Báo cáo khoa học: Crystal structures of open and closed forms of D-serine deaminase from Salmonella typhimurium – implications on substrate specificity and catalysis pptx

Crystal structures of open and closed forms of D-serine
deaminase from Salmonella typhimurium – implications on
substrate specificity and catalysis
Sakshibeedu Rajegowda Bharath
1
, Shveta Bisht
1
, Handanhal Subbarao Savithri
2
and
Mattur Ramabhadrashastry Narasimha Murthy
1
1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
2 Department of Biochemistry, Indian Institute of Science, Bangalore, India
Keywords
D-serine deaminase; open and closed
conformations; pyridoxal 5¢ phosphate
dependent Foldtype II enzyme; X-ray
diffraction; a, b elimination
Correspondence
M. R. N. Murthy, Molecular Biophysics Unit,
Indian Institute of Science, Bangalore 560
012, India
Fax: +91 80 2360 0535
Tel: +91 80 2293 2458
E-mail:
(Received 12 March 2011, revised 29 May
2011, accepted 7 June 2011)
doi:10.1111/j.1742-4658.2011.08210.x
Metabolism of D-amino acids is of considerable interest due to their key
importance in cell structure and function. Salmonella typhimurium

D-serine
deaminase (StDSD) is a pyridoxal 5¢ phosphate (PLP) dependent enzyme that
catalyses degr adation of
D-Ser to pyruvate and ammonia. The fi rst crystal
structure of
D-serine deaminase described here reveals a typical Foldtype II
or tryptophan synthase b subunit fold of PLP-dependent enzymes. Although
holoenzyme was used for crystallization of both wild-type StDSD (WtDSD)
and selenomethionine labelled StDSD (SeMetDSD), significant electron
density was not observed for the cofactor, indicating that the enzyme has a
low affinity for the cofactor under crystallization conditions. Interestingly,
unexpected conformational differences were observed between the two struc-
tures. The WtDSD was in an open conformation while SeMetDSD, crystal-
lized in the presence of isoserine, was in a closed conformation suggesting
that the enzyme is likely to undergo conformational changes upon binding of
substrate as observed in other Foldtype II PLP-dependent enzymes. Electron
density corresponding to a plausible sodium ion was found near the active
site of the closed but not in the open state of the enzyme. Examination of the
active site and substrate modelling suggests that Thr166 may be involved in
abstraction of proton from the Ca atom of the substrate. Apart from the
physiological reaction, StDSD catalyses a, b elimination of
D-Thr, D-Allothr
and
L-Ser to the corresponding a-keto acids and ammonia. The structure of
StDSD provides a molecular framework necessary for understanding differ-
ences in the rate of reaction with these substrates.
Introduction
Pyridoxal 5¢ phosphate (PLP) dependent enzymes
constitute a diverse family of proteins involved in the
metabolism of amino acids, amino sugars and amino

group containing lipids. A majority of them are key
enzymes in the metabolism of amino acids. The reac-
tions catalysed include the transfer of amino group,
decarboxylation, inter-conversion of l- and d-amino
acids and removal or replacement of chemical groups
at a, b or c positions [1]. Functionally, PLP-dependent
enzymes have been classified into three groups ( a, b
and c families) based on the carbon atom at which the
net reaction takes place [2]. Structurally, they have
been classified into five groups [3–6]: Foldtype I
enzymes that belong to the aspartate aminotransferase
Abbreviations
DNPH, 2,4-dinitrophenyl hydrazine; EcDSD,
D-serine deaminase from Escherichia coli; LSD, L-serine dehydratase; PLP, pyridoxal
5¢-phosphate; SeMetDSD, selenomethionine
D-serine deaminase; SpSR, Schizosaccharomyces pombe serine racemase; StDSD,
Salmonella typhimurium
D-serine deaminase; TRPSb, tryptophan synthase b; WtDSD, wild-type D-serine deaminase.
FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2879
family, Foldtype II that resemble tryptophan synthase
b, Foldtype III that are related to the alanine racemase
family, Foldtype IV enzymes related to the d-amino
acid aminotransferase family and Foldtype V or glyco-
gen phosphorylase family.
Most of the amino acid dehydratases belong to the
tryptophan synthase b (TRPSb) family or Foldtype II
PLP-dependent enzymes [5]. These enzymes catalyse
irreversible degradation of amino acids to the respec-
tive a-keto acids. l-serine and l-threonine dehydratases
have been purified and characterized both structurally

and biochemically from several organisms [7–9].
Comparison of the amino acid sequence of d-serine
deaminase from Salmonella typhimurium (StDSD) with
those of other PLP-dependent enzymes suggests that it
belongs to the Foldtype II or TRPSb family, although
the sequence identities are low (15–23%). However,
there are d-serine deaminases (unrelated in sequence to
StDSD) which are annotated in sequence databases as
Foldtype III or as members of the alanine racemase
family.
d-Serine deaminase from Escherichia coli (EcDSD;
EC 4.3.1.18) exhibits b-elimination activity with d-Ser,
d-Thr, d-Allothr and l-Ser with a pH optimum of 8.0
[10]. In most bacteria, DSD probably acts as a detoxi-
fying enzyme, carrying out degradation of d-Ser. Most
organisms fail to survive and propagate on d-Ser
containing nutrient media. This is attributed to the
formation of d-Ser activated aminoacyl tRNA leading
to toxicity and retardation of cell growth. d-Ser is a
co-agonist of NMDA channel receptors and therefore
EcDSD has been routinely included in the purification
of NMDA receptors from organotypic hippocampal
slices [11]. The Saccharomyces cerevisiae DSD has been
used in diagnostic laboratories for quantitative deter-
mination of d-Ser in human brain and urine [12]. The
E. coli dsdA gene has been found to be an excellent
marker for construction of strains for which the use of
antibiotic resistance genes as selective markers is not
allowed [13,14].
EcDSD (48 kDa) [15] and Klebsiella DSD (46 kDa)

[16] are known to be functional as monomers in
contrast to the majority of Foldtype II PLP-dependent
enzymes, which are dimers. However, DSD is a dimer
of 118 kDa in S. cerevisiae [17] and a heterodimer of
40 and 40.4 kDa subunits in chicken [18]. EcDSD has
been found to be activated by NH
þ
4
and K
+
and to a
lesser extent by Na
+
ions [10,19]. It was proposed that
K
+
is not involved directly in catalysis but is required
for stabilizing the active site geometry [20]. Although
crystals of EcDSD suitable for X-ray diffraction
studies have been obtained [21], its structure has not
been reported in the literature. Comparative studies on
monomeric StDSD and the more common dimeric
forms of Foldtype II PLP-dependent enzymes will
allow examination of the plausible role of oligomeric
state in these enzymes [22].
In this paper, we report the first crystal structure of
a Foldtype II d-serine deaminase and describe features
of the active site essential for catalysis. The crystal
structures reported are wild-type StDSD (WtDSD)
and selenomethionine incorp orated StDS D (SeM etDSD)

crystallized in the presence of isoserine. Although nei-
ther of the structures had density corresponding to the
cofactor, WtDSD was in an open conformation while
SeMetDSD was in the closed conformation, suggesting
that StDSD exhibits a domain movement similar to
those of rat liver l-serine deaminase (rat liver LSD) [9]
and serine racemase from Schizosaccharomyces pombe
(SpSR) [23]. Examination of the monomeric structure
of StDSD suggests that a dimeric structure similar to
those of other Foldtype II PLP-dependent enzymes
would lead to unacceptable van der Waals contacts
involving segments of StDSD that are insertions with
respect to other Foldtype II enzymes. Electron density
for a putative Na
+
ion was located close to the active
site of SeMetDSD but not of WtDSD. The active site
geometry allows identification of residues that may
play a key role in catalysis.
Results and Discussion
Biochemical studies on StDSD
Recombinant StDSD was expressed in E. coli as a
hexa-histidine tagged protein and purified by nickel
nitrilotriacetic acid affinity and size exclusion chroma-
tography. The purified protein was yellow in colour
with an absorbance maximum at 415 nm, indicating
the presence of PLP as an internal aldimine. As in
several other PLP-dependent enzymes, a small peak at
340 nm was also observed. The A
280

⁄ A
415
ratio was
close to 10. The peak at 415 nm was independent of
pH in the range 6.0–9.0. However, a small increase in
the peak at 330–340 nm was observed close to pH 6.0.
Similar observations have been reported for the
EcDSD by Dupourque et al. [10]. The peaks at 415
and 330 nm have been attributed to the ketoenamine
and enolimine forms, respectively, of the internal aldi-
mine [24].
StDSD was most active with d-Ser. The activities
of WtDSD (2.09 lmolÆmg
)1
Æmin
)1
) and SeMetDSD
(2.12 lmolÆmg
)1
Æmin
)1
) with d-Ser as the substrate
were comparable. It has been reported that the pres-
ence of Na
+
or K
+
ions enhances the activity of
EcDSD [20,24]. The enzymatic properties of StDSD
Crystal structure of D-serine deaminase S. R. Bharath et al.

2880 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS
were therefore examined in the presence of Na
+
or
K
+
ions and the results are tabulated (Table 1). K
m
and V
max
for d-Ser were about twice as high in the
presence of Na
+
than with K
+
. In the presence of
Na
+
, K
m
for d-Ser was higher than that of d-Thr.
However, V
max
was an order of magnitude higher for
d-Ser. In contrast, in the presence of K
+
ions, K
m
was
lower and V

max
was higher for d-Ser than for d -Thr.
The enzyme was much less active with d-Allothr and
l-Ser. With both d-Thr and d-Allothr, the enzyme was
more active in the presence of K
+
than Na
+
.
Structure and model quality
Crystal structures of SeMetDSD (1.9 A
˚
) and WtDSD
(2.4 A
˚
) were solved using four-wavelength anomalous
dispersion (4W-MAD) and molecular replacement
methods respectively. The data collection and structure
refinement statistics are given Tables 2 and 3 respec-
tively. In WtDSD, except for two short stretches
(68–71 and 234–239) electron density is of good
quality throughout the polypeptide main chain. In
SeMetDSD, electron density is absent for only two
C-terminal residues (439–440). A total of 17 and seven
residues have been truncated according to the extent of
observed electron density in WtDSD and SeMetDSD,
respectively. In both structures, the residues forming
the C-terminal hexa-histidine tag were not included in
the model due to absence of a well-defined electron
density. In SeMetDSD, 94.4% and 4.7% of residues

were in favoured and additionally allowed regions,
respectively, of the Ramachandran plot [25,26]. One
residue (Ile111) was in the disallowed region. The
WtDSD structure had 93.1% and 6.3% of the residues
in the favoured and additionally allowed regions and
two residues (Ile111 and His319) in the disallowed
region. Statistics of the Ramachandran plot obtained
from procheck [27] are given in Table 3. Interestingly,
a well-defined density for PLP was not observed in the
two structures.
The polypeptide fold of StDSD is illustrated in
Fig. 1. The secondary structural elements have been
assigned using dssp [28]. As in other PLP-dependent
enzymes of Foldtype II, the StDSD monomer consists
of a small domain (residues 43–75, 109–238) and a large
domain (residues 1–42, 76–108 and 239–440). The small
domain folds as an open twisted a ⁄ b structure consist-
ing of a four-stranded (S4–S7) parallel b-sheet sand-
wiched between one helix (H11) on the solvent facing
side and two helices (H9 and H10) on the other side.
Four more helices (H4, H5, H7 and H8) occur in this
domain. The core of the large domain contains a seven-
stranded mixed b-sheet surrounded by eight helices on
the solvent facing side (H6, H17, H18, H19 and H20 on
one side and H1, H2 and H3 on the other side) and six
helices that occur between the two domains (H12, H13,
H14, H15, H16 and H21). In the central b-sheet of the
large domain, all except two short strands at the peri-
phery (S1 and S2) are parallel. The strands of the cen-
tral b-sheet are strongly twisted. The N-terminal helices

H1 and H2 protrude away from the large domain.
StDSD structure was used as the template for identi-
fying other proteins with similar folds in the Protein
Data Bank (PDB) using the program dali [29] with the
view of identifying shared and unique structural fea-
tures of StDSD. There were 245 hits with Z-scores
higher than 20.0, of which 36 were unique structures.
The top hits corresponded to threonine deaminase,
serine racemase and l-serine dehydratase. Although the
overall fold of StDSD is similar to those of the PLP-
dependent Foldtype II enzymes, the helices H1, H2, H3
and H19 and the antiparallel b strand S1 in the large
domain and the helices H5 and H8 in the small domain
are significantly different and could be considered as
additions to the fold of the TRPSb family. These struc-
tural segments are shown in orange in Fig. 1.
Gel filtration studies indicate that StDSD is a mono-
mer in solution. This is consistent with the earlier
results obtained with EcDSD [10,15]. However, it is in
contrast to most of the enzymes belonging to the
TRPSb family, which are dimers in solution (except
for threonine synthase from E. coli (PDB code
1VB3)
and yeast (PDB code
1KL7), which are also mono-
mers). Modelling of StDSD resembling the dimeric
structures of other Foldtype II PLP-dependent
enzymes such as O-acetylserine sulfhydrylase (OASS)
or cystathionine b-synthase suggests that H5, H6 and
H21 prevent dimer formation by causing steric clashes.

Comparison between WtDSD and SeMetDSD
The rmsd upon superposition of corresponding
Ca atoms of WtDSD and SeMetDSD polypeptides is
Table 1. Kinetic parameters of StDSD with various substrates,
phosphate buffer pH 7.5. V
max
is expressed as micromoles of pyru-
vate formed per milligram of protein per minute of reaction.
Substrate Buffer K
m
(mM) V
max
D-Ser Na
+
phosphate 0.87 ± 0.28 90.98 ± 13.12
K
+
phosphate 0.42 ± 0.08 54.56 ± 6.51
D-Thr Na
+
phosphate 0.45 ± 0.10 6.28 ± 1.31
K
+
phosphate 0.53 ± 0.09 13.83 ± 1.62
D-Allothr Na
+
phosphate 0.63 ± 0.08 0.40 ± 0.08
K
+
phosphate 0.88 ± 0.11 0.63 ± 0.09

L-Ser Na
+
phosphate 11.78 ± 2.53 0.32 ± 0.06
K
+
phosphate 10.23 ± 3.70 0.47 ± 0.11
S. R. Bharath et al. Crystal structure of
D-serine deaminase
FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2881
1.32 A
˚
. Superposition of large and small domains of
StDSD with the corresponding domains of SeMetDSD
results in rmsd values of 0.53 and 1.64 A
˚
, respectively.
A number of local conformational changes in the small
domains of WtDSD and SeMetDSD are also observed.
These local changes lead to large rmsd in the compari-
son of the small domains. Superposition of the Ca
atoms of the large domains (Fig. 2A) in these struc-
tures leaves the small domains with a residual rotation
of 15°. Structures of WtDSD and SeMetDSD resemble
the open and closed forms, respectively, of other Fold-
type II PLP-dependent enzymes. The solvent accessible
surface areas of StDSD in the open and closed forms
are 16 994 and 16 540 A
˚
2
, respectively. Ligand-induced

movement of the small domain with respect to the
large domain has been observed in SpSR (Fig. 2B)
[23], serine racemase from Rattus norvegicus and
Homo sapiens [30], and OASS from S. typhimurium
[31]. In tryptophan synthase b from E. coli [32] and
l-serine dehydratase from Rattus norvegicus (rat liver
LSD) [9], a similar domain movement is observed
between apo and holo forms of the enzymes. Consider-
ing that SeMetDSD crystal was grown in the presence
of the inhibitor (isoserine), it is reasonable to assume
that SeMetDSD represents the ligand bound closed
form of the enzyme while the WtDSD represents the
unliganded open form of the enzyme. It is likely that,
upon formation of external aldimine with isoserine, the
enzyme undergoes conformational change to the closed
Table 2. Data collection statistics. Values in parentheses refer to the highest resolution shell. R
merge
=(R
hkl
R
i
|I
i
(hkl ) ) ÆI(hkl)æ|) ⁄ R
hkl
RIi(hkl ),
where I
i
(hkl) is the intensity of the ith measurement of reflection (hkl) and ÆI(hkl )æ is its mean intensity. R
pim

[46] = (R
hkl
[1 ⁄ N ) 1]
1 ⁄ 2
)
ÆI(hkl )æ|) ⁄ R
hkl
RI
i
(hkl ), where I
i
(hkl ) is the intensity of the ith measurement of reflection (hkl ), ÆI(hkl )æ is its mean intensity and N is the number
of measurements (redundancy). I is the integrated intensity and r(I) is the estimated standard deviation of that intensity.
Crystal Se-Met Se-Met Se-Met Se-Met Se-Met Wt
Wavelength (A
˚
) 0.97848
(peak)
0.97872
(inflection)
1.01876
(low remote)
0.97083
(high remote)
1.5418 (Cu Ka) 1.5418 (Cu Ka)
Cell parameters (A
˚
)
a 56.26 56.37 56.41 56.38 56.46 100.02
b 187.65 187.83 187.94 187.88 188.39 46.79

c 46.48 46.53 46.54 46.57 46.59 100.04
abc 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 93.75, 90
Space group P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2C2
Resolution range 45.1–2.2 (2.3–2.2) 37.6–2.0 (2.1–2.0) 41.9–2.1 (2.2–2.1) 35.3–1.8 (1.9–1.8) 48.4–1.9 (2.0–1.9) 49.9–2.4 (2.5–2.4)
R
merge
0.065 (0.145) 0.055 (0.226) 0.049 (0.157) 0.069 (0.304) 0.053 (0.174) 0.109 (0.492)
R
pim
0.023 (0.045) 0.036 (0.146) 0.028 (0.096) 0.038 (0.153) 0.019 (0.058) 0.070 (0.300)

Total measurements 367 430 (52 487) 143 786 (18 234) 129 720 (16 424) 262 803 (39 256) 329 613 (45 978) 72 766 (8757)
Unique reflections 25 862 (3704) 34 178 (4867) 30 465 (4311) 46 887 (6704) 36 067 (4560) 17 806 (2301)
ÆI ⁄ r(I)æ 30.4 (18.4) 13.5 (4.4) 15.8 (6.0) 11.7 (3.6) 28.02 (12.0) 6.0 (2.3)
Completeness 100 (100) 99.5 (98.7) 99.7 (99.0) 100 (100) 93.7 (82.5) 97.1 (87.8)
Multiplicity 14.2 (14.2) 4.2 (3.7) 4.3 (3.8) 5.6 (5.9) 9.1 (10.1) 4.1 (3.8)
Mosaicity 0.38 0.45 0.46 0.49 0.33 1.2
Wilson B-factor (A
˚
2
) 21.9 22.8 23.9 21.6 14.7 37.4
Anomalous
completeness
100 (100) 90.9 (81.1) 92.7 (83.9) 99.6 (100) 91.0 (79.4) 92.9 (78.4)
Anomalous
multiplicity
7.6 (7.4) 2.3 (2.2) 2.3 (2.2) 2.9 (3.0) 4.9 (5.3) 2.2 (2.1)
Table 3. Structure validation and refinement statistics.
R
work
=(R
hkl
|F
o
) F
c
|) ⁄ R
hkl
F
o
where F

o
and F
c
are the observed and
calculated structure factors. R
free
[47] is calculated as for R
work
but
from a randomly selected subset of the data (5%), which were
excluded from the refinement.
SeMet Native
R
work
0.18 0.22
R
free
0.21 0.27
RMSD bond length (A
˚
) 0.006 0.007
RMSD bond angle (
o
) 0.915 1.076
Ramachandran plot
Favoured region (%) 94.4 93.1
Additionally allowed region (%) 4.7 6.3
Generously allowed region (%) 0.6 0.0
Outliers (%) 0.3 0.5
Number of

Protein atoms 3307 3218
Water atoms 384 174
Non-water hetero-atoms 54 63
Average B-factor (A
˚
2
)
Protein atoms 14.3 27.5
Water atoms 26.9 29.9
Non-water hetero-atoms 22.4 50.6
Crystal structure of
D-serine deaminase S. R. Bharath et al.
2882 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS
form which is retained even after the removal of the
external aldimine under crystallization conditions or
from the crystals. Thus, the WtDSD and SeMetDSD
structures can be viewed as open and closed forms of
the enzyme.
In the WtDSD structure, two segments (68–71 and
234–239) were not built due to absence of significant
electron density. Of these, 68–71 stretch is involved in
crystal contacts and is 27 A
˚
away from the active site.
In SeMetDSD, this stretch is not involved in crystal
contacts and is ordered. Therefore, the disorder
observed in the WtDSD may be due to crystal pack-
ing. Residues 234–239 are close to the active site and
are ordered in SeMetDSD suggesting that this segment
may undergo disorder–order transition upon domain

closure. An aspartate residue (Asp236) occurring in
this segment might have a role in catalysis (see Impli-
cations for catalysis).
In the closed form of the structure (SeMetDSD), an
isolated electron density close to Cys276 that could
correspond to an Na
+
ion was observed. Although
Na
+
ions cannot be unambiguously distinguished from
water molecules on the basis of electron density, bind-
ing of an Na
+
or K
+
to EcDSD has been demon-
strated through NMR chemical shift data [20]. Also,
an equivalent site is known to bind divalent cations
(Mg
2+
or Mn
2+
)inSpSR [23], serine racemase from
Rattus norvegicus and Homo sapiens [30]. Therefore, an
Na
+
ion was built into the observed density. The
refined B-factor of the Na
+

(22.4 A
˚
2
) is about twice
that of the atoms in its close proximity. Figure 3
shows the residues that interact with the proposed
Na
+
ion. The charge on the Na
+
is neutralized by the
carboxylate of Glu303. Apart from Ser307 hydroxyl
and Cys309 sulfhydryl, the Na
+
is surrounded by
three main chain carbonyl groups. A similar geometry
has been observed around the bound K
+
in rat liver
LSD [9]. In contrast, no density that could correspond
to a bound ion was present at the equivalent position
in WtDSD indicating that the ion binds only to the
Fig. 1. Polypeptide fold of SeMetDSD showing secondary struc-
tural elements. a-helices are shown as cylinders, b-strands are
shown as ribbons. The two structural domains are coloured in dif-
ferent shades of teal and loops are shown in red. PLP–
D-Ser com-
plex at the active site is shown in ball and stick representation. The
Na
+

ion is shown as a yellow sphere. Secondary structural ele-
ments which are insertions with respect to most of the other Fold-
type II family of PLP-dependent enzymes are shown in orange. All
secondary structural elements are labelled.
Fig. 2. (A) Open and closed forms of
StDSD. Large domains of WtDSD and
SeMetDSD were superposed to depict the
relative movement of the small domain
between the two conformational states.
Open conformation of WtDSD is shown in
dark grey while the closed conformation of
SeMetDSD is shown in light grey. The small
domains are related by a residual rotation of
15°. The view is selected to highlight
domain movement. (B) Large domains of
open (dark grey; PDB code
1V71) and
closed (light grey; PDB code
2ZPU) forms of
SpSR are superposed illustrating similarity
to the conformational change observed in
StDSD (A).
S. R. Bharath et al. Crystal structure of
D-serine deaminase
FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2883
closed form of the enzyme. Similar observation has
been made in the apo form of rat liver LSD [9]. In
these enzymes, the metal ion does not appear to have
a catalytic role as it is not in direct contact with the
substrate.

Active site
The active site of Foldtype II PLP-dependent enzymes
is situated in a large crevice between the two domains.
Based on structural comparisons with other Foldtype II
PLP-dependent enzymes, PLP is expected to bind
StDSD as an internal aldimine covalently bonded to
the e-amino group of Lys116 situated at the beginning
of helix H7. However, significant density to fit an
intact PLP was not observed in the electron density
maps of either WtDSD (Fig. 4A) or SeMetDSD
(Fig. 4B) suggesting that there is a tendency for PLP
to diffuse away from the active site. It is known that
EcDSD is readily converted to the apo form when
incubated with l-Cys in the presence of EDTA [15].
However, l-Cys and ⁄ or EDTA were not present in
purification or crystallization steps. Also, the purified
protein was yellow in colour and was catalytically
active indicating that PLP is indeed bound. However,
the crystals were not yellow. Enzymatic assay carried
out with a dissolved SeMetDSD crystal showed low
activity, which increased by a factor of 5 upon addition
Fig. 3. (A) Cartoon diagram illustrating the residues of SeMetDSD that interact with Na
+
ion. (B) Relative position of Na
+
ion with respect to
the modelled PLP–
D-Ser complex is shown. Cys276 and Gly277, which are part of the glycine-rich loop (residues 276–282) anchoring phos-
phate of the cofactor, are close to the Na
+

ion.
Fig. 4. Electron density (2F
o
) F
c
contoured
at 1r corresponding to 0.41 electrons A
˚
)3
)
observed at the active site of (A) WtDSD
and (B) SeMetDSD. The carboxyl group of
modelled PLP–
D-Ser is in the density corre-
sponding to a bound ethylene glycol. A blob
of density that represents a bound sulfate
ion was observed at the site of phosphate
in both the structures although density was
absent for the cofactor. PLP in WtDSD and
PLP–
D-Ser external aldimine complex in
SeMetDSD modelled by comparison with
other Foldtype II enzymes are shown in ball
and stick representation.
Crystal structure of
D-serine deaminase S. R. Bharath et al.
2884 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS
of PLP (Fig. 5A). These results suggest that the struc-
tures described here most probably correspond to the
apo forms. As the purified enzyme samples had cova-

lently bound PLP, it is likely that the cofactor was
lost during crystallization. In order to examine this
possibility, 1 mgÆmL
)1
WtDSD and SeMetDSD were
dialysed extensively against crystallization condition.
Activities of these samples with d-Ser as substrate were
determined (Fig. 5B). The samples had only 20% of
the activity (0.38 and 0.42 lmolÆmg
)1
Æmin
)1
in WtDSD
and SeMetDSD, respectively) of undialysed samples
suggesting that crystallization condition leads to loss
of PLP.
The active site pocket in both WtDSD and
SeMetDSD had a blob of significant density (Fig. 4) at
a position corresponding to PLP of other Foldtype II
enzymes. A sulfate ion was fitted to this density as it
was a component of crystallization. The sulfate ion
could be refined to a B-factor (10 A
˚
2
in SeMetDSD
and 28 A
˚
2
in WtDSD) comparable with those of
the surrounding atoms (8–10 A

˚
2
in SeMetDSD and
17–26 A
˚
2
in WtDSD). It is worth noting that StDSD
is active in the presence of excess added sulfate and
hence absence of density for the PLP ring is unlikely
to be due to its displacement by sulfate.
As adequate density was not observed for the
cofactor, PLP (internal aldimine in WtDSD) and PLP-
isoserine, PLP-d-Ser, PLP-d-Thr, PLP-d-Allothr and
PLP-l-Ser (external aldimines in SeMetDSD) were
modelled at the active site based on the structure of
rat liver LSD [9]. The close similarity in the active sites
of these enzymes ensured that the modelling is reliable.
A significant density was present at the active site at a
position corresponding to the carboxylate of the mod-
elled substrate. This density most probably corre-
sponds to a bound ethylene glycol molecule. The
pyridine ring of PLP occupies a cavity between Leu338
and Ile115. These residues may limit the tilting of the
pyridine ring between the internal and external aldi-
mine forms [31]. The side chain of Asn168 and the
hydroxyl group of Thr422 are at hydrogen bonding
distances from O
3
and N
1

of PLP (2.2 and 2.8 A
˚
),
respectively. Thr422 is found to be replaced by Ser (in
threonine deaminase [8], O-acetylserine sulfhydrase [31]
and serine racemase [23]) and Cys (in l-serine dehydra-
tase [9]) in the other members of the TRPSb family of
PLP-dependent enzymes. It has been proposed that Ser
or Cys residue is important for maintaining the elec-
tronic state of the PLP–Schiff base conjugate [33]. Pre-
sumably, Thr422 fulfils the same role in StDSD. A
glycine-rich loop is conserved in all the PLP-dependent
enzymes and provides interactions for the binding of
PLP (PLP-binding cup) [34]. This loop located at the
N-terminal end of helix H12 in StDSD consists of resi-
dues Gly277, Val278, Gly279, Gly280, Gly281 and
Pro282. Most of these residues are conserved in all
known PLP-dependent enzymes. The metal ion may
stabilize the conformation of the PLP-binding loop by
its interaction with carbonyl groups of Gly277 and
Cys276.
The carboxyl group of the substrate is held by
hydrogen bonding to the Ser165 hydroxyl and amide
group of Leu169. Comparison of modelled PLP-d-Ser
and PLP-l-Ser complexes in SeMetDSD (Fig. 6) shows
that the Ca protons of the two external aldimine com-
plexes are in opposite orientations. The Ca proton in
the case of PLP-d-Ser complex faces the hydroxyl
group of Thr166 (2.5 A
˚

) whereas in PLP-l-Ser com-
plex it faces Lys116 (4.5 A
˚
) and Asp236 (4.2 A
˚
). Based
on the modelled PLP–isoserine external aldimine com-
plex, the hydroxyl group of Ser165 (2.7 A
˚
) and the
amide groups of Thr166 (3.3 A
˚
) and Leu169 (2.6 A
˚
)
appear to be important for stabilizing the external aldi-
mine (Fig. 7).
Earlier work on EcDSD has shown that modification
of a particular Cys residue leads to enzyme inactivation
[15]. This might correspond to Cys276 as it is conserved
and occurs near the phosphate binding loop.
Fig. 5. (A) Activity of dissolved SeMetDSD crystals without (C1)
and with (C2) added PLP (50 l
M); 5 mMD-Ser was used as the sub-
strate. (B) Activity of WtDSD and SeMetDSD under different condi-
tions as described in the text. W1 and W2 correspond to WtDSD
while S1 and S2 correspond to SeMetDSD. W1 and S1 correspond
to holo enzyme in 50 m
M HEPES pH 7.5. W2 and S2 correspond to
proteins dialysed against crystallization condition.

S. R. Bharath et al. Crystal structure of
D-serine deaminase
FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2885
Implications for catalysis
Degradation of d-Ser to pyruvate and ammonia by
StDSD involves two steps. In the first step, d-Ser is
converted to aminoacrylate by Ca proton abstraction
and protonation of the hydroxyl group of the substrate
resulting in the release of a water molecule. In a subse-
quent non-enzymatic step, aminoacrylate is converted
to ammonia and the a-keto acid, pyruvate.
In rat liver LSD [9], it has been observed that the
N1 atom of PLP is unlikely to be protonated in view
of its hydrogen bonding to the side chain S–H of
Cys303. These authors also note that the PLP is likely
to be in its less polarized form (HPO
À
4
) as no cation is
found in its vicinity and it is held in place only by
backbone amide groups of the residues from the gly-
cine-rich phosphate binding loop. Based on these
observations, they suggest that the Lys41 (which is
linked to PLP in the internal aldimine form of the
enzyme) may abstract the proton from the Ca atom of
the substrate. Elimination of the substrate hydroxyl
may be facilitated by the phosphate group acting as a
general acid. The active site geometry of SpSR [23] is
closely similar to that of rat liver LSD. Here, N1 is
Fig. 6. Stereodiagram of the superposition of active sites of SeMetDSD PLP-D-Ser (light grey) and PLP-L-Ser (dark grey) complexes. The Ca

proton of
D-Ser points towards Thr166 in SeMetDSD and that of L-Ser points towards Lys116 and Asp236.
Fig. 7. Stereodiagram of the active site geometry in WtDSD (dark grey) and SeMetDSD (light grey). PLP-D-Ser modelled in SeMetDSD is
also shown. The carboxyl group of modelled PLP-
D-Ser is held by hydrogen bonding with the hydroxyl group of Ser165 and the main chain
amide of Leu169. Lys116 is in different orientations in these structures. Ser165 and Thr166 are closer to the substrate in SeMetDSD com-
pared with WtDSD.
Crystal structure of
D-serine deaminase S. R. Bharath et al.
2886 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS
hydrogen bonded to the side chain of Ser308 and the
phosphate is held by the amide groups from the
glycine-rich phosphate binding loop. A two-base mech-
anism in which Ser82 and Lys57 are involved in the
abstraction of a proton from Ca of d-Ser and l-Ser,
respectively, has been proposed for the racemase reac-
tion. SpSR also exhibits a low level of a, b elimination
of d-Ser, for which Ser82 has been proposed as the
base in abstraction of the Ca proton.
Examination of the active site geometry in StDSD
suggests that the active site Lys116, unlike in rat liver
LSD, is not at a position suitable for proton abstrac-
tion (Fig. 6). Two residues, Thr166 and Tyr214, are
close to Ca of the modelled external aldimine (Fig. 7)
and hence might be suitable for proton abstraction.
Thr166 is part of the conserved loop which holds the
carboxyl group of the substrate and moves by 3.0 A
˚
when StDSD undergoes transition from the open to
the closed form. The modelled SeMetDSD–PLP-d-Ser

complex (Fig. 7) suggests that the Ca proton of the
substrate is close to Thr166 hydroxyl (2.5 A
˚
). Thr166
is structurally equivalent to Ser82 of SpSR. Therefore,
in StDSD Thr166 may fulfil the same role. The side
chain hydroxyl of Tyr214 is at a distance of 6.3 A
˚
(in
SeMetDSD) from the Ca atom of the substrate and is
disordered in WtDSD. It undergoes substantial dis-
placement between the open and closed forms and
hence may be involved in proton abstraction, although
it appears to be a less likely candidate than Thr166.
Further mutagenesis experiments need to be carried
out to clarify the role of these residues in catalysis. As
in rat liver LSD [9], PLP may be involved in the pro-
tonation of the substrate hydroxyl group leading
to the release of a water molecule and formation of
aminoacrylate.
It has been noted earlier that the amino group of
the incoming amino acid should be deprotonated to
make a nucleophilic attack on the C4¢ of PLP [23].
Occurrence of Tyr214 and Asp236 near the active
site of StDSD suggests that these residues might be
important for the initial formation of external
aldimine.
StDSD exhibits substantial activity with d-Thr. Mode-
lling of d-Thr as an external aldimine in SeMetDSD
shows no unacceptable contacts between the substrate

and protein atoms. The lower rate of degradation with
d-Thr may be because of a lower rate of protonation
by the phosphate group. StDSD has a low level of
activity with l-Ser. Modelling l-Ser at the active site
suggests that the Ca proton points towards Lys116
and Asp236 and not towards Thr166 or Tyr214.
Therefore, Lys166 or Asp236 may be involved in deg-
radation of l-Ser by St DSD.
Based on fluorescence energy transfer and CD stud-
ies in EcDSD [24], it was suggested that Trp197
(equivalent to StDSD Trp195) undergoes large dis-
placement during catalysis and hence could be a key
residue in catalysis. However, Trp195 is not close to
the active site and is unlikely to be important for the
catalytic function.
Conclusions
StDSD is a monomeric PLP-dependent enzyme that
catalyses a, b elimination of d-Ser, d-Thr, d-Allothr
and l-Ser to the corresponding keto acid and ammo-
nia. Structural data presented here suggest that StDSD
protomer has a fold similar to those of other Foldtype II
PLP-dependent enzymes and undergoes conforma-
tional change from an open unliganded state to a
closed liganded state. It has a low affinity for the co-
factor PLP under the conditions of crystallization. An
ion bound near the active site (most probably Na
+
)
may be essential to keep the PLP binding loop in a
conformation appropriate for cofactor binding and

hence for catalysis. The ion is unlikely to be directly
involved in the enzyme reaction. Differences in the cat-
alytic rates with respect to different substrates
(Table 1) in the presence of Na
+
and K
+
suggest that
these ions affect the conformation of the PLP binding
loop in subtle ways. The positioning of Thr166 in these
structures with respect to the substrate suggests that it
is suitable for abstraction of the proton from the Ca
atom of the substrate. Further structural and kinetic
studies with site mutants of residues at the active site
and determination of structures of ligand and inhibitor
complexes will provide a deeper understanding of the
catalytic mechanism of this Foldtype II PLP-dependent
enzyme.
Materials and methods
Cloning, overexpression and purification of
StDSD
The dsdA gene from S. typhimurium was amplified by PCR
using the following gene-specific primers: StDSD-sense pri-
mer
CATATGGCTAGC ATG GAA AAC ATA CAA
AAG CTC ATC; StDSD-antisense primer
GGATCC TTA
CTCGAG GCGTCC TTT TGC CAG GTA TTG. The
underlined bases correspond to restriction sites. The sense
primer had NdeI and NheI restriction sites, whereas the

antisense primer had BamHI and XhoI sites. The dsdA gene
was cloned into pET21b between the NheI and XhoI sites.
The cloning strategy was such that the expressed protein
had eight extra amino acids at the C-terminus
S. R. Bharath et al. Crystal structure of D-serine deaminase
FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2887
(LEHHHHHH) that included a hexa-histidine tag. The
clone obtained was confirmed by sequencing. The protein
was overexpressed in E. coli BL21 (DE3) Rosetta cells. The
cells were grown in LB medium containing 100 lgÆmL
)1
of
ampicillin at 37 °C until A
600
reached 0.5 and were then
induced with 1.0 mm isopropyl thio-b-d-galactoside (IPTG)
and grown at 25 °C for a further 6 h. The cells were pel-
leted by centrifugation at 4810 g for 10 min and resus-
pended in buffer A containing 50 mm Tris pH 8.0, 400 mm
NaCl, 30% glycerol and 50 lm PLP. After sonication and
centrifugation, 1 mL of Ni-nitrilotriacetic acid beads were
added to 30 mL of the soluble fraction and kept for end-
to-end rotation for 3 h. The unbound proteins from the
column were washed using buffer B containing 50 mm Tris
pH 8.0, 200 mm NaCl, 20% glycerol and 50 lm PLP. Non-
specifically bound proteins were removed by a wash with
buffer B containing 20 mm imidazole. In the last step, the
protein was eluted using buffer B containing 200 mm imid-
azole. The eluted protein was concentrated to 1 mL using
centricon tubes, loaded onto a Sephacryl S-200 preparative

column for a final round of purification and eluted using
50 mm Hepes buffer pH 7.5 containing 100 mm NaCl. The
purified protein, free of excess PLP was concentrated to
10 mg.mL
)1
in Centricon tubes and used for crystallization.
The purified protein corresponded to a size of 49 kDa on a
12% SDS ⁄ PAGE. The molecular mass was confirmed by
MALDI-TOF.
Selenomethionine incorporation
The plasmid containing dsdA gene was transformed into
BL21 (DE3) pLysS strain of E. coli. The cells were grown
in minimal medium. Methionine biosynthesis was inhibited
by the addition of 50 mgÆL
)1
of Leu, Ile, Val, Lys, Thr and
Phe half an hour before induction with 1.0 mm IPTG. Sele-
nomethionine was added at the time of induction. Purifica-
tion of the enzyme was carried out following the same
protocol as used for the native enzyme, except that all buf-
fers contained 5 mm b-mercaptoethanol. The selenomethio-
nine incorporation was confirmed by accurate mass
determination using ESI-MS.
Biochemical studies
a-keto acids released from d-Ser, d-Thr, d-Allothr and
l-Ser by the enzymatic action of StDSD was estimated
using the 2,4-dinitrophenyl hydrazine (DNPH) method [17].
The reaction mixture for the assay with d-Ser consisted of
50 mm sodium or potassium phosphate buffer (pH 7.5),
20 lm PLP, varying concentrations of d-Ser and 50 ng of

StDSD in a final volume of 50 lL. The reaction was
started by the addition of d-Ser and carried out at 37 °C
for 10 min. Then 50 lL of 0.1% DNPH in 2 m HCl was
added to stop the reaction and the mixture was incubated
at 37 °C for 2 min, followed by the addition of 150 lLof
0.4 m NaOH. After 5-min incubation at room temperature,
A
540
of the resultant hydrazone was measured. The result-
ing absorbance units corrected for enzyme-blank were plot-
ted against substrate concentration. The assay was again
carried out with d-Thr, d-Allothr and l-Ser following a
similar protocol. Activity measurements were also carried
out in the crystallization condition. The kinetic parameters
(K
m
and V
max
) were determined under two different condi-
tions (sodium phosphate buffer pH 7.5 and potassium
phosphate buffer pH 7.5). The amount of enzyme used for
determining K
m
and V
max
was different with each of the
substrates: 50 ng for the assay with d-Ser, 100 ng with
d-Thr, and 500 ng with d-Allothr and l-Ser. The concen-
trations of the substrates were varied between 0.2 and
9.5 mm. Biochemical data were analysed using graphpad

prism 5 (GraphPad software Inc, La Jolla, CA, USA). The
oligomeric state of the purified protein in solution was
determined using an analytical gel filtration column.
Crystallization and data collection
Crystallization trials were carried out with Hampton Crys-
tal screens 1 and 2, PEG-ion screen, and Index screens 1
and 2 using microbatch (under oil) as well as sitting-drop
vapour diffusion methods. The best crystals of StDSD were
obtained from 100 mm trisodium citrate, pH 6.1, containing
5mgÆmL
)1
StDSD, 0.8 m lithium sulfate and 0.4 m ammo-
nium sulfate in the hanging-drop vapour diffusion method.
Prior to crystallization, SeMetDSD (10 mgÆmL
)1
)in50mm
Hepes buffer pH 7.5, 100 mm NaCl, was incubated with
40 mmdl-isoserine for about an hour at 4 °C and n-octyl-
b-glucopyranoside was added to 0.1%. Using this sample,
crystals were obtained under the same condition as that of
the WtDSD crystals. The quality of these crystals was bet-
ter than the WtDSD crystals or WtDSD crystals obtained
in the presence of isoserine.
The crystals were mounted on a cryo-loop and frozen in
liquid nitrogen for X-ray diffraction data collection. Data
on a WtDSD crystal were collected to 2.4 A
˚
resolution using
a Bruker AXS Microstar rotating anode X-ray generator
and a MARRESEARCH image plate detector system. The

data were processed using mosflm and scala of the CCP4
suite [35]. These crystals belonged to the space group C2
with unit cell parameters a = 100.02 A
˚
, b = 46.80 A
˚
,
c = 100.04 A
˚
and b = 93.75°. The crystal asymmetric unit
was compatible with a monomer (solvent content 47.9%).
Data on SeMetDSD crystals were collected at four different
wavelengths, namely 0.97848 A
˚
(peak), 0.97872 A
˚
(inflection
point), 0.97083 A
˚
(high energy remote) and 1.01876 A
˚
(low
energy remote) at beamline 14 of ESRF, Grenoble, France.
The best of these data sets extended to 1.8 A
˚
resolution.
The data were processed using mosflm and scala of the
CCP4 suite. The crystals belonged to the orthorhombic
space group P2
1

2
1
2 with a = 56.4 A
˚
, b = 188.4 A
˚
and
c = 46.59 A
˚
. The asymmetric unit was compatible
Crystal structure of D-serine deaminase S. R. Bharath et al.
2888 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS
with a monomer with a solvent content of 50.9%
(V
M
= 2.5 Da A
)3
). A data set extending to 1.9 A
˚
resolu-
tion was also collected on an SeMetDSD crystal using home
source X-rays. Data collection statistics are given in Table 2.
Structure solution and refinement
The structure of SeMetDSD was determined using the
4W-MAD protocol of Auto-Rickshaw (the EMBL-
Hamburg automated crystal structure determination plat-
form) [36]. The input diffraction data were prepared and
converted for use in Auto-Rickshaw using programs of the
CCP4 suite [35]. Based on an initial analysis of the data, the
maximum resolution for substructure determination and ini-

tial phase calculation was set to 2.2 A
˚
. Twelve heavy atom
positions corresponding to the expected 13 Se atoms were
found using the program shelxd [37]. The correct hand for
the substructure was determined using the programs abs [38]
and shelxe [39]. The figure of merit based on the heavy
atom sites found was 0.59. Initial phases were improved by
density modification using the program shelxe [39]. arp ⁄
warp [40,41] could build 88.9% of the model into the result-
ing electron density map. The model obtained was refined
against the data set collected at high energy remote wave-
length. The SeMetDSD model was also refined to 1.9 A
˚
resolution using the data collected at home source with R
and R-free values of 0.18 and 0.21, respectively (Table 3).
WtDSD structure was determined by molecular replace-
ment using SeMetDSD as the starting model. The solution
obtained using phaser had RFZ and LLG scores of 28.4 and
502, respectively, for the rotation function and TFZ and
LLG scores of 24.8 and 834, respectively, for the translation
function. The native structure was refined to 2.4 A
˚
resolution
with R and R-free values of 0.21 and 0.27, respectively
(Table 3). Coordinates of WtDSD and SeMetDSD and
associated structure factors have been deposited in the
Protein Data Bank with codes
3R0Z and 3R0X.
Structure analysis

The geometry of the final models was examined using pro-
check [27]. Structural superposition of SeMetDSD and
WtDSD was achieved using the programs align [42] and
the ssm superpose [43] feature of coot [44]. Average B-fac-
tors for protein atoms, water molecules and ligands were
calculated using baverage of CCP4 suite. All figures were
prepared using pymol [45].
Acknowledgements
The intensity data for WtDSD and one data set of
SeMetDSD were collected using the X-ray facility for
structural biology at the Molecular Biophysics Unit
(MBU), Indian Institute of Science, supported by the
Department of Science and Technology (DST) and the
Department of Biotechnology (DBT), Government of
India. The other four data sets were collected at BM14
of ESRF, Grenoble, France. We thank the staff at the
X-ray laboratory in MBU and the beam line scientists
at BM14, ESRF, for their cooperation. MRNM and
HSS thank the DST and DBT for funding. SRB and
SB acknowledge the Council for Scientific and Indus-
trial Research, Government of India, for the award of
Research Fellowships.
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