Structural and biochemical studies on the role of active site Thr166 and Asp236 in the catalytic function of D-Serine deaminase from Salmonella typhimurium

Article abstract of DOI:10.1016/j.bbrc.2018.08.116

D-Serine deaminase (DSD) degrades D-Ser to pyruvate and ammonia. Uropathogenic bacteria survive in the toxic D-Ser containing mammalian urine because of DSD activity. The crystal structure of the apo form of Salmonella typhimurium DSD (StDSD) has been reported earlier. In the present work, we have investigated the role of two active site residues, Thr166 and Asp236 by site directed mutagenesis (T166A and D236L). The enzyme activity is lost upon mutation of these residues. The 2.7 ? resolution crystal structure of T166A DSD with bound PLP reported here represents the first structure of the holo form of StDSD. PLP binding induces small changes in the relative dispositions of the minor and major domains of the protein and this inter-domain movement becomes substantial upon interaction with the substrate. The conformational changes bring Thr166 to a position at the active site favorable for the degradation of D-Ser. Examination of the different forms of the enzyme and comparison with structures of homologous enzymes suggests that Thr166 is the most probable base abstracting proton from the Cα atom of the substrate and Asp236 is crucial for binding of the cofactor.

Full text of DOI:10.1016/j.bbrc.2018.08.116

Biochemical and Biophysical Research Communications 504 (2018) 40e45
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Structural and biochemical studies on the role of active site Thr166
and Asp236 in the catalytic function of D-Serine deaminase from
Salmonella typhimurium
a
a
b
Geeta Deka , Sakshibeedu R. Bharath , Handanhal Subbarao Savithri ,
a, *
Mathur Ramabhadrashastry Narasimha Murthy
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India
a
b
Biochemistry Department, Indian Institute of Science, Bangalore, 560012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
D-Serine deaminase (DSD) degrades D-Ser to pyruvate and ammonia. Uropathogenic bacteria survive in
the toxic D-Ser containing mammalian urine because of DSD activity. The crystal structure of the apo
form of Salmonella typhimurium DSD (StDSD) has been reported earlier. In the present work, we have
investigated the role of two active site residues, Thr166 and Asp236 by site directed mutagenesis (T166A
and D236L). The enzyme activity is lost upon mutation of these residues. The 2.7 Å resolution crystal
structure of T166A DSD with bound PLP reported here represents the first structure of the holo form of
StDSD. PLP binding induces small changes in the relative dispositions of the minor and major domains of
the protein and this inter-domain movement becomes substantial upon interaction with the substrate.
The conformational changes bring Thr166 to a position at the active site favorable for the degradation of
D-Ser. Examination of the different forms of the enzyme and comparison with structures of homologous
Received 7 August 2018
Accepted 17 August 2018
Available online 30 August 2018
Keywords:
Salmonella typhimurium
PLP binding
X-ray crystal structure
D-ser
Active site
enzymes suggests that Thr166 is the most probable base abstracting proton from the C
substrate and Asp236 is crucial for binding of the cofactor.
a atom of the
©
2018 Elsevier Inc. All rights reserved.
1
. Introduction
S. cerevisiae [9] and chicken [10]. Crystal structures of DSD from
E. coli (PDB code: 3SS7 [11]), P. xenovorans (PDB code: 3GWQ, Joint
Center for Structural Genomics) and S. typhimurium (PDB code:
3R0Z [12]) have been determined. The E. coli DSD (EcDSD) and
S. typhimurium DSD (StDSD) are monomeric proteins unlike most of
the fold type II PLP dependent enzymes such as 2, 3-
Diaminopropionate ammonia lyase (DAPAL [13]), O-acetylserine
sulfhydrylase (OASS [14]), which are dimers. Crystals of wild type
StDSD (Wt-StDSD, PDB code: 3R0Z) and SeMet incorporated DSD
co-crystallized in presence of isoserine (SeMet-StDSD, PDB code:
D-amino acids such as D-Ala and D-Glu are components of
bacterial cell walls [1]. Although D-Ser is toxic, most bacteria sur-
vive and propagate in D-Ser containing media by degrading D-Ser
to pyruvate and ammonia (Scheme 1), a reaction catalyzed by D-
Serine deaminase (DSD, encoded by dsdA gene). Uropathogenic
bacteria such as S. saprophyticus and E. coli (UPEC) survive in D-Ser
containing urine due to the action of DSD [2] [3]. D-Ser also plays an
important role in regulating the N-methyl-D-aspartate (NMDA)
subtype of glutamate receptor [4].
1 1
3R0X) belong to the space groups C2 and P2 2 2, respectively [12].
DSD (EC 4.3.1.18) belongs to fold type II or tryptophan synthase
b
The structures of both these crystal forms represent the apo form of
the enzyme. These structures suggested that the enzyme un-
dergoes a large conformational change from an open to a closed
conformation upon interaction with isoserine [12]. Based on these
structures, it was suggested that Thr166 might be responsible for
(
TRPS ) family of pyridoxal 5ʹ phosphate dependent (PLP) enzymes.
b
PLP is a versatile cofactor for a wide variety of enzymes that carry
out elimination, replacement, transamination, decarboxylation and
racemization reactions with amino acid substrates [5]. DSD has
been biochemically characterized from E. coli [6,7], Klebsiella [8],
abstraction of proton from Ca atom of D-Ser in the catalytic reac-
tion. It was also proposed that Asp236 might play a role in external
aldimine formation [12]. However, because of lack of crystal
structure of StDSD bound to the cofactor PLP, these proposals could
*
Corresponding author.
E-mail address: mrn@iisc.ac.in (M.R.N. Murthy).
https://doi.org/10.1016/j.bbrc.2018.08.116
006-291X/© 2018 Elsevier Inc. All rights reserved.
0

G. Deka et al. / Biochemical and Biophysical Research Communications 504 (2018) 40e45
41
and scaled using SCALA of the CCP4 suite [16]. The crystal belonged
to the space group C2 with unit cell parameters of a ¼ 108.31 Å,
ꢀ
b ¼ 46.14 Å, c ¼ 99.28 Å and
b
¼ 100.8 . Although the space group is
the same, the unit cell parameters for the T166A mutant were
different from those of the wild type enzyme (PDB code: 3R0Z) for
ꢀ
which a ¼ 100.02 Å, b ¼ 46.79 Å, c ¼ 100.04 Å and
b
¼ 93.75 .
Scheme 1. The degradation of D-Ser to pyruvate and ammonia by D-Serine deaminase,
a PLP dependent enzyme.
2.4. Structure determination and refinement
The structure of StDSD-T166A mutant was determined by mo-
lecular replacement using Wt-StDSD (PDB code: 3R0Z) as the
phasing model. All hetero atoms and water molecules of the
phasing model were removed to avoid model bias. The solution
obtained by PHASER [17] was first subjected to rigid body refine-
ment and then subjected to positional refinement using Refmac5 of
CCP4 suite [18]. Each cycle of refinement was followed by manual
model building using COOT [19].
not be validated further.
In this manuscript, we present the X-ray crystal structure of an
active site mutant (T166A) of StDSD; representing the first PLP-
bound (holo) form of the enzyme. We have carried out biochemical
studies on the T166A and D236L mutants of DSD to understand the
role of Thr166 and Asp236 in the function of DSD. The structural
features that are important for catalysis are discussed in the light of
these structural and biochemical investigations.
2.5. Spectral studies and activity assays
2
. Materials and methods
Spectral studies were carried out to monitor the formation of
2
.1. Cloning, over-expression and purification of active site mutants
internal aldimine as well as the product pyruvate using a Jasco
UVeVisible V-630 spectrophotometer. 1 mg/ml of protein in 50 mM
HEPES pH 7.5, 100 mM NaCl buffer were used for the study. The
reactions were initiated by addition of 1 mM of D-Ser and spectra
were recorded in the range of 300e550 nm as a function of time.
The specific activities of the Wt-StDSD as well as the two active
site mutants (T166A and D236L) were estimated using 2,4-
dinitrophenyl hydrazine (DNPH) method by measuring the a-keto
acid released during the reaction as described previously [12]. The
reaction mixture was composed of 50 mM potassium phosphate
The gene for StDSD cloned in pET21b vector was used as the
template for the generation of single site mutants [12]. Two active
site residues Thr166 and Asp236 were targeted for mutagenesis
(
shown in red italics) using single primer extension method [15].
The primers were designed so as to contain a restriction site
shown in green italics) by choosing appropriate degenerate co-
(
dons. The mutants were further confirmed by sequencing.
buffer (pH 7.5), 50
of enzyme.
mM PLP, 100 mM of the substrate D-Ser and 50 ng
3. Results
The two active site mutants of StDSD were over-expressed and
purified using the protocol found suitable for the wild type enzyme
3.1. X-ray crystal structure determination of StDSD-T166A
[
12]. The purified proteins corresponded to a size of 49 kDa when
examined on a 12% SDS-PAGE.
All the diffraction datasets collected for T166A-DSD mutant
crystals contained spots from polycrystalline contaminations
making meaningful data processing very difficult. Only a single data
set collected on a crystal of T166A which contained multiple ice
rings that could be processed (Supplementary Fig. S1) by excluding
the contaminating rings [20]. Because of the ice rings, the quality of
the processed data was low. This is reflected in the data statistics
(Table 1).
The structure of StDSD-T166A was determined by molecular
replacement using the wild type structure as the phasing model
from which all non-protein atoms were removed. The solution
obtained was refined to a final Rwork and Rfree of 28.91% and 34.85%,
respectively, using REFMAC5 of CCP4 suite [18]. The refinement
statistics is listed in Table 2.
2.2. Crystallization of StDSD
The purified proteins were incubated with 0.1% of n-octyl-b-
glucopyranoside prior to setting up crystallization. Initial trials of
crystallization were carried out at 298 K using the hanging drop
method under the conditions found suitable for the wild type
enzyme [12]. Crystals of mutant proteins (T166A and D236L) could
not be obtained under this condition or with small variations of the
condition. Therefore, screening for suitable conditions was carried
out using a number of commercially available screens including
Hampton Crystal screens 1 and 2, Index screens 1 and 2, Jena Basic
screens 1e4 and Jena classic screens 1e10. No hits were obtained
for D236L. However, crystals of T166A were obtained from 20% w/v
polyethylene glycol 4,000, 100 mM MES pH 6.5, 600 mM sodium
chloride (JBscreen classic 3/C2). These crystals were fragile and
difficult to mount. The diffraction data obtained from the best
crystal was used for structural studies.
3.2. Quality of the model
Despite the limited resolution (2.7 Å) and data quality, a model
(residues 2e439) of the mutant protein StDSD-T166A could be built
into the final electron density map with 99.4% of the residues in the
allowed regions of the Ramachandran map and only two residues
2.3. X-ray diffraction data collection
(0.60%) in the disallowed region. Continuous electron density could
A single crystal of StDSD-T166A was mounted on a cryo-loop
and frozen in liquid nitrogen. X-ray diffraction data extending to
.7 Å were recorded using a CCD image plate detector while
maintaining a temperature of 100 K at the BM14 beamline at ESRF,
Grenoble. The data were processed using the program iMOSFLM
be traced for most of the main chain as well as the side chain atoms
with the exception of a few residues (69e73, 212e227 and the C-
terminal hexa-histidine tag) where electron densities were either
missing or fragmented. These residues have not been included in
the final model.
2

42
G. Deka et al. / Biochemical and Biophysical Research Communications 504 (2018) 40e45
Table 1
Crystal data collection statistics for StDSD-T166A mutant.
3.3. Interactions of the cofactor (PLP) with the active site residues
PDB ID
Wavelength (Å)
Space group)
Unit cell parameters)
Resolution range (Å))
No. of observed reflections)
No. of unique reflections)
6AA9
0.95372
C2
The StDSD-T166A mutant was crystallized in its monomeric
form with one molecule in the crystal asymmetric unit. The overall
topology of the mutant, as expected, agrees closely with the wild
type as well as the SeMet-StDSD structures (RMSD ~1 Å). The
monomeric StDSD-T166A has a large (residues 1e42, 76e108 and
239e440) and a small domain (residues 43e75 and 109e238) like
the wild type enzyme. Significant electron density for a bound PLP
could be located at the active site. The PLP is bound at the beginning
of the helix H7 (Ile115-135Ala) at the interface of the two domains
a ¼ 108.31, b ¼ 46.14, c ¼ 99.28;
53.20e2.70 (2.83e2.70)
49,193 (6733)
13,514 (1791)
3.6 (3.8)
16.7 (22.1)
99.9 (100.0)
6.1 (4.6)
b
¼ 100.8ꢀ
Multiplicity)
a
R
merge (%) )
Completion (%))
I>/< I>)
<
s
3
ꢁ1
Mathews coefficient (Å Da ))
Solvent content (%))
Content of the asymmetric unit)
2.43
49.38%
Monomer
via a Schiff base linkage with the
pyridine ring of PLP is held in a hydrophobic cavity between Ile115
and Leu338. The hydroxyl group at C of PLP is hydrogen bonded
(3.22 Å) to Asn168 and the N of the pyridine ring of PLP is
3
3
Values in parentheses correspond to the highest resolution bin.
1
P
P
P
P
a
R
merge
th
¼
hkl
i
jI
i
(hkl) -<I(hkl)>j/ hkl
i
I
i
(hkl). where I
i
(hkl) is the intensity of
0
hydrogen bonded (2.90 Å) to Thr422. The 5 phosphate group of PLP
is stabilized by a Gly-rich loop or PLP binding loop as observed in all
other PLP dependent enzymes [21]. The PLP is anchored by
hydrogen bonding interactions between the main chain nitrogen
the i observation of reflection hkl and <I (hkl)> is the weighted average intensity.
Table 2
0
Refinement statistics for StDSD-T166A mutant.
atoms of Gly277, Val278, Gly279, Gly280, Gly281 and the 5 oxygen
atoms of the phosphate group of PLP. The interactions that stabilize
the PLP cofactor at the active site are shown in Fig. 1.
R
R
work (%)b
free (%)c
28.91
34.85
No of atoms
Protein
Water
Ligands
Average B-factor (Å )
Protein
Water
Ligands
RMSD from ideal values
Bond length (Å)
Bond angle ( )
Ramachandran plots (%)
Most favoured
Additionally allowed
Generously allowed
Disallowed
Structural superposition of the active sites of Wt-StDSD, SeMet-
StDSD and StDSD-T166A (Fig. 2) reveals that the orientations of
most of the active site residues that interact with the cofactor PLP
are preserved. In both the apo forms of DSD, a sulfate molecule was
3104
158
6
2
0
observed at a place corresponding to the 5 phosphate of PLP. A
19.08
19.70
32.39
movement of ~4.2 Å is observed in the Nε atom of Lys116 between
the Wt-StDSD and the SeMet-StDSD obtained in the presence of
isoserine. The active site is much less accessible to the solvent in the
closed form represented by SeMet-StDSD due to the well structured
segment 234e239 that partially blocks the active site. In the apo
form represented by Wt-StDSD, this segment is disordered and the
active site becomes much more accessible to PLP and the substrate.
Thus the substrate may enter the active site in the open confor-
mation while catalysis may take place in the closed conformation.
0.005
0.912
ꢀ
92.3
7.1
0.0
0.6
P
P
b
R
work (%) ¼ ( hkljF
o
-F
c
j)/ hkl
F
o
, F
o
and F
c
are observed
þ
A metal ion (Na ) bound close the active site was observed in
and calculated structure factor amplitudes.
c
SeMet-StDSD [12]. In StDSD-T166A crystal structure also electron
density at a corresponding position was observed. Based on ge-
ometry and interactions with nearby residues, the density has been
R
free (%) is defined in the same way as Rwork and refers
to randomly selected 5% of the reflections not used for
refinement.
þ
attributed to a Na ion. The metal ion is probably acquired from the
purification buffer. It is coordinated with a number of residues
Fig. 1. Stereodiagram showing interactions of the cofactor PLP (pink ball and stick) with the active site residues (cyan ball and stick) in StDSD-T166A. Inter atomic distances in Å are
shown on black dashed lines connecting the atoms. The electron density for PLP (2mFo-DFc contoured at 1.0
colour in this figure legend, the reader is referred to the Web version of this article.)
s) is shown as a pink mesh. (For interpretation of the references to

G. Deka et al. / Biochemical and Biophysical Research Communications 504 (2018) 40e45
43
Fig. 2. Stereodiagram showing structural superposition of the active sites of apo- StDSD (3R0Z, yellow ball and stick), SeMet-StDSD after interaction with isoserine (3R0X, pink ball
and stick) and holo-StDSD-T166A (green ball and stick). The change in the position and conformation of Lys116 between the open (apo- StDSD and StDSD-T166A) and closed (SeMet-
StDSD) forms is highlighted. In the apo-StDSD and SeMet-StDSD, a sulfate molecule (yellow ball and stick) occupies a position corresponding to 5ʹ phosphate (orange ball and stick)
of PLP in holo-StDSD-T166A. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
including Glu303 (3.08 Å), Cys309 (3.13 Å), Cys276 (3.16 Å), Gly277
2.87 Å), Leu338 (3.13 Å) and Val340 (3.47 Å) (Supplementary
surrounding the metal binding site as well as the PLP binding loop
were ordered even in the absence of the metal ion suggesting the
metal ion may not be required for stabilization of these segments.
(
2
Fig. S2). The metal ion was refined to a B-factor of 18 Å compara-
2
ble with the B-factor of the nearby residues (14e20 Å ). It was
proposed that the metal ion might stabilize the PLP binding loop
because of its interaction with a residue from the loop ie Gly277 and
nearby Cys276 [12]. However, in the WtDSD structure, residues
3
.4. Inter-domain movements
StDSD-T166A represents the first holo form of the enzyme. The
Fig. 3. A. Stereodiagram representing superposition of the Wt-StDSD (orange), SeMet-StDSD after interaction with isoserine (pink) and holo-StDSD-T166A (green) structures. Each
protomer has a large and a small domain. Conformational changes may be viewed as a movement of the small domain against the larger domain. B. Close up view of the residues
(
160e170) from the small domain that undergo structural transition upon open to close conformational change of the protein. In Wt-StDSD (orange ribbon; open form), the catalytic
base Thr166 (orange ball and stick) is far away from the active site. The position of Ala166 (green ball and stick) is unaltered in the T166A mutant (green ribbon; open form). In the
closed form (pink ribbon, SeMet-StDSD) the small domain undergoes conformational transition bringing the catalytic base Thr166 (pink ball and stick) closer to the cofactor (green
ball and stick). This movement is crucial for catalysis. C. Comparison of B-factors of Wt-StDSD (black), SeMet-StDSD (blue) and holo-StDSD-T166A (red). The highly disordered
residues were not included in the final polypeptide chain and hence B-factors are not available for them. (For interpretation of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)

4
4
G. Deka et al. / Biochemical and Biophysical Research Communications 504 (2018) 40e45
Table 3
The PLP binding affinity of wild type as well as mutant StDSD.
three different crystal structures is shown in Fig. 3B. The residues
12e227 are disordered in the T166A-StDSD and the equivalent
residues are ordered in the Wt-StDSD. However, they are associated
with higher B-factors (above 50 Å as compared to 20 Å of nearby
residues) in Wt-StDSD. These features make the active site acces-
sible to PLP and the substrate in the open conformation. The
2
Enzyme name
A
280 nm
A
412 nm
A280/A412
2
2
WtDSD
T166A
D236L
0.1299
0.1409
0.1283
0.0148
0.0132
0.006
8.7
10.67
21.38
equivalent residues are ordered in the SeMet-StDSD with B-factors
2
(
~30 Å ) comparable to those of the surrounding residues. Thus, the
two previously reported structures were in their apo forms (PDB
code: 3R0Z, 3R0X). StDSD (PDB code: 3R0Z) represents an open
form of the enzyme whereas the SeMet-StDSD crystallized in
presence of isoserine represents the closed form. The conforma-
tional transition could be described as a movement of the small
domain against the larger domain (Fig. 3A).
Superposition of the Wt-StDSD, SeMet-StDSD and holo-StDSD-
T166A revealed that the structures of Wt-StDSD and StDSD-T166A
are very similar and both of them show substantial differences from
SeMet-StDSD. Two segments corresponding to residues 160e170
and 180e234 undergo movements of about 5 and 10 Å, respec-
tively, during the structural transition from the open form in Wt-
StDSD and StDSD-T166A to the closed form in SeMet-StDSD
active site is effectively shielded from the solvent in SeMet-StDSD.
Normalized B-factors plot (Fig. 3C) and deviation plot
Supplementary Fig. S3A & B) suggest that the main conformational
changes are confined to the small domain. It appears that the small
domain undergoes a gradual movement from a more open to a less
open (upto 3 Å) conformation during apo to holo transition while it
undergoes a much larger transition to the closed conformation
C
a
(
(upto 10 Å) upon interaction with the substrate/inhibitor. The
closed conformation has the appropriate geometry to carry out
catalysis.
3.5. The role of Thr166 and Asp236 in catalysis
(
Fig. 3A). The deviations observed between Wt-StDSD structure and
the present PLP-bound structure in the equivalent regions
160e170 and 180e234) are small with a maximum deviation of
The wild type StDSD and the mutants (T166A, D236L) were
expressed in E. coli and purified as described in materials and
methods. The purified proteins were yellow in colour indicating the
presence of PLP in the form of an internal aldimine (absorbance
maximum 412 nm). The estimated PLP binding affinities of these
mutant enzymes are shown in Table 3. The absorbance peak at
(
about 3 Å (Fig. 3A). It was observed that the residue Thr166 (a part
of 160e170 segment) undergoes substantial movement when the
conformation changes from the open to the closed form. In apo-
Wt-StDSD and holo-StDSD-T166A structures, which are in the open
conformation, Thr166 is ~ 8e9 Å away from a modelled D-Ser-PLP
external aldimine complex. This distance is too large for Thr166 to
function as a proton abstracting residue. However, change of
conformation to the closed form as observed in SeMet-StDSD brings
4
12 nm was lower for D236L mutant as compared to Wt-StDSD and
T166A-StDSD indicating that the D236L mutant has a lower affinity
for PLP. The absorbance ratio A280/A412 also indicates lower PLP
binding affinity of the D236L mutant (Table 3).
This suggests that the residue Asp236 plays an important role in
the formation of internal aldimine between Lys116 and PLP. Asp236
Thr166 to a distance of 2.6 Å from the Ca position of the ligand,
which is favorable for proton abstraction. The relative position of
Thr166 with respect to the active site or the internal aldimine in the
0
is at about 5 Å away from the 5 phosphate of the internal aldimine
suggesting that it might play an important role in stabilizing the
Fig. 4. Spectral studies of Wt-StDSD, T166A-StDSD and D236L-StDSD upon addition of the substrate D-Ser. The absorbance peak at 412e415 nm indicates that the enzyme is bound
to PLP (internal aldimine). Appearance of an absorbance peak at 330 nm upon addition of the substrate D-Ser indicates the formation of the product pyruvate suggesting that the
enzyme is active. (A), (B) and (C) indicate the spectral profiles of Wt-StDSD, T166A and D236L, respectively, with respect to the substrate D-Ser. Black line corresponds to the
spectrum recorded in the absence of the substrate whereas the spectra recorded at 1 and 5 min after the addition of D-Ser are shown in blue and red, respectively. (D) Result of
DNPH assays carried out to measure the activity of Wt-StDSD and the two (T166A and D236L) active site mutants. Both T166A and D236L appear to be inactive enzymes as
suggested by both spectral studies and activity assays. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

G. Deka et al. / Biochemical and Biophysical Research Communications 504 (2018) 40e45
45
internal aldimine.
online at https://doi.org/10.1016/j.bbrc.2018.08.116.
Spectral studies and activity measurements using 2, 4-DNPH
assay were carried out to monitor the activity of the wild type and
the mutant enzymes (T166A, D236L) as described in materials and
methods. Addition of 1 mM of D-Ser to the Wt-StDSD enzyme re-
sults in the appearance of an absorbance peak at 330 nm due to the
formation of the product pyruvate (Fig. 4A). However, absence of
appearance of the corresponding peak upon addition of the sub-
strate D-Ser to T166A-StDSD (Fig. 4B) and D236L-StDSD (Fig. 4C)
indicated that the mutants were inactive. Measurement of per-
centage specific activity with respect to the Wt-StDSD again sug-
gests that both the mutants (T166A, D236L) are inactive (Fig. 4D).
Combining the available structural information and biochemical
data it is reasonable to suggest the substrate diffuses into the active
site in the open conformation of the enzyme. After the formation of
the external aldimine complex, the enzyme assumes the closed
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[
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conformation in which Thr166 is close to the substrate C
a atom.
[6] D. Dupourque, W.A. Newton, E.E. Snell, Purification and properties of D-serine
Thr166, which is probably activated by the Lys116, abstracts C
a
dehydrase from Escherichia coli, J. Biol. Chem. 241 (5) (1966) 1233e1238.
proton from the external aldimine complex leading to the forma-
tion of an aminoacrylate intermediate. In the homologous enzyme
serine racemase, Ser82 (structurally equivalent to Thr166 in StDSD)
has been proposed as the base abstracting proton from D-Ser [22].
Further non-enzymatic cleavage of the aminoacrylate intermediate
may lead to the formation of pyruvate and ammonia.
In conclusion, the crystal structure of an active site mutant of
StDSD (T166A) represents the first holo form of the enzyme from
Salmonella typhimurium. The holo form resembles the apo or open
conformation of the enzyme while the closed conformation is
observed upon interaction of the enzyme with isoserine. The active
site is accessible to the substrate in the open conformation while it
is shielded from the solvent in the closed conformation. The
movement of the small domain with respect to the large domain
and open to close conformational change is important to bring the
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a
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[
[
Acknowledgments
The authors thank Department of Science and Technology (DST,
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(
DBT, BT/PR7021/BRB/10/1142/2012), Government of India for
financial support. MRN and HSS gratefully acknowledge J.C. Bose
and INSA fellowships. GD and SRB were supported by fellowships
provided by IISc and CSIR, respectively. The authors also thank the
BM14 beamline scientists at ESRF, Grenoble, for collecting the
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.bbrc.2018.08.116.
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