
3-deoxy-D-ribo-heptulosonate 7-phosphate
acid formation by KDOP synthase.
It was surprising to identify the
D-ribo stereoisomer as the probable product of
E4P utilization by KDOP synthase in view of the similarity of its
reaction mechanism to that of the well-studied DAHP synthase (12,
19). The condensation of E4P and PEP by DAHP synthase
proceeds by a stereospecific addition across the si
face of C-3 of PEP and the re face of C-1 of E4P
(18). The resulting DAHP has an (R) configuration
at C-4 (Fig. 4 and
5), with the newly formed hydroxyl being
in trans to the C-5 hydroxyl. In the case of the KDOP
synthase reaction, based on the established structure of KDOP, the same
stereochemistry, 4(R), is generated at C-4 and the reaction
most likely proceeds with the same stereospecificity as that of the
DAHP synthase reaction. The configuration of the substrate (A5P)
disposes the C-5 in cis to the newly formed hydroxyl at C-4.
To form the 4(S)-isomer, as occurs when
3-deoxy-D-ribo-heptulosonic acid is formed by
N. gonorrhoeae KDOP synthase with E4P, the addition should
proceed across the si face of C-3 of PEP and the
si face of C-1 of E4P. E4P is presumably accommodated at the
active site such that the C-1 carbonyl presents the face opposite to
that across which PEP adds in E4P in the DAHP synthase reaction and across which PEP adds in A5P in the true KDOP synthase reaction. It is
assumed that the si face of PEP is consistently presented in
all of these reactions, since this appears to prevail in other reactions involving PEP, including those that do not involve C---O bond
cleavage (42). The D-ribo product
appears to be the predominant product of N. gonorrhoeae KDOP
synthase when offered PEP and E4P. This KDOP synthase thus lacks the
ability to function as a DAHP synthase. Whether the
D-ribo product might be produced in vivo for
some unknown function, as implied by the rather broad distribution of
2-keto, 3-deoxy sugars in nature (35), is an intriguing
possibility for future discovery.
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FIG. 5.
Representation of the mechanism of formation of DAHP by
DAHP synthase, KDOP by KDOP synthase and
3-deoxy-D-ribo-heptulosonate 7-phosphate (DRHP)
by KDOP synthase. The mechanism for the formation of DRHP is
hypothetical. In the case of the formation of DRHP, it is assumed that
the addition is across the si face of PEP by analogy.
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Substrate ambiguity and reversed modes of stereospecificity.
A
precedent exists in which another species of KDOP synthase exhibits
broad substrate specificity. KDOP synthase from spinach (13)
was able to use E4P as a substrate 24% as well as A5P. It also
catalyzed the condensation of PEP and E4P to the
D-ribo product, the reaction thus proceeding
with the opposite stereochemistry performed in condensing PEP and A5P
to form KDOP. The N. gonorrhoeae and higher-plant KDOP
synthases are thus far the only reported broad-specificity KDOP
synthases.
While most DAHP synthase enzymes are relatively specific (to the extent
that these characterizations have been made), one
species exhibits a
remarkable relaxation of substrate specificity.
A cytosolic enzyme
(denoted DS-Co) present in most if not all
higher plants (
14,
15) is able to condense PEP with an array
of aldoses that
include the phospho and dephospho counterparts
of glycolaldehyde,
glyceraldehyde, erythrose, threose, arabinose,
ribose, and glucose. The
efficiency of catalysis, as defined by
the
Vmax/
Km ratio, was
highest with glycolaldehyde, E4P, or glyceraldehyde-3-P.
Whereas E4P yielded a
trans C-4-C-5 product,
glyceraldehyde-3-P
and A5P produced
cis C-4-C-5 products.
The higher-plant DAHP synthase
(DS-Co) thus has a minor capability to
function as a KDOP synthase.
In contrast, the chloroplast-localized
DAHP synthase (denoted
DS-Mn) exhibits narrow substrate specificity
(
20).
2-Keto, 3-deoxy sugars are common in the cell wall, lipopolysaccharide,
and extracellular polysaccharides of bacteria (
35),
but
their biosynthetic routes are not known. It would be logical
to
envisage their synthesis by reactions analogous to those of
DAHP
synthase and KDOP synthase. From an evolutionary standpoint,
given the
existence of the broad catalytic ability to condense
PEP and aldoses,
selection for specific function could occur at
two levels: one at the
level of the stereospecificity of the reaction
which has a fourfold
freedom and the other at the level of specificity
for the appropriate
sugar substrates.
Gene families encoding AroA proteins.
The current database
contains two distinct classes of sequenced genes that specify DAHP
synthase. Walker et al. (49) defined type I DAHP synthases
as "E. coli-like" homologs having a subunit Mr of about 39,000, while type II DAHP synthases
were defined as "plant-like" homologs having a subunit
Mr of about 54,000. Our analysis shows that the
type I DAHP synthases belong to a family which further subdivides into
two subfamilies, as illustrated by the dendrogram presented in Fig.
6. Subfamily I consists entirely of
DAHP synthase proteins, whereas the previously unrecognized subfamily
I contains one group of DAHP synthase proteins (class ID) and one group of KDOP synthase proteins (class
IK). Proteins within subfamily I are 28.5 to 30.8 kDa, which is smaller than their subfamily I counterparts. The
levels of overall identities between individual members of I and
ID (e.g., 18% for E. coli AroAF
and Synechocystis sp. AroAF) or between
individual members of I and IK (e.g., 17% for
E. coli AroAF and E. coli KdsA) are sufficiently low that homology is not apparent. However, the multiple alignment given in Fig. 7
indicates that all three groups comprise a family of homologs which
share 17 invariant residues. Even though members of class
ID are functionally equivalent to members of subfamily
I in that they catalyze the DAHP synthase reaction, they exhibit
greater overall similarity to members of class IK, which
catalyze the closely related KDOP synthase reaction. The class
ID DAHP synthases possess 17 residues that are conserved with subfamily I DAHP synthases, but not with class
IK KDOP synthases. These residues may, therefore, be
important for specificity relationships which dictate E4P utilization.
The class ID DAHP synthases possess an additional 19 residues which are conserved with the KDOP synthases of class
IK, but not with the subfamily I DAHP synthases.
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FIG. 6.
Dendrogram (PILEUP) showing family I relationships.
Averaged percent identities of deduced amino acid sequences are
indicated at the node positions. The four E. coli paralogs
are highlighted in boldface. Abbreviations: Hin, Haemophilus
influenzae; Eco, E. coli; Ngo, N. gonorrhoeae; Ehe, E. herbicola; Bap, B. aphidicola; Cgl, C. glutamicum; Sty, Salmonella
typhimurium; Cal, Candida albicans; Sce, S. cerevisiae; Spo, Schizosaccharomyces pombe; Bsu,
B. subtilis; Sxy, S. xylosus; Spy,
Streptococcus pyogenes; Ssp, Synechocystis sp.;
Pha, Pasteurella haemolytica; Cps, Chlamydia
psittaci; and Ctr, Chlamydia trachomatis. Hypothetical
evolutionary events included acquisition of a binding pocket for
aromatic amino acids (A), fusion of genes encoding an unregulated AroA
(carboxy terminal) and chorismate mutase (N terminal) (B) and
acquisition of an N-terminal domain specifying allosteric control by
L-phenylalanine (C).
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FIG. 7.
Multiple alignment of family I DAHP and KDOP synthases.
Amino acid residues which are conserved between class ID
and either or both of the other two groupings are boxed. In a few
cases, a variant residue within a box is shaded. Invariant residues
that are restricted to one of the three groups are shaded. Possible
motif regions for DAHP synthase catalysis are indicated with heavy
overbars. Residues shown (40) to be essential for catalytic
activity of E. coli AroAW are indicated by
double asterisks within a given box. Residues shown to be important for
feedback inhibition by L-tryptophan (WR),
L-phenylalanine (FR), or L-tyrosine
(YR) are shown at the top, with the organism used for the
study (references 40, 22, and 50,
respectively) indicated in parentheses. A frameshift error between
residues 184 to 204 of C. albicans AroAF
(indicated by underlining) was corrected. Residue numbers are given at
the far right. Gaps used to optimize the alignment are indicated by
dots. Abbreviations are as indicated in the legend to Fig. 6.
|
|
The 30-kDa size of the class I
K proteins appears to
define the basic catalytic domain, and homology to this span of
residues
is apparent throughout family I. The approximate 40-residue
N-terminal
extensions of the differentially regulated isoenzyme types
within
subfamily I
show little identity with one another and
probably
function in allosteric control. N-terminal extensions in class
I
D specify catalytic domains for chorismate mutase which
also
serve as allosteric domains for DAHP synthase (
30) in
two cases.
In the
Synechocystis protein, the extension may
specify phenylalanine-mediated
allostery. The streptococcal protein
(
Streptococcus AroA) which
lacks an N-terminal extension
probably lacks allosteric control,
a feature noted for a number of
streptococcal DAHP synthases (unpublished
data).
Active-site motifs.
Several conspicuous motifs exist which may
correspond to active-site residues of DAHP synthase, but not KDOP
synthase. GPCS, KPRTS/T, and IGAR motifs are indicated in Fig. 7.
Missense mutants within the GPCS and IGAR motifs of E. coli
AroAW have been reported (40). (Interestingly,
eight family II [plant-type] DAHP synthases in the database possess a
KPRS motif in the same region of the primary sequence occupied by KPRT
in the enzyme members of family I.) Of the remaining nine conserved
residues which are potential active-site residues of DAHP synthase but
not KDOP synthase, three have been shown to be essential for activity
in E. coli AroAW (40), as indicated
in Fig. 7.
Subfamily I
DAHP synthases, as exemplified by studies with
E. coli isoenzymes (
46), are dependent on divalent metals
for
activity and are inactivated by EDTA treatment. The conserved
C-61
residue of
E. coli AroA
F has been shown to be a
catalytic
residue, and it has been noted that the nearby conserved H-64
provides an appropriate motif for a metal coordination center
(
47). If so, this center would not be available for
subfamily
I
members, which lack the H-64 residue. Consistent with
this,
B. subtilis AroA was unaffected by EDTA
(
30), and KDOP synthase
does not require divalent metals for
activity (
41).
B. subtilis Aro may lack an
active-site sulfhydryl, since sulfhydryl reagents
failed to inhibit
activity (
30).
Independently evolved regulatory domains.
Bacillus
subtilis and Staphylococcus xylosus possess putative
gene fusions yielding DAHP synthase and chorismate mutase as coexisting
domains of a bifunctional protein (AroGAroA). The chorismate-mutase
catalytic domain is also the regulatory domain governing allosteric
control by chorismate and prephenate (29). Since the AroG
domains belong to a different protein family, these have been excluded
from the multiple alignment of Fig. 7. The Synechocystis
AroAF protein is known to be sensitive to feedback inhibition by L-phenylalanine (24). It possesses
a unique N-terminal extension of 72 residues that also is not shown in
Fig. 7. We suggest that this might correspond to a regulatory
domain which governs feedback inhibition by
L-phenylalanine. A discrete domain governing sensitivity to
feedback inhibition of E. herbicola PheA by
L-phenylalanine which is similar in size has been
reported (52), although obvious homology is not apparent.
KdsA is not known to be sensitive to allosteric control. Thus, the
portion of sequences that align with class IK proteins
for the entire family I assemblage would appear to correspond to the
basic KDOP-DAHP synthase catalytic domains.
All members of subfamily I
are sensitive to feedback inhibition by
one of the aromatic amino acids (two in the case of Cgl
AroA
FY). A number of residues shown to be important for
feedback
inhibition in a given isoenzyme of
E. coli are
conserved in all
other members of subfamily I
, regardless of
substrate specificity,
e.g., P-18 and S-179 (numbered with reference to
E. coli AroA
W).
On the other hand, other
residues important for feedback inhibition
are uniquely conserved in
correlation with the specificity for
feedback inhibitor, e.g., V-160
for
E. herbicola AroA
W,
E. coli AroA
W, and
Buchnera aphidicola
AroA
W. Ray et al. (
40) concluded
that a common
ancestor evolved an aromatic binding pocket which
utilizes residues
scattered throughout the primary sequence. If
so, the DAHP synthase of
Corynebacterium glutamicum may be the
contemporary protein
that is closest to the ancestral DAHP synthase.
It further seems likely
that the N-terminal residues within subfamily
I
(which have no
counterparts in subfamily I
) interact with
other residues
interspersed throughout the primary sequence to
accomplish feedback
inhibition. Indeed, a conserved proline within
the N-terminal extension
(P-18 with reference to
E. coli AroA
W)
has been
shown to be essential for sensitivity to feedback inhibition
by
L-tryptophan (
40).
Evolutionary scenario.
The foregoing information indicates
that an ancient gene encoding a fundamental broad-specificity catalytic
entity duplicated to produce the initial ancestors of subfamily I
and subfamily I. Members of class IK exemplify
contemporary catalytic proteins which never acquired allosteric
regulation. Class ID proteins acquired allosteric
specificities by recruiting N-terminal allosteric domains through
fusion mechanisms. In subfamily I, acquisition of an N-terminal
extension may also have been crucial for an aromatic binding pocket
needed for allostery. Following gene duplication, alterations of key
residues narrowed specificity for a given aromatic amino acid, thus
yielding, for example, the three E. coli isoenzyme paralogs
AroAF, AroAY, and AroAW. Hence,
members of family I are homologs with respect to catalytic domain, but
not necessarily with respect to regulatory domains. Narrowed
specificity for inhibition of DAHP synthase by
L-phenylalanine occurred within subfamily I at least
twice (e.g., leading to E. coli AroAF and
Saccharomyces cerevisiae AroAF), but these
both were derived from the putative ancestral aromatic amino acid
binding pocket. In contrast, the phenylalanine-binding region acquired
by Synechocystis AroAF within subfamily I was
a completely independent event of domain acquisition.
The atomic relationships which dictate substrate specificity will be of
future interest at several levels. (i) An established
narrow-specificity DAHP synthase in class I
D (
B. subtilis AroA)
exhibits greater overall similarity to a known
narrow-specificity
KDOP synthase (
E. coli KdsA) than to a
known narrow-specificity
DAHP synthase homolog in subfamily I
(
E. coli Aro
F). (ii) Each
of the three homolog
groups considered in this report may prove
to contain both
broad-specificity and narrow-specificity members.
Established
broad-specificity DAHP synthases have not yet been
sequenced, and most
of the proteins encoded by sequenced genes
have not been characterized
for specificity.
| |
ACKNOWLEDGMENTS |
We appreciate the input of Anne Hendry (Hamilton General
Hospital), who initiated studies of N. gonorrhoeae in our
laboratories. We thank Carol A. Bonner for the generous provision of
the bifunctional dehydroquinaseshikimate dehydrogenase from
Nicotiana tabacum. We thank the Gonococcal Genome Sequencing
Project (University of Oklahoma) for availability of sequence data
prior to publication.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Cell Science, University of Florida, Gainesville, FL 32611. Phone: (352) 392-9677. Fax: (352) 392-5922. E-mail:
rjensen{at}micro.ifas.ufl.edu.
This paper is Florida Agricultural Station Series no. R-05902.
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J Bacteriol, January 1998, p. 119-127, Vol. 180, No. 1
0021-9193/98/$04.00+0
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Abstract
Introduction
