Establishment of an in vitro D-cycloserine-synthesizing system by using O-ureido-L-serine synthase and D-cycloserine synthetase found in the biosynthetic pathway

Article abstract of DOI:10.1128/AAC.02291-12

We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic, D-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which generates L-cysteine using O-acetyl-L-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-L-serine using O-acetyl-L-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase converting O-ureido-D-serine into D-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-L-serine and hydroxyurea, synthesis of D-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and DcsG are necessary for the syntheses of O-ureido-L-serine and D-cycloserine, respectively. DcsD was also able to catalyze the synthesis of L-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclic D-amino acid analogs, such as D-homocysteine thiolactone. Copyright

Full text of DOI:10.1128/AAC.02291-12

Establishment of an In Vitro D-Cycloserine-Synthesizing System by
Using O-Ureido-L-Serine Synthase and D-Cycloserine Synthetase
Found in the Biosynthetic Pathway
Narutoshi Uda, Yasuyuki Matoba, Takanori Kumagai, Kosuke Oda, Masafumi Noda, Masanori Sugiyama
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis
antibiotic, D-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the se-
quence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopi-
melate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which gen-
erates L-cysteine using O-acetyl-L-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-L-serine using
O-acetyl-L-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an
ATP-dependent synthetase converting O-ureido-D-serine into D-cycloserine. In the present study, to characterize the enzymatic
functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The
biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC),
high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demon-
strate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-L-serine and
hydroxyurea, synthesis of D-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and
DcsG are necessary for the syntheses of O-ureido-L-serine and D-cycloserine, respectively. DcsD was also able to catalyze the syn-
thesis of L-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also
form other cyclic D-amino acid analogs, such as D-homocysteine thiolactone.
he antituberculosis antibiotic D-cycloserine (D-CS) is pro- (16), respectively. These genes are apparently responsible for self-
T
duced by several Streptomyces species (1). D-CS, which is a
resistance to D-CS. On the basis of a sequence homology search of
the open reading frames that were found in the D-CS biosynthetic
gene cluster, our group revised the putative D-CS biosynthetic
pathway (14), as shown in Fig. 1.
cyclic analog of D-alanine, prevents the catalytic activities of both
alanine racemase (2, 3) and D-alanyl–D-alanine ligase (4, 5), which
are necessary for the biosynthesis of the bacterial cell wall; that is,
the antibiotic functions as an inhibitor of bacterial cell wall bio-
synthesis. D-CS is clinically used as a second line of defense against
Mycobacterium tuberculosis, especially when the bacterium is re-
sistant to other antituberculosis antibiotics (6, 7). Furthermore,
since D-CS was recently shown to be a partial agonist for the N-
methyl-D-aspartate receptor, the drug may be useful for the treat-
ment of various psychological dysfunctions (8–10).
The transfer of the acetyl group onto L-serine may be catalyzed
by DcsE, which is a homolog of L-homoserine O-acetyltransferase.
Hydroxyurea may be generated as the result of the hydrolysis of
␻
N -hydroxy-L-arginine by DcsB, an arginase homolog. The syn-
thesis of L-OUS from L-OAS and hydroxyurea may be catalyzed by
DcsD, since some O-acetylserine sulfhydrylases that display a high
level of similarity to DcsD can synthesize various derivatives of
␤-substituted L-alanine from L-OAS and a nucleophilic reagent
(17, 18). Based on the sequence similarity between DcsC and
known diaminopimelate epimerases, L-OUS might be configura-
tionally changed into D-OUS by DcsC functioning as a putative
racemase. Finally, an ATP- and Mg(II)-dependent enzyme may
catalyze the cyclization of D-OUS and the hydrolysis of the urea
moiety. Three proteins encoded by the D-CS biosynthetic gene
cluster (DcsG, DcsH, and DcsI) are suggested to be ATP- and
On the basis of evidence from substrate-feeding experiments
conducted by other research groups (11–13), a putative D-CS bio-
synthetic pathway has been proposed. The first step of D-CS bio-
synthesis is the transfer of an acetyl residue from acetyl coenzyme
A onto the hydroxyl group of L-serine (11, 13). The resulting O-
acetyl-L-serine (L-OAS) would be converted into O-ureido-L-ser-
ine (L-OUS) in the presence of hydroxyurea (13). Hydroxyurea
was found to be synthesized by the D-CS-producing microorgan-
ism (12). L-OUS might then be configurationally changed into
D-OUS by a racemase (13). Finally, to generate D-CS, the cycliza-
tion of D-OUS and the hydrolysis of the urea moiety would be
catalyzed by an ATP- and Mg(II)-dependent enzyme (13).
Recently, our group successfully cloned a D-CS biosynthetic
gene cluster from the genomic DNA of the D-CS-producing or-
ganism Streptomyces lavendulae ATCC 11924 (14). The gene clus-
ter consists of 10 open reading frames, designated dcsA to dcsJ.
Our group has previously found that the dcsI and dcsJ genes from
another D-CS-producing microorganism encode a D-CS-resistant
D-alanyl–D-alanine ligase (15) and a putative D-CS efflux pump
Received 13 November 2012 Returned for modification 12 December 2012
Accepted 18 March 2013
Published ahead of print 25 March 2013
Address correspondence to Masanori Sugiyama, sugi@hiroshima-u.ac.jp.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.02291-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.02291-12
June 2013 Volume 57 Number 6
Antimicrobial Agents and Chemotherapy p. 2603–2612
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Uda et al.
lase and cystathionine ␤-synthase, the amino group of the first
substrate, either L-OAS or L-serine, is covalently attached to PLP
instead of to the amino group of the Lys residue at the active site.
The Lys residue then acts as a base to catalyze an eliminative dou-
ble-bond formation, resulting in the generation of an ␣-aminoac-
rylate intermediate, which shows an absorption maximum at
about 470 nm. The final step is that a nucleophilic reagent (sulfide
or L-homocysteine) attacks the ␤-carbon of the intermediate to
release either L-cysteine or cystathionine, respectively.
In contrast, sequence homology searches for DcsG suggested
that this protein is a member of the ATP-grasp superfamily (19,
20), which includes various enzymes, such as D-alanyl–D-alanine
ligase, glutathione synthetase, biotin carboxylase, and carbamoyl
phosphate synthetase. In general, sequence similarities among the
ATP-grasp enzymes are low. All of the ATP-grasp enzymes cata-
lyze an ATP-dependent ligation between a carboxyl group carbon
of one substrate and an amino or imino group nitrogen or a thiol
group sulfur of the second substrate. In the initial step of the
reaction mechanism, the acylphosphate intermediates are
formed, accompanied by the conversion of ATP to ADP. A cova-
lent bond is then formed, along with the release of phosphate.
Although DcsG has been suggested to be an ATP-grasp enzyme, it
has been difficult to predict the accurate function of DcsG due to
the absence of clear sequence similarity to any functionally de-
fined proteins.
The present study reveals that D-CS is formed when a mixture
of purified recombinant DcsC, DcsD, and DcsG is incubated to-
gether with L-OAS and hydroxyurea as substrates. Enzymatic ki-
netic analyses were also performed to characterize DcsD and
FIG 1 Predicted biosynthetic pathway of D-CS in S. lavendulae ATCC 11924.
CoA, coenzyme A.
Mg(II)-dependent enzymes with an ATP-grasp fold motif (19, DcsG.
20). However, DcsI has been demonstrated by our studies to be a
MATERIALS AND METHODS
D-CS-resistant D-alanyl–D-alanine ligase (15). In addition, our
group observed that a dcsG disruption mutant from the D-CS-
producing organism S. lavendulae does not produce D-CS (14).
These results indicate that DcsG is the best candidate for the cy-
clization and hydrolysis of D-OUS.
Our group has already confirmed the catalytic functions of
DcsB and DcsE (14). Based on our putative biosynthetic pathway
of D-CS (14), another research group recently confirmed that the
Reagents. Most of the reagents used in this study were of analytical grade
and purchased from Wako Chemical, Japan. Reagents from other suppli-
ers are indicated in parentheses. L-OUS was chemically synthesized from
L-CS (Sigma), and D-OUS and ␤-aminooxy-D-alanine were synthesized
from D-CS, according to methods described previously (27, 28).
DNA manipulations. Genomic DNA of S. lavendulae ATCC 11924
was isolated according to a standard protocol (29). Plasmid DNA was
isolated from Escherichia coli by using the Wizard Plus Minipreps DNA
racemization of OUS is catalyzed by DcsC (21). Furthermore, our purification system (Promega).
Preparations of DcsC, DcsD, and DcsG. C-terminally His6-tagged
recent mutagenesis analysis suggested that DcsA, which exhibits
heme-binding-protein-like properties, catalyzes the hydroxyla-
tion of L-arginine to yield the substrate for DcsB (22), although the
catalytic function of DcsA has not been demonstrated in vitro. In
the present study, we focus on the catalytic functions of DcsD and
DcsG.
DcsD has high sequence similarity with O-acetylserine sulfhy-
drylase and cystathionine ␤-synthase. O-Acetylserine sulfhydry-
lase is an enzyme that catalyzes L-cysteine synthesis using L-OAS
DcsC, DcsD, and DcsG were expressed in E. coli and purified to near
homogeneity. Recombinant proteins were prepared as described in the
supplemental material. Each protein concentration was determined by
measuring the absorbance at 280 nm using the molar extinction coeffi-
cients 20,700, 26,300, and 35,900 MϪ1 cmϪ1, respectively.
Analysis of molecular mass. The molecular mass of the purified en-
zymes was estimated by high-performance liquid chromatography
(HPLC), using a Superdex 200 10/300 GL column (GE Healthcare) at 0.75
ml minϪ1 with 100 mM sodium phosphate buffer (pH 7.5) containing
and sulfide (23, 24), which is the last step in L-cysteine biosynthe- 0.15 M NaCl. The protein standards used for the calibration were as fol-
lows: ferritin (440 kDa), bovine serum albumin (67 kDa), ovalbumin (43
kDa), and RNase A (13.7 kDa). The proteins were detected at 280 nm by
using a multiwavelength detector (MD-2010; Jasco). To estimate the mo-
lecular mass of the purified proteins, three repetitions of the experiments
were done.
Absorption spectroscopy. Prior to spectroscopic analysis, DcsD was
dialyzed against 250 mM potassium phosphate buffer (pH 8.0) containing
1 ␮M PLP. The absorption spectrum of DcsD (15 ␮M) in the absence or
the presence of the given concentrations of L-OAS (Sigma) was recorded
at 20°C in a 1-cm-path-length cuvette using a V-550 spectrophotometer
sis, whereas cystathionine ␤-synthase synthesizes cystathionine
using L-serine and L-homocysteine in the transsulfuration path-
way from L-methionine to L-cysteine (25, 26). These enzymes
commonly require pyridoxal 5=-phosphate (PLP) as a cofactor to
express the catalytic activity. PLP is covalently bound to the amino
group of the Lys residue at the active center of the enzyme. The
absorption spectrum of the PLP-bound enzyme displays a local
maximum at about 412 nm, in addition to a local maximum at
about 280 nm derived from the Tyr and Trp residues in the en-
zyme. In the first step of the catalysis of O-acetylserine sulfhydry- (Jasco).
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Two Novel Enzymes Found in D-CS Biosynthetic Pathway
In vitro D-CS synthesis. DcsC, DcsD, and DcsG (0.1 mg mlϪ1 each) the ratio of methanol was linearly increased from 15 to 49% in 20 min. For
were incubated for 2 h at 30°C in 100 mM Tris-HCl buffer (pH 7.8) the kinetic analysis, peak areas on the HPLC chromatogram were used as
containing 1 mM L-OAS, 1 mM hydroxyurea, 10 mM KCl, 5 mM MgCl2, indicators.
and 2 mM ATP. When L- or D-OUS was used as a starting material, the
Next, the kinetic response toward increasing concentrations of L-OAS
reagent was added at a concentration of 1 mM instead of L-OAS and was measured in the presence of 50 mM hydroxyurea at pH 8.0. A reaction
hydroxyurea. Negative-control experiments without addition of an en- mixture consisting of 250 mM potassium phosphate (pH 8.0), 5 to 300
zyme, ATP, or Mg(II) were also performed. Enzymatic reactions were mM L-OAS, 50 mM hydroxyurea, and 2.0 ␮g mlϪ1 DcsD was incubated at
stopped by boiling at 100°C for 5 min, and solutions were filtered through 30°C for 10 min. To determine the kinetic parameters for hydroxyurea, a
a membrane filter (0.2-␮m pore size; Advantec). The productivity of D-CS reaction mixture that consisted of 250 mM potassium phosphate (pH
was analyzed by HPLC. Aliquots (10 ␮l) were injected into a Senshu Pak 8.0), 100 mM L-OAS, 1 to 100 mM hydroxyurea, and 2.0 ␮g mlϪ1 DcsD
SCX-1251-N column (250 by 4.6 mm; Senshu Scientific) and eluted with was incubated at 30°C for 10 min. The amount of L-OUS formed in the
an isocratic mobile phase of 10 mM ammonium acetate buffer (pH 4.6). reaction mixture was determined by HPLC analysis.
The flow rate was set to 0.8 ml minϪ1. D-CS was detected at 210 nm by
The enzymatic activity of DcsD to synthesize L-cysteine, S-sulfo-L-
using a multiwavelength detector (MD-2010; Jasco), and its configuration cysteine, or cystathionine was determined by a colorimetric method es-
was conveniently confirmed by using a chiral detector (OR-2090; Jasco). tablished previously (31–33). Experimental procedures are described in
To further confirm the configuration of the product, a 50-␮l portion of detail in the supplemental material.
the reaction mixture was injected into a Senshu Pak SCX-1251-N column
Analysis of the enzymatic activity of DcsC. A reaction mixture con-
and eluted with ammonium acetate buffer (pH 4.6). A peak correspond- sisting of 50 mM potassium phosphate (pH 8.0), 1 mM L-OUS or D-OUS,
ing to D-CS was collected and then lyophilized. The sample was mixed and 1.0 ␮g mlϪ1 DcsC was incubated at 30°C for 20 min. HPLC analysis
with 10 ␮l of a 1 mM L-alanine solution as an internal control and 5 ␮l of was carried out after derivatization with O-phthaldialdehyde and
a derivatization solution (30) containing O-phthaldialdehyde and N-acetyl-L-cysteine, as described above.
N-acetyl-L-cysteine. Aliquots were injected into an ODS-A column (100
Analysis of the enzymatic activity of DcsG. Before the measurement
by 4.6 mm; YMC) equilibrated with 85% solvent A (50 mM sodium ace- of enzyme activity, stock solutions of D-OUS, L-OUS, ␤-aminooxy-D-
tate at pH 5.2) and 15% solvent B (methanol). The ratio of solvent B was alanine, D-2,4-diaminobutyrate (2HCl form; Watanabe Chemical), D-or-
linearly increased from 15 to 66% in 20 min. The flow rate was set to 1.0 ml nithine (HCl form), D-homocysteine (Chemcube), D-homoserine, D-ala-
minϪ1. A derivative from the product was detected by the absorbance at nine, D-asparagine, and ATP (2Na form) were newly prepared. The pH of
340 nm using a multiwavelength detector. Generation of D-CS was further the stock solutions was adjusted to approximately 7 with HCl or NaOH.
confirmed by a coinjection experiment using authentic D-CS. Coinjection The enzyme product was analyzed by TLC, HPLC, or ESI-TOF MS anal-
experiments were performed in each of the cases in the present study.
ysis. The conditions for TLC analysis were the same as those applied for
Analysis of the enzyme activity of DcsD. Before the measurement of the detection of intact ␤-substituted L-alanine. The conditions for HPLC
enzyme activity, stock solutions of L-OAS (HCl form), hydroxyurea, so- analysis to detect products other than D-CS were similar to those applied
dium sulfide, sodium thiosulfate, and L-homocysteine (Biosynth) were for the detection of intact D-CS. In this case, however, the elution solution
newly prepared, and the pH of the stock solutions was adjusted to ca. 7 was changed to 100 mM ammonium acetate, and the products were de-
with HCl or NaOH. The reaction mixture, consisting of 50 mM MES tected by the absorbance at 240 nm.
(2-morpholinoethanesulfonic acid)-KOH (pH 6.5), 1 mM EDTA, 5 mM
The pH dependence of DcsG activity to synthesize D-CS from D-OUS
L-OAS, 5 mM a second substrate (hydroxyurea, sodium sulfide, sodium was measured. The reaction mixture, which consisted of 250 mM MES-
thiosulfate, or L-homocysteine), and 20 ␮g mlϪ1 DcsD, was incubated at KOH (pH 5.0 to 7.5) or Tris-HCl (pH 7.5 to 10.0), 10 mM KCl, 5 mM
30°C for 2 h. As a control, the same reaction mixture without DcsD was MgCl2, 5 mM ATP, 1 mM D-OUS, and 5 ␮g mlϪ1 (at pH 7.0 to 9.0) or 50
used. The enzymatic reaction was stopped by boiling at 100°C for 5 min, ␮g mlϪ1 (at pH 5.0 to 6.5 and 9.5 to 10.0) DcsG, was incubated at 30°C for
and any precipitate was removed by centrifugation. The supernatant was 10 min. HPLC analysis to detect the amount of D-CS formed was per-
used for product analysis. The formation of the enzymatic product was formed as described above. HPLC analysis was also performed to detect
conveniently analyzed by thin-layer chromatography (TLC) using silica the amount of liberated ADP. Aliquots (10 ␮l) were injected into a Hy-
gel 60 F254 (Merck). In the case of detection of L-cysteine, the supernatant drosphere C18 column (100 by 4.6 mm; YMC) and eluted with the iso-
was mixed with an equivalent volume of a 5 mM N-ethylmaleimide solu- cratic mobile phase of 100 mM potassium phosphate buffer (pH 5.5). The
tion and incubated at room temperature for 20 min before TLC analysis. flow rate was set to 1.0 ml minϪ1. ATP and ADP were detected at 260 nm
The solvent for the chromatogram was 2-butanone–pyridine–water (18: by using a multiwavelength detector.
5:10). Amino acids were visualized by ninhydrin. Furthermore, the enzy-
The kinetic parameters of DcsG for the synthesis of D-CS from D-OUS
matic products were analyzed by electrospray ionization-time of flight were determined by an ADP release-coupled assay (34). A reaction mix-
mass spectrometry (ESI-TOF MS) using a QSTAR XL spectrometer (Ap- ture consisting of 250 mM Tris-HCl (pH 8.0), 10 mM KCl, 5 mM MgCl2,
plied Biosystems). It should be noted that although L-OAS in solution was 2.5 mM potassium phosphoenolpyruvate, 0.3 mM NADH, 1 mM dithio-
slowly hydrolyzed to L-serine and acetate, the generation of L-serine from threitol, 45 U mlϪ1 pyruvate kinase, 20 U mlϪ1 lactate dehydrogenase
L-OAS was increased by the boiling treatment. Therefore, the L-serine that (Oriental Yeast), D-OUS and ATP at the given concentrations, and 18 ␮g
was detected by the TLC and MS analyses appears to have been generated mlϪ1 DcsG was incubated at 30°C. The kinetic parameters for D-OUS
by boiling treatment rather than by any enzyme reaction.
were determined in the presence of a fixed concentration (5 mM) of ATP
We measured the pH dependence of the L-OUS-synthesizing activity and variable concentrations (0.5 to 100 mM) of D-OUS. On the other
of DcsD. A reaction mixture consisting of 250 mM potassium phosphate hand, the kinetic parameters for ATP were determined in the presence of
(pH 5.0 to 9.0), 100 mM L-OAS, 2 mM hydroxyurea, and 5 ␮g mlϪ1 DcsD a fixed concentration (50 mM) of D-OUS and variable concentrations (4
was incubated at 30°C for 10 min. The reaction was started by the addition to 500 ␮M) of ATP. The rate of the decrease in absorbance at 340 nm of
of hydroxyurea after preincubation for 10 min and stopped by heat treat- NADH (⑀ ϭ 6,220 MϪ1 cmϪ1) was monitored by using a V-550 spectro-
ment at 100°C for 5 min. DcsD enzyme activity was analyzed by HPLC photometer. The kinetic response of DcsG toward the other amino acids
after derivatization with O-phthaldialdehyde and N-acetyl-L-cysteine. A as a substrate was also analyzed by the ADP release-coupled assay.
10-␮l aliquot of the reaction mixture (diluted, if necessary) was mixed
Enzymatic kinetic study. In general, an appropriate amount of en-
with 10 ␮l of a 1 mM L-threonine solution as an internal control and 5 ␮l zyme was added to the reaction mixture. The amount of DcsD and DcsG
of a derivatization solution (30). Aliquots were injected into an ODS-A added in the present study was adjusted so that the amount of the enzy-
column and eluted in a way similar to that described above. In this case, matic products became less than 30% of the maximum amount, which
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Uda et al.
tion column demonstrated that the molecular masses of DcsC,
DcsD, and DcsG are 34 Ϯ 3, 126 Ϯ 2, and 26 Ϯ 4 kDa, respectively,
indicating that DcsC and DcsG are monomers, whereas DcsD is a
tetramer. Diaminopimelate epimerases and enzymes with an
ATP-grasp fold, in general, are known to be monomeric proteins.
It should be noted, however, that O-acetylserine sulfhydrylases are
dimeric or tetrameric. The difference of molecular mass between
the observed and predicted values for DcsG was 8 kDa, which
accounts for 24% of the predicted molecular mass. Although the
reason for the large difference is unclear, DcsG may take the com-
pact structure.
To synthesize D-CS in vitro, a mixture composed of the DcsC,
DcsD, and DcsG proteins was incubated together with L-OAS and
hydroxyurea as substrates. The synthesis of D-CS was detected
when Mg(II) ion and ATP were added into a protein mixture
supplemented with L-OAS and hydroxyurea as substrates
(Fig. 3B). For D-CS production, all three enzymes, Mg(II) ion, and
ATP are necessary (Fig. 3C to F). Note that whether D-CS or L-CS
was produced from these reactions cannot be distinguished by
HPLC analysis using the Senshu Pak SCX-1251-N column, which
was used to determine the product being produced, since both
compounds are eluted at the same retention time. However, the
product appeared to be D-CS, since it showed a positive chirality.
The configuration of the product was further confirmed by HPLC
analysis after derivatization with O-phthaldialdehyde and
N-acetyl-L-cysteine (Fig. 4).
To verify the enzyme reaction in the D-CS biosynthetic path-
way, we prepared L-OUS and D-OUS chemically from L-CS and
D-CS, respectively (27, 28). When DcsC and DcsG were incubated
with Mg(II), ATP, and either L-OUS or D-OUS as the substrate,
D-CS was synthesized (Fig. 5A and B). D-CS was synthesized from
D-OUS, even without DcsC (Fig. 5D), but not from L-OUS
(Fig. 5C). To synthesize D-CS using D-OUS, Mg(II) and ATP were
necessary. These results provide additional evidence in support of
FIG 2 SDS-PAGE analysis. Purities of DcsC (lane 1), DcsD (lane 2), and DcsG
(lane 3) were analyzed by SDS-PAGE. Gels were stained by using Coomassie
blue.
was estimated from the initial concentrations of two substrates. In the
ADP release-coupled assay applied for DcsG, the product concentration
could be monitored over time. In this case, the amount of the enzyme was
adjusted so that the rate of the decrease in absorbance at 340 nm became
less than 1 minϪ1. To determine the activity at each data point, three sets
of experiments were done. The initial reaction rates at different substrate
concentrations were analyzed by nonlinear regression fit to the Michaelis-
Menten equation. When the Km value was estimated to be very high, only
the kcat/Km value was calculated from the slope of the linear line in the
kinetic graph.
RESULTS
In vitro D-CS synthesis. In the present study, DcsC, DcsD, and
DcsG were overexpressed in E. coli and purified to near homoge-
neity (Fig. 2). The molecular masses of DcsC, DcsD, and DcsG
monomers are 31, 36, and 34 kDa, respectively, based on the
amino acid sequences. However, HPLC analysis using a gel filtra- the D-CS biosynthetic pathway proposed previously by our group
FIG 3 HPLC profile of products formed after incubation of reaction mixtures consisting of L-OAS and hydroxyurea with DcsC, DcsD, and DcsG in the presence
of MgCl2 and ATP. (A) HPLC profile of authentic D-CS (1 mM). (B) HPLC profile of the incubation mixture consisting of L-OAS, hydroxyurea, MgCl2, and ATP,
which was incubated together with DcsC, DcsD, and DcsG. (C) Incubation without added DcsG. (D) Incubation without DcsD. (E) Incubation without DcsC.
(F) Incubation without ATP and MgCl2. Arrows indicate the peak corresponding to D-CS. mAU, milliabsorbance unit.
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Two Novel Enzymes Found in D-CS Biosynthetic Pathway
Evaluation of DcsD activity. The ability of DcsD to synthesize
L-OUS was conveniently examined by TLC analysis. However, the
accurate evaluation of DcsD activity was performed by HPLC
analysis after derivatization with O-phthaldialdehyde and
N-acetyl-L-cysteine. The derivatization method (30) employed in
this experiment has the advantage of being able to determine the
product configuration, whereas determining the configuration by
TLC analysis is difficult. In the present study, DcsD was confirmed
to catalyze the synthesis of L-OUS, but not D-OUS, by using L-OAS
and hydroxyurea as substrates (Fig. 6). Furthermore, when L-ser-
ine was used instead of L-OAS, no reaction products were gener-
ated.
Like O-acetylserine sulfhydrylase and cystathionine ␤-syn-
thase, purified recombinant DcsD was yellow. When DcsD was
preincubated with hydroxylamine, L-OUS-synthesizing activity
was completely lost, based on the HPLC analysis. However, the
activity of DcsD preincubated with hydroxylamine was restored
by the addition of PLP after the dialysis, indicating that DcsD is
evidently a PLP-dependent enzyme. The UV-visible (UV-vis)
spectrum of DcsD showed an absorption maximum at 412 nm
(solid line in Fig. 7A). The addition of sufficient L-OAS (100 mM)
quickly caused a change in the spectrum due to the formation of
an ␣-aminoacrylate intermediate (dotted line in Fig. 7A), as found
for other O-acetylserine sulfhydrylases.
When the concentration of the substrate L-OAS was increased
to evaluate the kinetic response of DcsD for L-OUS or L-cysteine
synthesis, the Michaelis constant (Km) for L-OAS was determined
to be Ͼ200 mM. Since the Km values of the known O-acetylserine
sulfhydrylase enzymes for L-OAS are in the low-millimolar range,
the Km value of DcsD is 1 to 2 orders of magnitude higher than that
FIG4 Configuration analysis of the product after derivatization. (A and B) HPLC
profile of authentic derivatives from D-CS (A) and L-CS (B) (1 mM each). (C)
HPLC profile of the derivative that was formed by incubation with DcsC, DcsD,
and DcsG. Derivatives from D- and L-CS have retention times of about 15.5 and
16.5 min, respectively. The peak eluting with a retention time of about 13.5 min
corresponds to a derivative from L-alanine used as an internal control.
(Fig. 1); that is, DcsD catalyzes the biosynthesis of L-OUS using of O-acetylserine sulfhydrylases.
L-OAS and hydroxyurea. We also confirmed that DcsC catalyzes
The absorption spectra of the DcsD solution (15 ␮M) were
the conversion from L-OUS to D-OUS. DcsG was necessary to recorded after the addition of various concentrations of L-OAS
catalyze the synthesis of D-CS from D-OUS in an Mg(II)- and (Fig. 7B). The rate of the increase in absorbance at 470 nm, which
ATP-dependent manner.
is due to the formation of an ␣-aminoacrylate intermediate, was
FIG 5 HPLC profile of D-CS formed after incubation of OUS with DcsC and DcsG. (A and C) L-OUS; (B and D) D-OUS. In the case of panels C and D, DcsC was
eliminated from the reaction mixture. Arrows indicate the peak corresponding to D-CS.
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Uda et al.
FIG 6 HPLC profile of the reaction mixture of DcsD after derivatization. (A
and B) Authentic derivatives from L-OUS (A) and D-OUS (B) (1 mM each).
(C) The product by DcsD was analyzed after incubation with 100 mM L-OAS
and 20 mM hydroxyurea. Derivatives of D- and L-OUS eluted at about 9.5 and
11 min, respectively. The peak eluting at a retention time of about 14 min
corresponds to a derivative of L-threonine used as an internal control. The
peak at a retention time of about 7 min corresponds to a derivative of L-serine.
FIG 7 Absorption spectrum of DcsD. (A) Absorption spectrum of solutions
containing 15 ␮M DcsD in the absence (solid line) or the presence (dotted
line) of 100 mM L-OAS. (B) Time course of the increase of absorption at 470
nm. The increase of absorption, which is produced by the formation of the
␣-aminoacrylate intermediate, was monitored after the addition of a given
concentration (1, 3, 10, and 30 mM) of L-OAS to a solution containing DcsD.
(C) Changes in the spectrum after the addition of 1 mM L-OAS to a solution
containing DcsD. The bold line represents the spectrum immediately after the
addition of L-OAS. The spectrum was recorded every 3 min. AU, arbitrary
units.
found to be dependent on the concentration of added L-OAS. In
addition, the absorbance at 470 nm in the plateau phase was also
dependent on the concentration of added L-OAS at lower concen-
trations. The absorbance in the plateau phase was scarcely altered
for concentrations of L-OAS over 10 mM, indicating that all of the
enzyme molecules were converted into ␣-aminoacrylate interme-
diates in this concentration range. However, the rate of increase in
absorbance at 470 nm at 30 mM was much higher than that at 10
mM. The increasing rate (k) should be expressed as follows (35):
fhydrylases. This may be another reason for the high Km value of
DcsD toward L-OAS.
kmax · [L-OAS]
k ϭ
K ϩ [L-OAS]
When assayed in 250 mM potassium phosphate buffer (pH 5.0
where kmax is the value of k at a saturating L-OAS concentration to 9.0) in the presence of 100 mM L-OAS, L-OUS-synthesizing
and K is the dissociation constant of the complex between PLP- activity increased with an increase of pH (Fig. 8A). Alkaline pH
bound DcsD and L-OAS. Our spectroscopic analysis indicates that conditions may be useful to increase the nucleophilicity of hy-
K is very high, which may be one of the reasons for the high Km droxyurea via deprotonation. The kinetic parameters of DcsD for
value of DcsD toward L-OAS.
When L-OAS (1 mM) was added to the DcsD solution, the mM potassium phosphate buffer at pH 8.0 (Table 1).
absorbance at 470 nm initially increased to a maximum within a Substrate specificity of DcsD. In general, cystathionine ␤-syn-
the synthesis of ␤-substituted L-alanine were determined in 250
few minutes, whereas the absorbance at 412 nm decreased to a thase uses L-homocysteine as a nucleophilic reagent, whereas O-
minimum (Fig. 7C). Following this, the absorbance at 470 nm acetylserine sulfhydrylase uses sulfide. These enzymes are known
gradually decreased again as the ␣-aminoacrylate intermediate to have broad specificity for the second substrate and have been
degraded, whereas the absorbance at 412 nm gradually increased. suggested to catalyze the synthesis of various ␤-substituted L-ala-
This change occurred within 20 min, and the resulting spectrum nine derivatives (17, 18, 26). O-Acetylserine sulfhydrylases are
corresponds with that of the enzyme-bound PLP. Compared with classified into two types, A and B, based on their amino acid se-
the results from similar experiments using O-acetylserine sulfhy- quences. In particular, O-acetylserine sulfhydrylase B is known to
drylases (36), the lifetime of the ␣-aminoacrylate intermediate in have high activity for the synthesis of S-sulfo-L-cysteine when
DcsD is obviously shorter than that in normal O-acetylserine sul- thiosulfate is used as a nucleophilic reagent.
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Two Novel Enzymes Found in D-CS Biosynthetic Pathway
FIG 8 The pH dependencies of DcsD and DcsG. (A) D-OUS-synthesizing
activity of DcsD; (B) D-CS-synthesizing activity of DcsG. Potassium phosphate
buffer was used for the pH range from 5.0 to 9.0 in panel A, while MES-KOH
and Tris-HCl buffers were used for the pH ranges from 5.0 to 7.5 and from 7.5
to 10.0, respectively, in panel B. Bars indicate the standard deviations.
FIG 9 HPLC profile of reaction mixtures of DcsC after derivatization. DcsC
was incubated with L-OUS (A) or D-OUS (B).
Using recombinant DcsD, we examined the specificity toward
the second substrate. The reagents tested were hydroxyurea, so- ADP. By using HPLC analysis, the stoichiometric ratio of synthe-
dium sulfide, sodium thiosulfate, and L-homocysteine, and the sized D-CS to ADP was found to be approximately 1:1. In the first
expected products were L-OUS, L-cysteine, S-sulfo-L-cysteine, and reaction step, DcsG is predicted to catalyze the formation of a
cystathionine, respectively. The TLC and MS analyses demon- D-OUS–phosphate intermediate accompanied by hydrolysis of
strated that the expected products were formed in reaction mix- ATP to ADP. Next, the N-␦ atom of the D-OUS–phosphate inter-
tures consisting of L-OAS, the second substrate, and DcsD. The mediate would attack its own carbonyl carbon to form a five-
kinetic parameters for each substrate are summarized in Table 1. membered ring, with the release of phosphate. It is worth noting
Based on the kcat/Km values (turnover rates), DcsD most prefers that in the catalysis step, due to the activity of DcsG, a covalent
sulfide as the second substrate, followed by hydroxyurea, L-homo- bond is formed intramolecularly (Fig. 1) but not intermolecularly,
cysteine, and thiosulfate.
Evaluation of DcsC activity. DcsC was recently shown to be a D-CS, hydrolysis of the urea moiety must occur (Fig. 1).
cofactor-independent racemase that strictly reacts with OUS (21). Substrate specificity of DcsG. When the synthesis of D-CS
as found in normal ATP-grasp enzymes. In addition, to synthesize
The activity of DcsC was confirmed through the measurement of from D-OUS by DcsG was measured at various pH values, activity
the exchange of ␣-hydrogen of the substrate to deuterium or the was highest at pH 8.0 (Fig. 8B). To examine the substrate specific-
change of the circular dichroism spectrum of the reaction mix- ity of DcsG, an ADP release-coupled assay was performed with
ture. In the present study, to evaluate DcsC activity, HPLC analy- various substrates at pH 8.0 and 30°C (Table 2). The kcat/Km for
sis was conducted after derivatization with O-phthaldialdehyde D-OUS was 2.1 ϫ 105Ϯ 0.3 ϫ 105 MϪ1 sϪ1. Furthermore, we
and N-acetyl-L-cysteine, which is the same method used for the confirmed that L-OUS was not a substrate for DcsG, suggesting
evaluation of L-OUS synthesis by DcsD. We confirmed that DcsC that DcsG reacts exclusively with the D-amino acid.
can catalyze the configurational conversion of OUS in both the L-
to D- and D- to L-directions (Fig. 9).
Since D-alanyl–D-alanine ligase is classified with ATP-grasp en-
zymes, like DcsG, we investigated whether DcsG reacts with D-al-
Evaluation of DcsG activity. As described above, DcsG cata- anine, but no activity was detected with this substrate. When DcsG
lyzes the conversion of D-OUS into D-CS in an Mg(II)- and ATP- reacted with D-OUS, D-CS was formed, which is a cyclic D-amino
dependent manner (Fig. 5D). Since the sequence homology search acid analog with a five-membered ring and an amide linkage. We
for DcsG suggested that the protein has an ATP-grasp motif, the tried to clarify whether DcsG catalyzes the synthesis of cyclic D-
reaction is expected to be coupled with the conversion of ATP to amino acid analogs from other D-amino acids. ␤-Aminooxy-D-
TABLE 1 Kinetic parameters of DcsD
app
app
Mean Km
Mean kcat
Mean kcat/Km
(MϪ1 sϪ1) Ϯ SD
Mean kcat/Km for L-OAS
(MϪ1 sϪ1)b Ϯ SD
2nd substrate
Product
(mM)a Ϯ SD
(sϪ1)a Ϯ SD
Hydroxyurea
Na2S
Na2S2O3
L-OUS
L-Cysteine
S-Sulfo-L-cysteine
Cystathionine
9.0 Ϯ 2.1
0.12 Ϯ 0.02
22 Ϯ 2
NDc
11 Ϯ 1
12 Ϯ 1
0.72 Ϯ 0.03
NDc
1,200 Ϯ 300
100,000 Ϯ 20,000
33 Ϯ 3
150 Ϯ 10
140 Ϯ 20
L-Homocysteine
190 Ϯ 10
a Kmapp and kcatapp were determined in the presence of 100 mM L-OAS as the first substrate.
b kcat/Km for L-OAS was determined in the presence of 50 mM hydroxyurea or 1 mM sodium sulfide.
c Since Kmapp was too high, Kmapp and kcatapp were not determined (ND).
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Uda et al.
TABLE 2 Kinetic parameters of DcsG
Mean Km
(mM)a Ϯ SD
Mean kcat
(sϪ1)a Ϯ SD
Mean kcat/Km
(MϪ1 sϪ1) Ϯ SD
Mean Kmapp for ATP
(␮M)b Ϯ SD
Substrate
D-OUS
10 Ϯ 1
NDc
2,200 Ϯ 200
NDc
210,000 Ϯ 30,000
14,000 Ϯ 1,000
2,100 Ϯ 100
670 Ϯ 40
45,000 Ϯ 9,000
6,100 Ϯ 300
73 Ϯ 8
37 Ϯ 3
45 Ϯ 7
45 Ϯ 2
23 Ϯ 6
54 Ϯ 10
␤-Aminooxy-D-alanine
D-2,4-Diaminobutyrate
D-Ornithine
D-Homocysteine
D-Homoserine
NDc
NDc
NDc
19 Ϯ 3
NDc
NDc
860 Ϯ 60
NDc
a Km and kcat were determined in the presence of 5 mM ATP.
b Kmapp for ATP was determined in the presence of 50 mM D-amino acid.
c Since Km was too high, Km and kcat were not determined (ND).
alanine, which lacks the carbamoyl group in D-OUS, was synthe- including L-OUS. The kcat/Km value of DcsD for L-cysteine synthe-
sized chemically by hydrolyzing D-CS (27). TLC, HPLC, and MS sis is the highest, 80-fold higher than that for L-OUS synthesis
analyses showed that DcsG synthesizes D-CS from ␤-aminooxy- (Table 1). The DcsD protein exhibits lower activities for the syn-
D-alanine. However, the kcat/Km value for this compound was thesis of S-sulfo-L-cysteine and cystathionine than for L-OUS syn-
shown to be an order of magnitude lower than that for D-OUS thesis. These results are in agreement with the fact that the amino
(Table 2).
Other reagents tested were D-asparagine, D-2,4-diaminobu-
acid sequence of DcsD is more homologous to that of O-acetyl-
serine sulfhydrylase A than to those of O-acetylserine sulfhydry-
lase B and cystathionine ␤-synthase. In fact, when a sequence ho-
mology search for DcsD was done against structurally defined
proteins, in order of similarity, O-acetylserine sulfhydrylase A,
O-acetylserine sulfhydrylase B, and cystathionine ␤-synthase were
retrieved. For example, the amino acid sequence of DcsD has 48%
identity with that of Mycobacterium tuberculosis O-acetylserine
sulfhydrylase A, 43% identity with that of E. coli O-acetylserine
sulfhydrylase B, and 38% identity with that of the catalytic domain
of human cystathionine ␤-synthase.
Based on the kinetic parameters shown in Table 1, DcsD would
preferentially synthesize L-cysteine if hydroxyurea and sulfide co-
existed at the same low concentrations. Although previous whole-
genome sequence analyses suggested that Streptomyces species
should possess a sulfate-reducing pathway to generate sulfide (37,
38), it has been reported that bacteria from this genus display low
activity for the reduction of sulfate to sulfide (39). Therefore, in
cells of the D-CS-producing organism S. lavendulae, the concen-
tration of sulfide might be kept at a low level, such that DcsD
would catalyze the synthesis of L-OUS to produce D-CS.
The O-acetylserine sulfhydrylase enzymes from plant sources
were reported previously to display L-OUS-synthesizing activity
(40–45). However, L-OUS-synthesizing activity is generally low
even in the presence of a high concentration of hydroxyurea. Rel-
ative velocities of the enzymes from Allium tuberosum (45), Bras-
sica juncea (42), Citrullus vulgaris (42), Lathyrus latifolius (44), and
Leucaena leucocephala (43) for the synthesis of L-OUS are less than
1% of the velocity to synthesize L-cysteine. Exceptionally, al-
though the enzyme from Spinacia oleracea (40) has a high rate of
synthesis of L-OUS, it is only approximately 10% of its L-cysteine-
synthesizing velocity. However, the rate of synthesis of L-OUS by
DcsD in the presence of a high concentration of hydroxyurea is
comparable to the rate of synthesis of L-cysteine. Therefore, we
tyrate, D-ornithine, D-homoserine, and D-homocysteine. In the
case of D-asparagine, the formation of D-amino-succinimide,
which has a five-membered ring with an amide linkage, like D-CS,
is expected. In the case of D-2,4-diaminobutyrate and D-ornithine,
five- and six-membered rings with amide linkages would be pro-
duced, respectively. However, if DcsG reacts with D-homocysteine
and D-homoserine, five-membered rings with ester and thioester
linkages will be produced, respectively. On the basis of the ADP
release-coupled assay, D-asparagine was not a substrate for DcsG,
probably due to the low nucleophilicity of the amide group nitro-
gen. However, the assay indicated that the other compounds serve
as substrates of DcsG. Furthermore, MS analysis demonstrated
the generation of the monodehydrate products, probably due to
ring formation. Ring formation in products was further con-
firmed by TLC and HPLC analyses, and the configuration was
confirmed by using a chiral detector. The kcat/Km value for these
compounds is lower than that for D-OUS (Table 2). These results
indicated that DcsG most prefers D-OUS as a substrate, followed
by D-homocysteine, ␤-aminooxy-D-alanine, D-homoserine,
D-2,4-diaminobutyrate, and D-ornithine.
DISCUSSION
DcsD. The DcsD protein has a high level of sequence similarity
with O-acetylserine sulfhydrylase and cystathionine ␤-synthase.
The first substrate for O-acetylserine sulfhydrylase is L-OAS,
whereas that for cystathionine ␤-synthase is L-serine. Both en-
zymes are known to be able to synthesize various derivatives of
␤-substituted L-alanine, depending on their specificities toward
the second substrate (17, 18, 26). Cystathionine ␤-synthase,
whose normal function is to generate cystathionine using L-ho-
mocysteine as the second substrate, is also known to synthesize
L-cysteine by using sulfide (26), although the detailed substrate
specificity of the enzyme has not been reported. In contrast, O- concluded that DcsD must be a specific enzyme for the synthesis
acetylserine sulfhydrylases have been reported to exhibit their of L-OUS in the D-CS biosynthetic pathway.
highest activities for the synthesis of L-cysteine among the ␤-sub-
stituted L-alanine derivatives (17, 18).
The kinetic and spectroscopic analyses of DcsD indicate that
the enzyme has a low affinity for L-OAS and that the lifetime of the
Our present study shows that DcsD uses L-OAS, but not L-ser- ␣-aminoacrylate intermediate formed in DcsD is short. These ob-
ine, as the first substrate. Furthermore, we found that DcsD cata- servations suggest that the active site of DcsD is more accessible to
lyzes the synthesis of various ␤-substituted L-alanine derivatives, a solvent than that of O-acetylserine sulfhydrylases. Therefore,
2610 aac.asm.org
Antimicrobial Agents and Chemotherapy

Two Novel Enzymes Found in D-CS Biosynthetic Pathway
DcsD may have a binding preference for the bulky compounds as thetic operon are unknown at this moment, but no obvious role is
the second substrate. In other words, DcsD may be better able to apparent for either protein in D-CS biosynthesis. The results of
react with hydroxyurea, which is bulkier than sulfide, due to the sequence homology searches of DcsF suggest that the protein is a
greater accessibility of the substrate to the active site.
dehydrogenase harboring an NAD-binding motif. In contrast,
DcsG. The DcsG protein catalyzes the synthesis of D-CS from DcsH seems to have an ATP-grasp fold motif, like DcsG and DcsI,
D-OUS, and the reaction includes covalent bond formation and and DcsH displays low sequence similarity with DcsG (16%) and
hydrolysis of the urea moiety. At present, it is uncertain whether DcsI (15%). To clarify the functions of DcsF and DcsH, the ex-
the urea moiety is hydrolyzed before or after the formation of pression of the recombinant proteins in E. coli and gene disrup-
the covalent N-C bond. However, since nucleophilic attack on the tion analyses are now under way.
carbonyl carbon by the N-␦ atom of D-OUS is not favored, the
By using the methods described here, we can now synthesize
urea moiety of D-OUS may be hydrolyzed before the formation of D-CS in vitro using commercially available L-OAS and hy-
the covalent N-C bond. Although DcsG also catalyzes the synthe- droxyurea as substrates (Fig. 3B). Considering that many chemi-
sis of D-CS from ␤-aminooxy-D-alanine, the kcat/Km value for this cal or pharmaceutical companies have successfully synthesized
compound was 1 order of magnitude lower than that with D-OUS D-CS in large quantities, our enzymatic D-CS production method
(Table 2). The carbamoyl group attached in D-OUS may be an may seem insignificant. However, by modifying the enzymes in
important factor for DcsG to catalyze the reaction effectively. Spe- the D-CS biosynthetic pathway, it may be possible to generate
cifically, the Km for ␤-aminooxy-D-alanine was estimated to be novel antibacterial compounds with the D-CS-like structure. Fur-
much higher than that for D-OUS, suggesting that the affinity of thermore, our present study provides a new route for the produc-
DcsG toward ␤-aminooxy-D-alanine is lower than that toward tion of amino acids and their analogs by using DcsC, DcsD, and
D-OUS. The side chain of ␤-aminooxy-D-alanine is expected to DcsG.
exhibit a positive charge under physiological pH conditions,
In recent years, unnatural amino acids have been used fre-
whereas the side chain of D-OUS is uncharged. The low Km for quently as building blocks for the development of new pharma-
D-OUS indicates that DcsG would preferentially bind to the D- ceutical drugs (18, 46). As shown in this study, although the sub-
amino acid having a neutral charge at the side chain. In addition, strate specificity of DcsC has been reported to be limited to OUS
it indicates that the carbamoyl group may form hydrophilic inter- (21), those of DcsD and DcsG are very broad (Tables 1 and 2).
actions with the atoms in DcsG.
DcsD may be useful to produce unnatural L-amino acids, while
DcsG can catalyze the formation of five- and six-membered DcsG may be available as an enzyme to produce cyclic D-amino
rings with amide linkages, from D-2,4-diaminobutyrate and D-or- acid analogs and to separate L-amino acids from racemic mix-
nithine, respectively. The kcat/Km values for these compounds are tures. For example, by using DcsG together with inexpensive ra-
2 to 3 orders of magnitude lower than that for D-OUS. In partic- cemic homocysteine, it may be possible to produce both L-homo-
ular, D-2,4-diaminobutyrate was less reactive than ␤-aminooxy- cysteine and D-homocysteine thiolactone at a much lower cost
D-alanine, indicating that reactivity of the terminal amino than is available by current means.
groups is dependent on their adjacent atoms (carbon in D-2,4-
DcsG is expected to possess a novel structure, since the protein
diaminobutyrate and oxygen in ␤-aminooxy-D-alanine). Fur- has a low level of sequence similarity to structurally elucidated
thermore, since D-ornithine was less reactive than D-2,4-di- proteins of related function. The determination of the crystal
aminobutyrate, the normal function of DcsG is likely to form a structure of DcsG is under way. However, we can predict the
five-membered ring. Similarly, the kcat/Km values for D-homocys- structures of DcsC and DcsD. Mutation analysis based on the
teine and D-homoserine are 1 and 2 orders of magnitude lower predicted structure could lead to the identification of the amino
than that for D-OUS, respectively, but higher than that for D-2,4- acid residues that are important for substrate specificity. Further-
diaminobutyrate. The terminal group of D-homocysteine displays more, by introducing site-specific variations based on the struc-
a neutral or negative charge at physiological pH, while that of tural information, more useful enzymes exhibiting improved sub-
D-homoserine is neutral. In contrast, the terminal group of D-2,4- strate specificity characteristics may be created. Mutation analyses
diaminobutyrate exhibits a positive charge at physiological pH. based on the predicted structures of DcsC and DcsD are now
Therefore, it was also suggested that DcsG appears to bind prefer- under way.
entially to substrates in which the terminal group does not display
a positive charge.
ACKNOWLEDGMENTS
Conclusion. In the present study, we were able to determine
the enzymatic functions of the DcsC, DcsD, and DcsG proteins,
which are responsible for D-CS biosynthesis. Another research
group recently showed that DcsC is a cofactor-independent race-
mase utilizing OUS as the substrate (21). Based on the experimen-
tal evidence presented in this study, we conclude that DscD and
DscG are an O-ureido-L-serine synthase and a D-cycloserine syn-
thethase, respectively. Since the DcsG protein needs ATP as an
energy source, it may be right to call it a synthetase.
This study was funded by the Institute of Fermentation, Osaka (IFO),
Japan (Y.M.), and by a grant-in-aid for scientific research from the Min-
istry of Education, Science, and Culture of Japan (T.K.).
DNA sequence determination and MS analysis were carried out at the
Analysis Center of Life Science, Hiroshima University.
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