The structure of mammalian serine racemase: Evidence for conformational changes upon inhibitor binding

Article abstract of DOI:10.1074/jbc.M109.050062

Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5′-phosphate-dependent enzyme is involved both in the reversible conversion of L- to D-serine and serine catabolism by α,β-elimination of water, thereby regulating D-serine levels. Because D-serine affects NMDAR signaling throughout the brain, serine racemase is a promising target for the treatment of disorders related to NMDAR dysfunction. To provide a molecular basis for rational drug design the x-ray crystal structures of human and rat serine racemase were determined at 1.5- and 2.1-A resolution, respectively, and in the presence and absence of the orthosteric inhibitor malonate. The structures revealed a fold typical of β-family pyridoxal 5′-phosphate enzymes, with both a large domain and a flexible small domain associated into a symmetric dimer, and indicated a ligand-induced rearrangement of the small domain that organizes the active site for specific turnover of the substrate.

Full text of DOI:10.1074/jbc.M109.050062

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 17, pp. 12873–12881, April 23, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
The Structure of Mammalian Serine Racemase
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EVIDENCE FOR CONFORMATIONAL CHANGES UPON INHIBITOR BINDING
Received for publication, August 7, 2009, and in revised form, January 5, 2010 Published, JBC Papers in Press, January 27, 2010, DOI 10.1074/jbc.M109.050062
Myron A. Smith1, Volker Mack, Andreas Ebneth, Isabel Moraes, Brunella Felicetti, Michael Wood, Dorian Schonfeld,
Owen Mather, Andrea Cesura, and John Barker
From the Department of Structural Biology and Biology, Evotec, 114 Milton Park, Abingdon, Oxon OX14 4SA, United Kingdom
Serine racemase is responsible for the synthesis of D-serine, an
endogenous co-agonist for N-methyl-D-aspartate receptor-type
glutamate receptors (NMDARs). This pyridoxal 5ꢀ-phosphate-
dependent enzyme is involved both in the reversible conversion
of L- to D-serine and serine catabolism by ␣,␤-elimination of
water, thereby regulating D-serine levels. Because D-serine
affects NMDAR signaling throughout the brain, serine race-
mase is a promising target for the treatment of disorders related
to NMDAR dysfunction. To provide a molecular basis for ratio-
nal drug design the x-ray crystal structures of human and rat
transmission and impaired synaptic plasticity in the absence of
D-serine (6, 7).
D-serine is produced by enzymatic conversion of L- to D-ser-
ine mediated by the pyridoxal 5Ј-phosphate (PLP)-containing
enzyme serine racemase (SR). Mammalian SR was first purified
from rat brain and functionally characterized in 1999 by Wolo-
sker and colleagues (8). The enzyme is expressed in glial cells
and neurons and constitutes the sole endogenous source for
D-serine in mammals (9). Both human and rodent SR have been
purified and extensively studied to generate comprehensive
knowledge of the enzymatic characteristics and allosteric mod-
ulation by a number of agents such as ATP and Mg2ϩ ions.
In addition to serine isomerization, the enzyme catalyzes the
␣,␤-elimination of water from L- and D-serine to produce pyruvate
and ammonia, thus, offering the possibility for SR not only to ele-
vate but also to reduce the level of D-serine (5), a role that previ-
ously has mainly been attributed to D-amino acid oxidase (10).
Recent data from SR-deficient mice strongly implicate this
enzyme in the process of NMDAR-dependent plasticity and
neurotoxicity. Knock-out mice were shown to have severely
reduced D-serine levels with the consequence of impaired
NMDAR-mediated transmission, long term potentiation, and
attenuated NMDA- and A␤1–42-induced toxicity in the hip-
pocampus and cerebral cortex (5, 11). These results emphasize
the importance of SR as a pharmacological target to treat neu-
ropathological conditions associated with dysfunction in
NMDAR neurotransmission. Overactivation of NMDARs is
involved in acute and progressive neurodegenerative diseases
such as stroke, amyotrophic lateral sclerosis, and Huntington,
Alzheimer, and Parkinson diseases (12). In addition, consider-
able evidence was found for enhanced NMDAR-mediated
transmission and plasticity as a potential cause for pain condi-
tions, e.g. neuropathic pain (13).
˚
serine racemase were determined at 1.5- and 2.1-A resolution,
respectively, and in the presence and absence of the orthosteric
inhibitor malonate. The structures revealed a fold typical of
␤-family pyridoxal 5ꢀ-phosphate enzymes, with both a large
domain and a flexible small domain associated into a symmetric
dimer, and indicated a ligand-induced rearrangement of the
small domain that organizes the active site for specific turnover
of the substrate.
N-Methyl-D-aspartate receptor-type glutamate receptors
(NMDARs)2 are a key component in glutamatergic transmis-
sion implicated in the development, function, and plasticity of
the nervous system. In addition to the neurotransmitter gluta-
mate, the activation of NMDARs requires the binding of either
of the two endogenous co-agonists, glycine or D-serine, to the
NMDAR “glycine modulatory site” (1, 2). Although both amino
acids have similar potency as co-agonists, they display regional
differences in modulating NMDAR function (3, 4). According
to its distribution within the CNS, D-serine exerts its function
predominantly in corticolimbic brain structures (5). This piv-
otal role of D-serine is supported by several experimental find-
ings, including depletion studies in neuronal tissue prepara-
tions, indicating a strong reduction in NMDAR-mediated
Pharmacological intervention with SR activity constitutes an
innovative strategy to treat neurological disorders. To pave the
way for rational drug design by means of fragment-based
screening and related in silico approaches we wished to deter-
mine the x-ray crystal structure of mammalian SR. In this study
we provide the first crystal structures of mammalian SR both as
a holoenzyme and complexed with an inhibitor. This informa-
tion will be important for the rational design of novel drugs for
the treatment of diverse neurological diseases.
The atomic coordinates and structure factors (codes 3L6B, 3L6R, 3L6C, and
3HMK) have been deposited in the Protein Data Bank, Research Collaboratory
for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
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The on-line version of this article (available at http://www.jbc.org) contains
additional crystallographic data figures.
1 To whom correspondence should be addressed. Tel.: 44-1235-838-864; Fax:
44-1235-838-931; E-mail: myron.smith@evotec.com.
2 The abbreviations used are: NMDAR, N-methyl-D-aspartate receptor-type
glutamatereceptor;PLP, pyridoxal5Ј-phosphate;SR, serineracemase;hSR,
human serine racemase; rSR, rat serine racemase; SDH, serine dehydratase;
CHES, 2-(cyclohexylamino)ethanesulfonic acid; Bis-Tris, 2-[bis(2-hydroxy-
ethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; r.m.s.d., root mean
square deviation.
EXPERIMENTAL PROCEDURES
Human and Rat SR Cloning, Expression, and Purification—
Two point mutations (C2D and C6D) were introduced by site-
directed mutagenesis (QuikChange II, Stratagene) into a wild-
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Inhibitor Binding Changes Serine Racemase Conformation
type pET24a construct. After DNA sequence verification, ammonia, and hydrogen peroxide. The generated hydrogen
plasmid DNA was transformed into Escherichia coli Rosetta 2 peroxide was quantified by addition of AmpLite, which reacts
(DE3) cells for expression at 37 °C in 9 liters of 2ϫYT medium in a 1:1 stoichiometry with hydrogen peroxide in the presence
(16 g of Tryptone, 10 g of yeast extract, and 5 g of NaCl in 1 liter of horseradish peroxidase to produce the highly fluorescent
of H2O) supplemented with 50 ␮g/ml kanamycin and 0.01% oxidation product, resorufin.
pyridoxine. The cells were grown to an optical density at 600
The activity of 100 nM purified SR was determined in 100 mM
nm of 0.6., at which point isopropyl 1-thio-␤-D-galactopyrano- CHES (pH 9) containing 1 mM MgCl2, 1 mM ATP, 10 ␮M PLP,
side was added to a final concentration of 0.5 mM, and the cells 0,05% Pluronic F-127, and 0.5% bovine serum albumin. For
were incubated for a further 16 h at 25 °C. The cells were har- D-serine detection 1.5 units/ml D-amino acid oxidase and 0.25
vested by centrifugation at 5,000 ϫ g for 10 min and stored at unit/ml horseradish peroxidase were added. The reactions
Ϫ80 °C. Next the cell pellet was lysed by sonication and cen- were incubated at 37 °C, and fluorescence emission of resorufin
trifuged for 60 min at 4 °C at 16,500 rpm. All the purification was measured at 585 nm in a Tecan Saphire device. The D-ser-
steps were performed at 4 °C. Because all the proteins had ine concentration present in a given sample was determined by
been expressed with a histidine tag purification of each pro- correlating the fluorescence with D-serine calibration curves.
tein was initially performed by affinity chromatography with
Crystallization and Data Collection—The hSR double mu-
a Talon column. The fractions were analyzed by SDS-PAGE, tant concentrated to 10 mg/ml was crystallized by sitting drop,
and the protein-containing samples were collected and vapor diffusion with 25% polyethylene glycol 3350, 200 mM
loaded onto a Superdex 200 (16/60) column equilibrated sodium malonate, and 50 mM MnCl2 as the reservoir solution.
with 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% glycerol, 50 Crystals appeared overnight and grew to 70 ␮m within 4 days.
␮M PLP, 2 mM MgCl2, 5 mM dithiothreitol. The fractions that The crystal used for the diffraction experiment was cryoprotected
contained Ͼ98% pure SR, after SDS-PAGE analysis, were col- by reservoir solution to which glycerol was added to make 20% v/v
lected, pooled, concentrated to 10 mg/ml, flash frozen in liquid concentration cryoprotection solution. X-ray data were collected
nitrogen, and stored at Ϫ80 °C. Selenomethionine-labeled pro- at the Diamond Light Source using the Area Detector Systems
tein was expressed using B834(DE3) cells (Novagen), and puri- Corp. (ADSC) Quantum 315 charge-coupled device detector in
fication followed the same protocol as human SR mutant.
beamline I04.
Oligomeric Analysis of Recombinant SR—A Superdex 200
The selenomethionine-labeled hSR crystals grew under sim-
5/150 column was calibrated with proteins, including Dextran ilar conditions and cryoprotection was also with 20% glycerol.
blue (2000 kDa), aldolase (156.8 kDa), conalbumin (75 kDa), X-ray data were collected at Diamond Light Source on beam-
ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and ribonu- line I04.
clease A (13.7 kDa). Aliquots of each enzyme (50 ␮l at 10
Rat SR (rSR) double mutant concentrated to 35 mg/ml was
mg/ml) were loaded onto the column and equilibrated with 20 crystallized in 55% v/v Tacsimate (1.8305 M malonic acid, 0.25 M
mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% glycerol, 50 ␮M PLP, ammonium citrate tribasic, 0.12 M succinic acid, 0.3 M DL-malic
2 mM MgCl2, 5 mM dithiothreitol. The flow rate was kept at 0.3 acid, 0.4 M sodium acetate trihydrate, 0.5 M sodium formate,
mg/ml. The elution volume of each molecular weight standard and 0.16 M ammonium tartrate dibasic), pH 8.0, 100 mM Bis-
was recorded, and a calibration curve was obtained by plotting Tris propane, pH 7.8. Crystals grew to 50 ␮m after 10 days. The
the elution volume of the protein (milliliters) against the log of crystal used for the diffraction experiment was cryoprotected
the molecular mass. An aliquot of human SR (hSR) mutant with by reservoir solution to which 20% glycerol was added. X-ray
Cys-2 and Cys-6 mutated to Asp (hSR_C2,6D_CHis, 50 ␮l at 2 data of the rSR malonate complex were collected in-house on a
mg/ml) was also loaded onto the same column under the same Rigaku RUH3R generator and an RAXIS IVϩϩ image plate
conditions, and the elution volume was recorded. Dynamic detector, and the rSR holoenzyme was collected at Diamond
light scattering experiments were performed with a Viscotek Light Source.
802 system on SR samples of 1 and 5 mg/ml concentration in 20
mM Tris, pH 8.0, 100 mM NaCl, 10% glycerol, 50 ␮M PLP, 1 mM selenomethionine crystal were integrated and scaled using
MgCl2, 5 mM dithiothreitol at 22 °C. d*trek, and the selenium atoms were located using SHELX (14).
Structure Determination—The diffraction images of the hSR
Cross-linking experiments with hSR_C2,6D_CHis. Aliquots After solvent flattening with SOLOMON (15) initial inspection
of hSR_C2,6D_CHis (20 ␮l, 10 mg/ml in buffer B) were diluted of the maps revealed excellent density for the protein side
1:10 with phosphate-buffered saline buffer, pH 7.4. Each aliquot chains, and the covalently linked PLP molecule was also easily
was incubated with different concentrations of glutaraldehyde visible. 95% of the structure was built using ARP/WARP (16),
ranging from 0.0005% to 1%. Each sample was incubated for 5 and the final model building was done manually with iterative
min at 37 °C. The reactions were stopped by adding 2 ␮l of 1 M refinement using REFMAC5 (17).
Tris-HCl, pH 8.0.
The diffraction images of hSR were integrated using MOSFLM
Enzyme Assay—To determine the activity of purified SR, a (18) and scaled and truncated using SCALA (19, 20). The struc-
fluorometric assay was used with L-serine as a substrate, cou- ture was determined using PHASER (21) molecular replace-
pling the formation of D-serine to D-amino acid oxidase and ment software, and a model of human serine racemase was
horseradish peroxidase (Sigma-Aldrich), plus the peroxidase previously determined by selenomethionine single anomalous
substrate AmpLite (10-acetyl-3,7-dihydroxyphenoxazine, Bio- diffraction (Table 1). rSR integration and scaling were per-
quest). The D-serine produced during the incubation period formed with D*TREK (22), and the structure was solved with
was degraded by D-amino acid oxidase, generating keto acid, PHASER (21) using the refined human SR model.
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Inhibitor Binding Changes Serine Racemase Conformation
Refinement—After iterative cycles of rebuilding and refine- petitive inhibitor malonate (28). Inhibition by malonate, deter-
ment with REFMAC5 (17), the R-factor of hSR dropped 19.6%, mined at a substrate concentration of 5 mM L-serine, showed
while the free R-factor decreased 20.8%. TLS refinement was the predicted concentration-dependent behavior and revealed
included toward the end using eight TLS groups and the final inhibition constants (Ki) of 59 and 111 ␮M on wild-type and
R-factor/Rfree was 16.7/17.7%. The stereochemistry was good mutant human SR, respectively, in agreement with previously
with 92.1% of residues in the most favored region of the Ram- published data (28). The affinity of malonate for rSR appeared
achandran plot and 7.9% of residues in additionally allowed substantially lower with a Ki of 568 ␮M on the wild-type and
regions with none in the generously allowed or disallowed 1599 ␮M on the mutant enzyme.
regions. The rSR refinement was performed with REFMAC5
Crystal Structure—Human and rat holoenzymes were used
afterwhichthefinalR-factor/Rfree ratiowas22/26%.Thestereo- for protein crystallization and yielded crystals diffracting to as
chemistry was similar to human with 93% of residues in the high as 2.2 Å. Initially the S. pombe structure (PDB code 1V71)
most favored and 7% in the additionally allowed regions of the of serine racemase was used as the model for molecular replace-
Ramachandran plot. Structure alignments were performed ment. Unfortunately, the density for a significant region span-
with COOT (23) and PyMOL (24).
ning residues 70–155 of the human SR sequence, previously
defined as the enzyme’s small domain, was not interpretable.
Further crystal screening was performed to improve crystal
RESULTS
Recombinant hSR and rSR—Expression of wild-type SR in quality and to identify new crystal forms. Co-crystallizations
E. coli was found to yield very low levels of soluble protein. To with the substrate L-serine, glycine, the orthosteric inhibitor,
overcome this issue a homology model of hSR (data not shown) malonate, and other small molecules were explored to deter-
based on the Schizosaccharomyces pombe SR structure (PDB mine if crystal quality could be improved. However, only mal-
code 1V71) was constructed. This model identified two N-ter- onate resulted in highly diffracting co-crystals. In addition, sel-
minal surface-exposed cysteines, Cys-2 and Cys-6, which were enomethionine-labeled protein was produced to generate
hypothesized to affect protein solubility. Indeed, mutation of crystals for experimental phasing. After single anomalous dif-
these two cysteines into aspartic acids did improve protein fraction data collection the structure was solved to 1.7 Å and
expression and enabled the production of sufficient amounts of was used as a model for subsequent structure solutions. The
soluble enzyme. Biophysical and enzymatic activity character- data collection and refinement statistics are shown in Table 1.
ization of the Cys-2 and Cys-6 mutant confirmed comparable Further optimization and screening experiments led to the
properties to wild-type SR. Further stabilization was achieved crystallization of ligand-free rSR with significantly improved
by fusion of a His tag to the C terminus of SR. The optimized resolution of the small domain. The supplemental data show
expression construct yielded soluble, stable, highly pure (Ͼ98% the electron density for human and rat SR contoured at 1␴, as
as judged by SDS-PAGE analysis), and homogeneous protein. well as the Ramachandran plots for each structure.
Both the human and rat mutants were expressed and purified
following similar protocols.
The structures of human and rat SR have an overall fold
typical of ␤-family PLP-dependent enzymes. The secondary
For further characterization of the quaternary protein struc- structure of the mammalian racemase is detailed in Fig. 1 high-
ture we subjected the purified SR to size-exclusion chromatog- lighting the large and small domains. For simplicity all the num-
raphy. Upon gel-filtration chromatography on a Superdex 200 bering used throughout this discussion will correspond to the
5/150 column (Amersham Biosciences), the purified SR prepa- mammalian numbering, unless stated otherwise. The first 2 and
ration gave a clear symmetric peak corresponding to a theoret- last 17 residues of the hSR construct were not well defined in
ical mass of ϳ74 kDa, likely corresponding to the SR dimer. the electron density. In addition, the residues 69–73, those
Subsequently, this observation was confirmed by cross-linking before the start of the small domain, were found to be disor-
experiments with glutaraldehyde. The covalently linked dimer, dered. These residues were not included in the final model.
visualized on SDS-PAGE, was the most abundant species, and
PLP enzymes are categorized into five groups by similarity of
formed at glutaraldehyde concentrations as low as 0.0005%. their secondary structure. SR, as well as the closest homologue
Dynamic light scattering experiments correlated well with the for which a crystal structure is available, serine dehydratase
size-exclusion chromatography results with calculated masses (SDH) (PDB codes 1PWH, 1PWE, and 1P5J) (29, 30), belong to
for the hSR and rSR of ϳ75 kDa and a polydispersity index of the fold-type II group containing a large and small domain.
21%.
Human SR and human SDH have 23% sequence identity and
The activity of wild-type human and rat enzymes, as well as are structurally ϳ90% similar (Fig. 1). Both domains have the
the corresponding C2DC6D-mutant forms was determined by typical open ␣/␤ architecture characteristic of this family. The
a fluorometric assay based on the synthesis of D-serine and ana- large PLP-containing domain has at its core a seven-stranded
lyzed using Michaelis-Menten kinetics. All SR preparations twisted ␤-sheet, with all but the first strand parallel, surrounded
were active and Km values obtained were in accordance with by ten helices. The small domain also has a twisted ␣/␤ archi-
previously published data (8, 26, 27). Wild-type and mutant tecture with four central parallel ␤-sheets, and three ␣-helices
enzymes of the same species showed comparable Km values, arranged so that two (helices 4 and 5) are on one side of the
whereas the Km for rSR (rSR, Km of 3.7 mM; rSR C2DC6D, Km of sheet close to the domain interface, and one (helix 6) is on the
4.5 mM) was slightly lower than for human SR (hSR, Km of 6.5 other solvent-exposed side.
mM; hSR C2DC6D, Km of 7.7 mM). As previously reported, the
For SDH, the boundary between large and small domains
activity of human and rat SR was strongly reduced by the com- is described differently with helix 3 included in the small
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Inhibitor Binding Changes Serine Racemase Conformation
TABLE 1
Crystallographic statistics of human and rat serine racemase
Human selenomethionine
Human
Rat
Rat holo
Data collection
Space group
Cell dimensions
a, b, c (Å)
P21212
P21212
P3
P212121
54.54, 84.21, 70.38
90.00, 90.00, 90.00
45.78 (1.70)
9.4 (28.1)
54.57, 84.29, 69.98
90.00, 90.00, 90.00
38.3 (1.50)
110.59, 110.59, 47.92
90.00, 90.00, 120.00
55.3 (1.95)
48.26, 102.96, 120.92
90.00, 90.00, 90.00
47.4 (1.8)
␣,␤,␥ (°)
Resolution (Å)
R
sym or Rmerge
4.9 (27.5)
6.0 (31.1)
6.9 (19.5)
I/␴I
16.1 (4.5)
13.9 (4.4)
9.4 (2.1)
10.1 (3.5)
Completeness (%)
Redundancy
97.1 (78.9)
98.63 (99.97)
3.7 (3.7)
95.9 (71.3)
94.8 (74.8)
12.45 (5.44)
2.5 (1.6)
3.17 (1.95)
Phasing
Selenium atoms found by SHELX
Mean FOM
4
0.45
0.65
Mean FOM after DM
Refinement
Resolution (Å)
No. reflections
45.8-1.7
33,537
16.8/20.2
1
36.1-1.5
49,358
16.7/17.7
1
47.9-2.2
30,285
21.6/26.7
2
41.1-2.1
32,657
24.1/28.9
2
R
work/Rfree
No. molecules in asu
No. atoms
Protein
2,472
23
2,441
23
4,779
46
4,844
32
Ligand/ion
Water
305
244
80
202
B-factors
Wilson B-factor
Protein
18.8
21.2
15.7
23.4
15.9
8.8
38.2
47.8
36.6
36.8
23.8
29.6
19.7
28.0
Ligand/ion
Water
10.0
33.2
r.m.s.d.
Bond lengths (Å)
Bond angles (°)
0.03
2.0
0.009
1.2
0.015
1.6
0.007
1.0
domain. The SDH small domain, therefore, has four ␣-heli- neighboring A monomer. Leu-72 has a water-mediated inter-
ces and four ␤-sheets (29, 30). Importantly, helix 3 in the SR action with Lys-221, and Glu-73, Gly-74, and Lys-75 are
structure precedes a mobile hinge region (69–77) connecting involved in interactions with Lys-221, Leu-222, and Tyr-218 of
the small and the large domains. This helix, as will be discussed the symmetry-related monomer A.
later, is not involved in the rearrangement of the small domain.
The second monomer (Monomer B) of the rat malonate
Based on our observations of the x-ray crystal structure of ser- complex was less well defined in the electron density with
ine racemase we have included helix 3 (residues 55–67), con- higher overall B-factors. In Monomer B residues 1–3 and 324–
taining the catalytic lysine (Lys-56), into the large domain (Fig. 333 were disordered (consistent with Monomer A), further-
1). Thus, the large PLP-containing domain of SR is constituted more, the loop residues 67–75 that were ordered in monomer A
of residues 1–68 and 157–340, and the small domain spans could not be resolved. The corresponding loop region was dis-
residues 78–155 (Fig. 2). A flexible loop region connecting both ordered in the malonate complex of human structure as well
domains is comprised of residues 68–77 and 145–149. The and constitutes a highly mobile region of mammalian SR. In the
essential PLP cofactor is linked covalently to Lys-56 forming a holo-form of rSR residues 3–323 were visible with the excep-
Schiff base, and the PLP site motifs 54(SXKIRG)59, tion of small domain residues 128–137 and 144–150, which
313(SXGN)316, and 185(GGGG)188 are conserved as found in were disordered in both Monomers A and B. The rat holoen-
other members of the ␤-family of PLP enzymes (29). A manga- zyme and malonate complex of serine racemase have two mol-
nese ion is octahedrally coordinated by the acid groups of resi- ecules associated into a dimer. The dimer interface of rSR is
dues Glu-210 and Asp-216, the backbone carbonyl oxygen of conserved in the malonate complex of hSR via a 2-fold crystal-
Ala-214, and three water molecules. The side-chain amine lographic axis and indicates a consistent dimeric form for mam-
group of Arg-135 and the carboxylic acid group of the malonate malian serine racemase that is in agreement with other bio-
ligand form a salt bridge type ionic interaction in a compact physical data reported herein. An analysis of the structural
active site. Arg-135 may perform a similar role to the “arginine alignment of the human and rat monomers shows that they are
finger” seen in the active site of G-proteins in stabilization of the well aligned with a C r.m.s.d. of 0.62 Å2. However, when the
reaction transition state to enhance the reaction rate (31, 32).
human and rat dimer␣s are aligned, using a single monomer for
The crystal structure of the rat enzyme was determined in the the superimposition, the average r.m.s.d. of the second mono-
ligand-free and malonate-bound form at 2.1- and 2.2-Å resolu- mer rises to 1.71 Å. Alignment of the uncomplexed rat dimer
tion, respectively (Fig. 2, b and c). For the malonate complex, with the rat malonate complex increases the r.m.s.d. even fur-
one monomer (Monomer A) was well ordered, and all residues ther to 2.19 Å indicating greater conformational differences
except for 1 and 324–333 were well defined, including a sol- between the free and complexed structures. The human dimer
vent-exposed loop region of the small domain (residues has a buried monomer-monomer surface area of 840.6 Å2, the
69–73). The ordering of residues 69–73 in monomer A is rat complex 736.8 Å2, and the rat uncomplexed dimer 827 Å2.
favored by stabilizing crystal lattice contacts with helix 9 of the Although the role of crystal packing forces on the dimer inter-
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Inhibitor Binding Changes Serine Racemase Conformation
FIGURE 1. Multiple-sequence alignment of human, rat, and yeast racemases with human and rat serine dehydratase. Human SR and rat SR are 91%
identical, and the sequence of human SR shares 23% identity with human SDH. There is ϳ90% structural similarity between SR and SDH. The small domain
secondary structure of mammalian SR (residues 78–155) is in gray.
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Inhibitor Binding Changes Serine Racemase Conformation
FIGURE 2. a, schematic representation of the rSR-malonate complex symmet-
ric dimer with malonate (orange) and PLP (green) at the dimer interface. The
large domain is colored yellow, and the small domain is light brown. b, rSR
holoenzyme dimer. The large and small domains are colored orange and pink.
c, schematic structure of human SR dimer. The small domains are displayed in
light blue, and the catalytic domains are in dark blue. The human dimer is
generated by a crystallographic axis of symmetry and is consistent with the
rat dimer.
FIGURE 3. Structural alignment of rat holo (gray) and complexed human
(blue) serine racemase. The small domain of each molecule has a different
face cannot be discounted, the variation observed in the structures
reported suggests a high degree of flexibility at the dimer interface. orientation with respect to the large domain (gray). Curved arrows at the base
of H5 and H6 indicate the direction of movement from unbound (open) to
ligand-bound (closed) conformation.
DISCUSSION
Comparison of Mammalian and Yeast Crystal Structures—
Human and rat SR are 90% identical in sequence and are struc-
turally almost indistinguishable. The malonate complex
formed by either orthologue can be compared with the ligand-
free rat structure with similar results. Using a monomer of mal-
onate-bound human and holo rat enzymes for a structural
alignment over all the residues shows that the large domains
superimpose almost identically with an r.m.s.d. of 0.288 Å.
There are, however, large differences in the small domain ori-
entation as indicated by elevation of the r.m.s.d. to 0.711 Å with
the small domains included. The small domain is shifted away
from the PLP in the ligand-free structure with the exception of H4
(residues 85–98), which remains in the same position in both mol-
ecules with only a slight shift at the top of the helix (residue 98)
when compared with the malonate complex (Fig. 3).
conserved, and the yeast has a deletion of three amino acids
when compared with the mammalian sequence.
Comparison of the inhibitor-bound human structure with
the rat holo and yeast structure indicate a closed conformation
for the human complex and an open conformation for the yeast
and rat holo-SR. There is also a large degree of flexibility and
accommodation at the intradomain interface that allows the
small domain to adopt different orientations in each of the crys-
tal structures. The range of motion of the small domain may be
larger, and is likely to be continuous and fluid rather than the
three discrete poses captured, to date, by the crystal structures.
This is suggested by one of our human holo structures where
the small domain was fully disordered, highlighting the mobil-
ity in this region.3 The motion of the domains in the context of
the reaction mechanism will be discussed in the next section.
Reaction Mechanism—The SR-mediated reaction mecha-
nisms are displayed in Fig. 4, according to the proposed scheme
by Yoshimura and Goto (33, 34). For isomerization the L-serine
C␣-hydrogen is replaced by the hydrogen from the hydroxyl
group of Ser-82 of the human SR, which attacks the C␣ from
the opposite side to where the C␣-hydrogen was removed. The
elimination reaction requires the removal of water with the
hydroxyl group from C␤ of L-serine and hydrogen from the lysyl-
NH3 as leaving groups. After the water is removed, lysine replaces
the aminoacrylate, which is then transformed to pyruvate.
There is 40% sequence identity between the S. pombe SR
sequence and the human and rat. A comparison of the S. pombe
crystal structures (1V71 and 1WTC) with the human and rat
structures reveals strong overall structural similarity. The yeast
structure with and without ATP was determined by Goto and
colleagues to 1.7 and 1.9 Å, respectively, both having accessible
PLP sites. The large domains superimpose well with the mam-
malian structures with an r.m.s.d. of 1.30 Å, but differences are
obvious in the orientation of the small domain as the r.m.s.d.
increases to 1.76 Å for the whole structure (Fig. 3). In this region
of the protein there is a distance of 4.5 Å between the ends of
helix 6 (residues 131–147) in the yeast and the mammalian
malonate-bound orthologs where the Arg-135 side chain is ori-
entated in toward the PLP within the catalytic site. The small
domain of the yeast is also rotated away from the PLP site with
respect to the large domain exposing a channel into the PLP.
In the crystal structure of the rat malonate complex the loop
residues (68–77) that constitute the flexible hinge region in the
A monomer are ordered and adopt an extended loop confor-
mation that may be typical of the orientation of these residues
in mammalian SR enzymes. In the open yeast structure the loop
(67–75) is ordered into an ␣-helix, and this is one of the areas of
divergence from the mammalian SR. This protein region is less
Previous structural studies have provided information on the
key features that allow the convergently evolved PLP enzymes
to have similar reaction mechanisms. Comparisons of the SR
and its closest homologue, SDH, which have structural similar-
ity of Ͼ90%, reveal identical transition states with respect to
their catalyzed reactions. The hSR crystal structure provides a
rationale for its capability to perform two different reactions.
Both enzymes can catalyze the elimination of water from serine,
whereas only the SR can mediate the conversion of L-serine to
its D-isomer and vice versa. The isomerization of L-serine is
3 M. Smith, V. Mack, A. Ebneth, I. Moraes, B. Felicetti, M. Wood, D. Schonfeld,
O. Mather, A. Cesura, and J. Barker, unpublished results.
12878 JOURNAL OF BIOLOGICAL CHEMISTRY
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Inhibitor Binding Changes Serine Racemase Conformation
FIGURE 4. The racemase and dehydratase reaction mechanism of serine racemase. The racemase can attack the L-serine with either Ser-84 or Lys-56. The
Ser-84 hydrogen from the hydroxyl side-chain attacks the C␣ from the opposite side to where the L-serine C␣-hydrogen was removed and racemization occurs
resulting in D-serine. Or the lysyl-NH3-group and the hydroxyl-group from C␤ of L-serine lose H and OH, respectively, to form water and follow the dehydratase
reaction path. In the transition state a planar sp2-configuration of the L-serine C␣ is created by removal of the C␣-hydrogen at which point the racemase can
proceed with either reaction.
dependent on the serine residue at position 84, which donates dence suggesting that the small domain of the racemase, upon
the proton to mediate this particular reaction. In contrast SDH, binding to the substrate or inhibitor, undergoes a major struc-
which has an alanine at this position, is devoid of racemase activity tural change placing Ser-84 into close proximity to the sub-
and can, therefore, also not use the D-isomer as substrate.
strate and in an ideal orientation to donate the proton required
Mutagenesis studies reported by Yoshimura and Goto (33) for isomerization.
showed the essential role of Ser-84 for the isomerization of
First, it can easily be seen that the catalytically active residue
serine and the consumption of D-serine. They found that a Ser-84 in the mammalian racemase is in close proximity to the
Ser-82 SR mutant from D. discoideum lost racemase and D-ser- C␣ of L-serine (2.6 Å) to allow the hydroxyl group to drive
ine dehydratase activity but retained its capability to act as an the transition state into racemization. The corresponding resi-
L-serine dehydratase. This provides evidence for the crucial due Ala-65 in the dehydratase is almost identically positioned
function of Ser-82 for racemization, which appears to be although being catalytically inactive. The yeast racemase
responsible for shuttling the transition state either into racemi- ortholog, free of substrate and inhibitor, shows the small
zation of L-serine or allowing the D-serine to pass the transition domain helix (H6, residues 132–144) to be tilted away from the
state toward dehydratation (Fig. 4).
catalytic site by almost 4.5 Å as compared with the correspond-
Ligand-induced Shift of Small and Large Domains of the ing helices of the human (131–147) and rat complexed race-
Racemase—It is interesting to compare the new structures of mases (131–147). The small domain of the yeast structure is
mammalian serine racemase complexed with malonate to the also differently orientated with respect to the corresponding
yeast open structure and to the serine dehydratase bound to the helix of SDH (residues 113–126), which reflects the closed con-
inhibitor L-serine O-sulfate. We provide several lines of evi- formation of mammalian SR. The catalytically active Ser-82 in
APRIL 23, 2010•VOLUME 285•NUMBER 17
JOURNAL OF BIOLOGICAL CHEMISTRY 12879

Inhibitor Binding Changes Serine Racemase Conformation
upon release of the enzyme from its
membrane anchor phosphatidyl-
inositol (4,5)-bisphosphate. Indeed,
it has been shown that serine race-
mase seems to be kept in an inacti-
vated state when associated to
membranes and becomes activated
only upon release into the cytosol.
This process seems to be intimately
linked to NMDAR activity, thus,
providing feedback control to avoid
excessive NMDAR activation (35).
A similar small domain movement
as described above has been sug-
gested by Chen et al. to occur also in
the PLP-dependent bacterial aspar-
tate decarboxylase upon substrate
binding (25). However, this hypoth-
esis is based on the three-dimen-
sional structure of the holoenzyme
only and with no substrate-bound
enzyme available. This may suggest
a highly conserved reaction mecha-
nism within this family of enzymes.
In summary the x-ray crystal
FIGURE 5. Overlay of the PLP site of human serine racemase (blue), holo rat serine racemase (orange),
yeast serine racemase (magenta), and rat serine dehydratase (green). The catalytic serine in yeast SR and
rat SDH is too far from the malonate or O-methyl serine (5.29 and 9.31 Å, respectively) for any reactions to occur
in its open conformation. The human SR and rat SDH active sites overlay well both ligands in similar confor-
mation. The catalytically inactive dehydratase Ala-65 is superbly aligned with the catalytic racemase Ser-84.
the yeast structure is shifted to a position where it cannot par- structures reported here have confirmed that the domains of
ticipate in the turnover of L-serine, because it is too distant to serine racemase are connected by a flexible linker and can adopt
allow the hydroxyl group to donate the proton (5.29 Å). In addi- different orientations with respect to each other. The crystal
tion, Ser-82 is tilted away by the helix displacement and, there- structures correspond to biophysical data and confirm the
fore, its hydroxyl group does not point to the catalytically active dimeric state of the enzyme. Based on the motion of the
site (Fig. 5). The catalytic Ser-84 of the holo rat is positioned domains observed between the unbound and ligand-bound
even further away from the ligand and represents an even structures we have redefined the domain boundaries of serine
greater opening between the large and small domains, into the racemase in relation to the closely related type II PLP-depen-
active site than for the yeast structure.
dent enzyme, SDH. The orthosteric inhibitor, malonate, adopts
Second, it is evident from the closed structures of the mal- the binding pose of the cognate substrate, serine, in the active
onate-bound racemases that the access of L-serine to the cata- site of the closed structures. The racemization reaction is crit-
lytic site is hardly possible because of the very tight cleft ically dependent on the proximity of the catalytic Ser-82 to the
between the large and the small domains. Hence, it appears substrate as shown by the closed ligand-bound structures. The
likely that both domains need to move apart to create a gateway structures of SR we have reported represent starting points for
for the substrate and that binding of substrate or inhibitor trig- rational drug design and suggest the possibility of modulating
gers an induced shift of the small domain toward the catalytic the enzymatic activity possibly by blocking the movement of
site, which puts Ser-84 into the correct position toward the the small domain.
L-serine. This mechanism suggests that both subdomains are
Acknowledgments—We thank Dr. Young-Hwa Song for supporting
our crystallographic efforts and for technical input. We also thank
Dr. John Kemp for his advice, discussion, and critical review of the
manuscript.
linked via a flexible hinge region.
Recent publications provide further support for the hypoth-
esis that the small domain has to undergo an induced shift to
allow the binding of the substrate. It has been shown that the
racemase can be inhibited by binding to phosphatidylinositol
(4,5)-bisphosphate, which is accomplished by several con-
served lysine residues (33). Three of these residues (Lys-75, Lys-
77, and Lys-96) shown to be involved in phosphatidylinositol
(4,5)-bisphosphate-mediated inactivation of SR, reside in or
close to the small domain, and two of those in direct vicinity
of the catalytically active Ser-84 in the loop region (Lys-75
and Lys-77). It may be of interest that this loop is unique to
mammalian racemases and absent in all other orthologs.
Hence, it might be speculated that this region controls the acti-
vation of SR by allowing the substrate induced fit to occur only
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APRIL 23, 2010•VOLUME 285•NUMBER 17
JOURNAL OF BIOLOGICAL CHEMISTRY 12881

Protein Structure and Folding:
The Structure of Mammalian Serine
Racemase: EVIDENCE FOR
CONFORMATIONAL CHANGES UPON
INHIBITOR BINDING
Myron A. Smith, Volker Mack, Andreas
Ebneth, Isabel Moraes, Brunella Felicetti,
Michael Wood, Dorian Schonfeld, Owen
Mather, Andrea Cesura and John Barker
J. Biol. Chem. 2010, 285:12873-12881.
doi: 10.1074/jbc.M109.050062 originally published online January 27, 2010
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