Metal ion dependency of serine racemase from Dictyostelium discoideum

Article abstract of DOI:10.1007/s00726-012-1232-z

d-Serine is known to act as an endogenous co-Agonist of the N-methyl-d-Aspartate receptor in the mammalian brain and is endogenously synthesized from l-serine by Apyridoxal 5′-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the d-serine metabolism, such as serine racemase, d-Amino acid oxidase, and d-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of d-serine such as Arole in cell development. As part of the elucidation of the role of d-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na⁺ in addition to Mg²⁺ and Ca²⁺, which are well-known activators for the mammalian serine racemase. Mg²⁺ or Na⁺ binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg²⁺ and Na⁺ were determined to be 1.2 μM and 2.2 mM, respectively. In the l-serine dehydrase reaction, Mg²⁺ and Na ⁺ enhanced the k _cat value without changing the K _m value. Alanine mutation of the residues E207 and D213, which correspond to the Mg²⁺-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg²⁺- and Na⁺-dependent stimulation. These results suggest that Mg²⁺ and Na⁺ share the common metal ion-binding site.

Full text of DOI:10.1007/s00726-012-1232-z

Amino Acids (2012) 43:1567–1576
DOI 10.1007/s00726-012-1232-z
ORIGINAL ARTICLE
Metal ion dependency of serine racemase
from Dictyostelium discoideum
•
Tomokazu Ito Hirotaka Murase Motoki Maekawa
•
•
•
•
Masaru Goto Shuhei Hayashi Hajime Saito
•
•
Masatoshi Maki Hisashi Hemmi Tohru Yoshimura
•
Received: 11 November 2011 / Accepted: 23 January 2012 / Published online: 5 February 2012
Ó Springer-Verlag 2012
Abstract D-Serine is known to act as an endogenous
co-agonist of the N-methyl-D-aspartate receptor in the
mammalian brain and is endogenously synthesized from
L-serine by a pyridoxal 50-phosphate-dependent enzyme,
serine racemase. Though the soil-living mycetozoa
Dictyostelium discoideum possesses no genes homologous
to that of NMDA receptor, it contains genes encoding
putative proteins relating to the D-serine metabolism, such
as serine racemase, D-amino acid oxidase, and D-serine
dehydratase. D. discoideum is an attractive target for the
elucidation of the unknown functions of D-serine such as a
role in cell development. As part of the elucidation of the
role of D-serine in D. discoideum, we cloned, overex-
pressed, and examined the properties of the putative serine
racemase exhibiting 46% amino acid sequence similarity
with the human enzyme. The enzyme is unique in its
stimulation by monovalent cations such as Na? in addition
to Mg2? and Ca2?, which are well-known activators for
the mammalian serine racemase. Mg2? or Na? binding
caused two- to ninefold enhancement of the rates of both
racemization and dehydration. The half-maximal activa-
tion concentrations of Mg2? and Na? were determined to
be 1.2 lM and 2.2 mM, respectively. In the L-serine
dehydrase reaction, Mg2? and Na? enhanced the kcat value
without changing the Km value. Alanine mutation of the
residues E207 and D213, which correspond to the Mg2?
-
binding site of Schizosaccharomyces pombe serine race-
mase, abolished the Mg2?- and Na?-dependent stimula-
tion. These results suggest that Mg2? and Na? share the
common metal ion-binding site.
Keywords D-Serine ꢀ Serine racemase ꢀ
Dictyostelium discoideum ꢀ Metal ion activation
Abbreviations
SR
Serine racemase
DdSR
PLP
Serine racemase of Dictyostelium discoideum
Pyridoxal 50-phosphate
2,4-DNP 2,4-Dinitrophenyl hydrazine
T. Ito, H. Murase, and M. Maekawa contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-012-1232-z) contains supplementary
material, which is available to authorized users.
Introduction
In the mammalian brain, D-serine acts as an endogenous co-
agonist of the N-methyl-D-aspartate (NMDA) receptor and
modulates brain function by binding to the ‘‘glycine site’’ of
its NR1 subunit (Wolosker et al. 2008). The NMDA receptor,
a key excitatory neurotransmitter receptor, is involved in
central nervous functions, including memory, synaptic
plasticity, and development. The hyper- and hypofunction of
NMDA receptor brought by D-serine signaling dysfunction is
thought to be involved in diseases, such as schizophrenia,
T. Ito ꢀ H. Murase ꢀ M. Maekawa ꢀ S. Hayashi ꢀ H. Saito ꢀ
M. Maki ꢀ H. Hemmi ꢀ T. Yoshimura (&)
Department of Applied Molecular Biosciences,
Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya, Aichi 464-8601, Japan
e-mail: yosimura@agr.nagoya-u.ac.jp
M. Goto
Department of Biomolecular Science,
Faculty of Science, Toho University,
Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
123

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T. Ito et al.
Alzheimer’s disease, and amyotrophic lateral sclerosis
(ALS) (Nishikawa 2005; Sasabe et al. 2007). Reduced levels
of D-serine in cerebrospinal fluid and serum were reported in
schizophrenia patients. Sasabe et al. (2007) proposed that
elevation of D-serine concentration is the key determinant of
glutamate toxicity in ALS. Excessive amounts of D-serine
are thought to cause glutamate toxicity and lead to
motoneuronal cell death. In addition to neurotransmission,
D-serine is thought to be related to cell development. D-Serine
concentration has been reported to change along with the
developmental stage of some tissues (Hamase et al. 2002).
D-Serine content reaches 270 nmol/g in the newborn rat
cerebellum but rapidly decreases and is almost diminishes in
2-week-old rats. We found that the D-serine concentration in
the silkworm Bombyx mori transiently increases during
specific stages of metamorphosis, including pupation and
eclosion (our unpublished results).
that are homologous to NMDA receptors. D. discoideum is
an attractive target for the elucidation of the unknown
functions of D-serine such as its role in cell development. In
the current work, we studied the properties of DdSR,
focusing on the effect of metal ions and nucleotide phos-
phate. DdSR showed 46, 47, and 38% sequence similarity
to that of human, mouse, and S. pombe SRs, respectively.
DdSR is a dimer that catalyzes both serine racemization
and dehydration. Like other SRs, DdSR is regulated by
some divalent metal ions and metal ion-nucleotide com-
plexes such as Mg2? and MgATP, respectively. Unlike
other SRs, DdSR is stimulated by Na? in addition to the
divalent metal ions. Our data indicate that the Na? and
divalent metal ions bind to the same binding site of the
enzyme. We also demonstrated that the binding site of the
MgATP complex differs from that of the Na? and divalent
cations. Binding of MgATP brings conformational change
to the enzyme and enhances its catalytic activity.
D-Serine is synthesized from L-serine by a serine race-
mase (SR), a member of the fold-type II group of pyridoxal
50-phosphate (PLP)-dependent enzymes (Wolosker et al.
2008; Yoshimura and Goto 2008). SR is recognized to be
responsible for D-serine biosynthesis in mammals because
D-serine levels in SR-deficient mice were reported to be
*10% of that of wild-type mice (Basu et al. 2009). In
addition to serine racemization, the enzyme catalyzes ser-
ine dehydration (a,b-elimination) to yield pyruvate and
ammonia (De Miranda et al. 2002). The rate of serine
dehydration is comparable to that of serine racemization
(Strisovsky et al. 2005).
Materials and methods
Materials
PLP, nicotinamide adenine dinucleotide (NADH), isopro-
pyl-b-D-thiogalactoside (IPTG), and lactic dehydrogenase
from pig heart were obtained from Wako Pure Chemicals
(Osaka, Japan). ATP was purchased from Sigma-Aldrich
Japan (Tokyo, Japan). D- and L-serine were obtained from
Kyowa Hakko (Tokyo, Japan). All other chemicals were of
the highest grade commercially available.
Because of the physiological importance of D-serine, the
regulation of intrinsic D-serine has attracted interest. SR
possibly modulates the level of D-serine not only by its
synthesis but also by its decomposition. The enzymatic
activities of SRs were reported to be modulated by various
agents (Kim et al. 2005; Mustafa et al. 2007; Balan et al.
Cloning and expression of the DdSR
The serine racemase genes of D. discoideum were ampli-
fied by KOD-plus DNA polymerase (TOYOBO, Tokyo,
Japan), using the following primers: DdSR-FW, 50-cac
catggaacccatggcaacagtaac-30 and DdSR-RV, 50-aatttgga
tcctcaatgatgatgatgatgatgatttaaaatctttgaaattgaac-30. The EST
clone of the serine racemase (DDB0230209, ID of Dicty-
base) obtained from Japan National Bio Resource Project
(NBRP-nenkin) was used as a template to amplify the
target gene. The resultant PCR product was treated with
NcoI and BamHI followed by ligation with pET-16b vector
(Novagen, Madison, WI, USA) and introduced into Esch-
erichia coli Top10 cells (Invitrogen, Gaithersburg, MD,
USA). The resultant expression vector pDdSR was
designed to express DdSR with a C-terminal 6-histidine
tag. The sequence of the inserted gene was confirmed by
DNA sequencing using a BigDye Terminator v1.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA,
USA).
2009). Among them, divalent cations, such as Mg2?, Ca2?
,
Mn2?, and Mg-adenosine triphosphate (MgATP) are
common activators of SRs.
In this work, we carried out enzymological studies of the
SR of Dictyostelium discoideum (DdSR). D. discoideum is
a soil-living mycetozoa that is used in developmental
studies because it has a unique life cycle consisting of a
unicellular ameba phase and a multicellular body at the
sporulation stage. D. discoideum contains genes that are
homologous to those of the serine racemase (De Miranda
et al. 2002; Strisovsky et al. 2005; Goto et al. 2009),
eukaryotic D-serine dehydratase (DdDSD) (Ito et al. 2008),
and D-amino acid oxidase (DdDAO) (Pollegioni et al.
2007), and expresses their cognate mRNA (Dicty Base,
http://www.dictybase.org). The presence of a set of D-ser-
ine metabolizing enzymes implies that D. discoideum
synthesizes and metabolizes D-serine, yet it has no genes
123

Metal ion dependency of serine racemase from D. discoideum
1569
Construction of DdSR mutants
substrate was assayed spectrophotometrically at 340 nm
derived from the NADH oxidation coupled with the con-
version of pyruvate to lactate (Ito et al. 2007, 2008).
Pyruvate was also assayed using the 2,4-dinitrophenyl
hydrazine (2,4-DNP) method as described previously (Ito
et al. 2007, 2008).
Mutations of the E207 and D213 residues of DdSR to
alanine residue (the resultant mutant enzymes were named
E207A and D213A, respectively) were carried out by PCR
using the following synthetic primers and their compli-
mentary oligonucleotides: 50-gtatttgctgctgcaccattaggtgct-
gatg-30 for E207A and 50-ccattaggtgctgatgctacttatcgttcac-30
for D213A with KOD-plus DNA polymerase and a pDdSR
as a template. The reaction mixture was then treated with
DpnI to degrade the parental plasmid and the E. coli Top10
cells were transformed with the resultant mutated plasmids.
Sequences of DNA inserts of the plasmid constructs
encoding the mutant enzymes were verified by DNA
sequencing.
Serine racemase activity was assayed by measuring the
amount of each antipodal serine formed from D- or L-serine.
Enantioselective determination of the serine was performed
with a high performance liquid chromatography (HPLC)
after the serine was derivatized to a fluorescent diastereomer.
The reaction was performed at 30°C for 30 min in a reaction
mixture (200 lL) consisting of 50 mM buffer (Tris–HCl or
borate–NaOH buffer, pH 8.5), 20 lM PLP, 50 mM sub-
strate, and 3.5 lM of DdSR. The reaction was stopped by the
addition of 200 lL of 10% trichloroacetic acid (TCA) to the
reaction mixture followed by incubation of the mixture at
4°C for 15 min. After three times extraction with water-
saturated diethyl ether, the aqueous layer was collected. The
solution was diluted appropriately and a 10 lL portion was
mixed with 190 lL of borate–NaOH buffer (pH 9.0) and
50 lL of BOC-L-Cys-OPA, a mixture of 5 mg of t-butoxy-
carbonyl-L-cysteine and 5 mg o-phthalaldehyde mixture in
500 lL of methanol. Separation and quantification of the
derivatized D- and L-serine were performed as described
previously (Hashimoto et al. 1992).
Expression and purification of the enzymes
For the expression of DdSR, E. coli Rosetta II (DE3) cells
(Novagen, Madison, WI, USA) harboring plasmids were
grown in LB medium containing 100 lg/mL of ampicillin
and 30 lg/mL of chloramphenicol at 30°C. The protein
expression was achieved by the addition of isopropyl-b-D-
thiogalactoside (IPTG) to the medium at the mid-log phase
to a final concentration of 0.1 mM and the cells were har-
vested after 13-h incubation. The cells were suspended in a
binding buffer (20 mM Tris–HCl, 500 mM NaCl, and
20 mM imidazole, pH 7.9) and sonicated and centrifuged at
20,0009g for 30 min. The supernatant was applied to a
Ni2?-affinity column equilibrated with the binding buffer.
The column was washed with washing buffer (20 mM Tris–
HCl buffer containing 500 mM NaCl and 80 mM imidaz-
ole, pH 7.9) and the DdSR was eluted with elution buffer
(20 mM Tris–HCl buffer containing 500 mM NaCl and
500 mM imidazole, pH 7.9). The eluted protein was passed
through an Econo-Pac 10DG desalting column (Bio-Rad,
Hercules, CA, USA) with 10 mM Tris–HCl buffer (pH 8.5)
containing 20 lM PLP and stored with 10% glycerol at
-80°C until use. The N-terminal amino acid sequence of
the purified enzyme was confirmed with an Applied Bio-
systems Procise HT. The concentration of protein was
determined by measuring the absorbance at 280 nm using
Gel-filtration chromatography
To estimate the molecular weight of DdSR, the purified
enzyme (2.5 mg) was subjected to gel-permeation chro-
matography with a HiLoad 16/60 Superdex 200 pg column
(GE-Healthcare, Waukesha, WI, USA) equipped with
FPLC system (Pharmacia, Uppsala, Sweden). DdSR was
eluted with 10 mM borate–NaOH (pH 8.5), 20 lM PLP,
200 mM NaCl, and 1 mM EDTA at a flow rate of 1 mL/
min at 4°C. The column was calibrated with catalase
(molecular mass, 232 kDa), aldolase (158 kDa), bovine
serum albumin (66 kDa), egg albumin (45 kDa), and
chymotrypsin (25 kDa).
Spectrophotometric measurements
the molar absorption coefficient of 15,950 M-1cm-1
.
The UV–visible spectrum of the enzyme (22 lL) was
recorded in 10 mM borate buffer (pH 8.5) with a Shimadzu
UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan).
Fluorescence spectra were taken with a HITACHI F-4500
fluorescence spectrophotometer (HITACHI, Hitachi,
Japan) at 4°C using 5.0 nm of excitation and emission
bandwidths at a protein concentration of 0.1 mg/mL in
50 mM Tris–HCl buffer (pH 8.5). The excitation wave-
length was 295 nm.
Assay of enzyme activities
Serine dehydrase activity was assayed as follows. The
reaction mixture (1 mL) contained 50 mM buffer (Tris–
HCl or borate–NaOH buffer, pH 8.5), 20 lM PLP, 50 mM
D- or L-serine, 0.3 mM NADH, 10 units of lactic dehy-
drogenase, and 10–370 lg of DdSR. The reaction was
carried out at 30°C and the pyruvate released from the
123

1570
T. Ito et al.
Fig. 1 Alignment of the
deduced amino acid sequences
of serine racemases (SR) and rat
L-serine dehydratase. The
deduced amino acid sequences
of the D. discoideum SR
(DdSR), human SR
(SR_HUMAN), mouse
SR (SR_MOUSE), S. pombe
SR (SR_SCHPO), and the
rat L-serine dehydratase
(SDHL_RAT) were aligned
using the Clustal W program.
The numbers on the left are the
residue numbers of each amino
acid sequence. The Mg2? and
K
? binding residues of the yeast
SR and rat L-serine dehydratase,
respectively, are shown by
closed circles. In the yeast SR,
Leu182, Gly183, Ala237, and
Thr239 were indirectly involved
in the Mg2? binding (Goto et al.
2009)
Results
74.4 kDa by gel-permeation chromatography (Fig. 2c). The
calculatedmolecularmassofDdSRis35 kDa, suggestingthat
the enzyme exists as a dimer in solution. As shown in Fig. 3,
the L-serine dehydrase and racemase activities of DdSR
increased as the buffer pH increased. Similar pH dependency
was obtained in Britton–Robinson buffer system (pH 6–10.5)
(Supplementary Fig. 1). The enzyme was stable within the pH
range of 6–10.5 (data not shown). The reasons for the low
catalytic activities in Tris–HCl and potassium phosphate
buffers (KPB) are discussed later. We used buffers (borate or
Tris) of pH 8.5 during the course of the study.
Cloning and expression of DdSR
To examine the roles of D-serine and its metabolism in
D. discoideum, we constructed an E. coli expression system
of the putative serine racemase of D. discoideum (DdSR).
DdSR showed 46, 47, and 38% sequence similarity to
human, mouse, and S. pombe SRs, respectively (Fig. 1). The
recombinant protein with an additional C-terminal 6-histi-
dine tag was successfully obtained in the soluble fraction of
the E. coli cell lysate and purified to homogeneity using
Ni2?-chelating column chromatography. DdSR showed a
single protein band on SDS-PAGE gel with an estimated
molecular mass of 35 kDa (Fig. 2a) and exhibited absorption
maxima at around 280 and 420 nm (Fig. 2b, solid line). The
peak at around 420 nm was decreased by the reduction with
NaBH4 with a concomitant increase at around 320 nm
(Fig. 2b, dotted line), which is a well-known phenomena that
occurs upon reduction of the Schiff base between PLP and an
e-amino group of the lysyl residue of the enzyme.
The steady-state kinetic parameters of racemization and
dehydration (a,b-elimination) catalyzed by DdSR for both
L-serine and D-serine as substrates were obtained in borate
buffer and are shown in Supplementary Table 1. The Km
and kcat values in the serine racemization were determined
to be 30.4 mM and 87.4 min-1 for L-serine and 34.0 mM
and 69.5 min-1 for D-serine. The kcat/Km values of serine
racemization were similar in both directions. In the
dehydrase reaction, the Km and kcat values for L-serine
were 40.1 mM and 57.9 min-1, while those for D-serine were
20.7 mM and 13.2 min-1, respectively. These values were
obtained in the absence of known activators for SRs, such
as metal ions and nucleotides.
Enzymatic characterization of DdSR
We compared the enzymatic properties of DdSR to those of
other SRs. The molecular mass of DdSR was deduced to be
As reported previously, the racemization and dehydra-
tion catalyzed by SRs from mammals and S. pombe are
123

Metal ion dependency of serine racemase from D. discoideum
1571
Fig. 2 a SDS-PAGE analysis
of DdSR. The purified DdSR
(A) (kDa)
(2.4 lg) was analyzed by 10%
SDS-PAGE gel and stained with
coomassie blue. b Absorption
spectra of DdSR. The solid line
represents the purified enzyme
(22 lM). The dotted line
illustrates the spectrum of the
enzyme mixed with 0.5 mM
NaBH4 at 5 min after mixing.
The measurements were taken
in a 1-cm light pass cell at 25°C
in 10 mM borate–NaOH buffer
(pH 8.5). c Gel-filtration
chromatography of DdSR.
Elution profile of DdSR is
shown. The inset figure shows
the calibration curve obtained
with a set of molecular mass
standard
97
66
0.4
0.3
0.2
0.1
0
(B)
45
31
250
300
350
400
450
500
Wavelength (nm)
(C)
5.6
5.4
5.2
5
DdSR
4.8
4.6
4.4
4.2
4
60
65
70
75
80
85
90
Retention time (min)
Vo
0
20
40
60
80
100
120
Retention time (min)
stimulated by the addition of Mg2? or MgATP. In addition
to Mg2?, other divalent cations such as Ca2? and Mn2?
also act as SR activators. Mg2? and MgATP enhance the
mouse enzyme by five- to tenfold in racemization and
dehydration, and the addition of EDTA abolishes the
enzyme activities (De Miranda et al. 2002). In the presence
of MgATP, the Km and kcat values of the mouse enzyme in
the racemization with L-serine as a substrate were reported
to be 3.8 mM and 45.5 min-1, while those with D-serine
were 14.5 mM and 113 min-1, respectively. Under the
same conditions, the Km and kcat values in the L-serine
dehydration were 4.0 mM and 81.3 min-1 and those in the
D-serine dehydration were 3.2 mM and 8.8 min-1 (Stri-
sovsky et al. 2005). The kcat values in both racemization
and dehydration were similar to those of DdSR obtained
without the use of Mg2? or MgATP. In contrast, the Km
values of DdSR were about one order of magnitude higher
than those of the mouse SR.
Unlike the mouse and yeast SRs, DdSR was inhibited by
Mg2?, Ca2?, and MgATP under the assay conditions using
borate–NaOH buffer (Fig. 4a). Presence of 1 mM Mg2?
and Ca2? decreased the racemase and dehydrase activities
by around 50%. Addition of free ATP and EDTA showed
neither stimulatory nor inhibitory effects. Treatment of the
enzyme with EDTA also had no effect on the enzyme when
it was assayed in the borate–NaOH buffer (data not
shown).
Na? acts as a DdSR activator
X-ray crystallographic studies revealed the Mg2? and
MgATP-binding site of the human, rat, and yeast SRs
123

1572
T. Ito et al.
160
140
120
100
80
120
100
80
(A)
(a) racemase
(b) dehydrase
60
40
20
60
40
20
900
800
700
600
500
400
300
200
100
(B)
(a) racemase
(b) dehydrase
0
5
6
7
8
9
10
11
pH
Fig. 3 The pH dependency of DdSR. The effect of pH on the L-serine
dehydration (continuous line) and racemization (dotted line) catalyzed
by DdSR was studied using the following buffers: potassium
phosphate (pH 6–8), HEPES–NaOH (pH 7–8), Tricine–NaOH
(pH 7.5–8.5), Tris–HCl (pH 7.5–9), borate–NaOH (pH 8.5–10), or
CAPS–NaOH (pH 10–11). The L-serine dehydrase reaction (100 ll)
was performed with each 50 mM buffer, 20 lM PLP, 50 mM L-serine,
and 0.8 lM of DdSR at 30°C for 30 min. The pyruvate formed was
assayed with 2,4-dinitrophenyl hydrazine (2,4-DNP). Racemase activ-
ities were assayed with HPLC. Error bars indicate standard deviation
0
Fig. 4 The effect of metal ions and MgATP on DdSR activity.
Racemization with L-serine as a substrate and L-serine dehydration
catalyzed by DdSR were assayed in borate–NaOH buffer (a) or Tris–
HCl buffer (b) with the indicated additives (1 mM MgCl2, 1 mM
CaCl2, 1 mM EDTA, 1 mM MgATP, 20 mM NaCl, or 20 mM KCl).
The reaction mixture (a) consisting of 100 lL of 50 mM borate–
NaOH buffer (pH 8.5), 20 lM PLP, 50 mM L-serine, and each 1 mM
additive was incubated at 30°C for 30 min. The assay for (b) was
carried out under the same conditions as (a), except that Tris–HCl
buffer (pH 8.5) was used instead of borate–NaOH buffer. Racemase
activities were assayed with HPLC. Dehydratase activities were
determined with 0.3 lM of DdSR using a 2,4-DNP assay according to
Ito et al. (2007, 2008). The data are represented as percentages of
activity, and the values without additives were assumed to be 100%.
The mean values of at least three independent experiments are
represented. Error bars represent SD from the measurements
(
SD) from the average of three independent measurements
(Goto et al. 2009; Smith et al. 2010). The structure of the
S. pombe enzyme bound with an ATP analog, 50-adenylyl
(b, c-methylene)diphosphonate (AMP-PCP), reveals that
the SR binds to Mg2?. One Mg2? binds with the nucleotide
complex (Mg-AMP-PCP) at a groove formed between the
domain and the subunit interface, which changes the rela-
tive orientation of the two subunits. The other Mg2?
locates beside the glycine-rich region and interacts with
Glu208, Asp214, Gly212, and three water molecules. The
three water molecules are supported by the backbone car-
bonyl oxygens of Leu182, Gly183, Ala237, and Thr239
(Goto et al. 2009). As shown in Fig. 1, the residues in the
Mg2?-binding site are well conserved in most of the SRs
including DdSR. It is, therefore, interesting that the diva-
lent cations had no activation effects on DdSR.
D-serine dehydrase, and serine racemase activities were
activated about 6-, 2-, and 2.5-fold, respectively (Fig. 4b).
Without divalent cations, both serine racemase and
dehydrase activities of DdSR in the Tris–HCl buffer were
less than one-fourth of those in the borate–NaOH buffer
(Figs. 3, 4b). As shown in Fig. 3, DdSR activities in the
potassium phosphate and Tris–HCl buffers were lower than
those in the HEPES, Tricine, borate, and CAPS buffers.
Because the buffers giving higher activities contained
NaOH as a pH adjuster, we speculated that Na? activates
DdSR. As shown in Fig. 4b, 20 mM Na? enhanced serine
racemase activity by fourfold in the Tris–HCl buffer.
L-Serine and D-serine dehydrase activities were also
enhanced by seven- and fourfold, respectively, by the
addition of 20 mM NaCl. In all cases, the enhanced
activities were comparable to the DdSR activities in
We further studied the effect of divalent cations on
DdSR and found that the enzyme was activated by Mg2?
when the enzyme reaction was performed in Tris–HCl
buffer instead of borate–NaOH buffer. In Tris–HCl buffer,
the serine racemase activity (L- to D-serine) was stimulated
about three- to fourfold by the addition of 1 mM Mg2? or
Ca2? (Fig. 4b). Likewise, L-serine dehydrase activity was
also stimulated about threefold by the addition of 1 mM
Mg2? or Ca2? in Tris–HCl buffer (Fig. 4b). The dehy-
dration of D-serine was enhanced about fourfold by the
addition of 1 mM Mg2? (data not shown). MgATP also
stimulated DdSR in Tris–HCl buffer: L-serine dehydrase,
123

Metal ion dependency of serine racemase from D. discoideum
1573
Table 1 Effect of Na?, Mg2? and MgATP on the kinetic parameters
borate–NaOH buffer (Fig. 4b). The borate–NaOH buffer
used in the DdSR assay (50 mM, pH 8.5) was calculated to
contain 11.2 mM Na?. In addition to Na?, Li? partly
stimulates the enzymatic activity (data not shown). On
the other hand, K? did not elicit enzymatic activation
(Figs. 3, 4b). K? probably does not bind to the enzyme
because the reaction in the borate–NaOH buffer was nei-
ther inhibited nor activated by the addition of 100 mM KCl
(data not shown). This is probably because the ionic radius
of K? (138 pm) is much larger than that of Na? (102 pm).
The difference between the activation effects by NaCl and
that by KCl rule out the possibility that Cl- is an effector
molecule. These results indicate that DdSR is activated by
monovalent and divalent cations. We concluded that the
high reactivity in borate buffer is derived from the Na? in
the buffer.
Additive
Km (mM)
kcat (min-1
)
kcat/Km
(min-1/mM)
Wild type (None)
(Na?)
53.0 6.6
7.4 0.4
0.14
1.48
0.37
5.86
0.20
42.3 3.3 62.5 2.1
36.9 4.4 13.7 0.7
6.7 1.1 39.3 1.5
(Mg2?
)
(MgATP)
(None)
E207A
47.4 3.2
9.5 0.3
(MgATP) 24.0 0.4 64.6 0.38 2.69
The rates of L-serine dehydration were determined by lactic dehy-
drogenase coupling assay method with 50 mM Tris–HCl (pH 8.5),
20 lM PLP, 0.3 mM NADH, 2, 5, 20, 50, or 100 mM substrate, 10 U
lactic dehydrogenase, 0.5–3.2 lM DdSR at 30°C for 5 min, and the
indicated additive. The additives used were 20 mM NaCl, 1 mM
MgCl2, or 1 mM MgATP. The Km and kcat values were determined by
fitting data points to the Michaelis–Menten equation using the
Kaleida Graph program
To examine the effective concentration of these metal
ions, ratios of the activation of L-serine dehydration were
plotted against the concentrations of Na? (Supplementary
Fig. 2A), Mg2?, and Ca2? (Supplementary Fig 2B). The
half-maximal activation concentration of Na?, Mg2?, and
Ca2? determined from the plots was estimated to be
2.2 mM, 1.2 lM, and 1.2 lM, respectively. The half-
maximal activation concentrations of Mg2? and Ca2? were
about three orders of magnitude lower than that of Na?. In
contrast, the maximum activation ratio with Na? was about
(kcat/Km) of L-serine dehydration was stimulated about
40-fold. Change in the Km value supports the speculation
that MgATP causes the conformational change of DdSR
(Supplementary Fig. 3). With the S. pombe SR, MgATP
has been reported to cause conformational change:
Mg-AMP-PCP changed the relative orientation between
the two subunits without altering subunit conformation
(Goto et al. 2009).
two to three times higher than that with Mg2? and Ca2?
.
Mg2? and Na? are recognized by an identical site
Effect of Na?, Mg2?, and MgATP on DdSR catalysis
and structure
We found that the activation effect on L-serine dehydration
by 20 mM Na? was partially canceled by the presence of
1 mM Mg2? (Supplementary Fig. 4). L-Serine dehydrase
activity with 200 mM Na? and 1 mM Mg2? was nearly the
same as that with 20 mM Na? and no Mg2?. These results
suggest that Na? competes with Mg2? in DdSR activation
and that the cations share a binding site. As described pre-
viously, amino acid residues consisting of the Mg2?-binding
site of the S. pombe SR (Glu208, Asp214) are conserved in
DdSR. We mutated the corresponding residues of DdSR,
Glu207, and Asp213 to alanine residues. As shown in
Table 2, the E207A and D213A mutant enzymes retained
their racemase and dehydrase activities, but the mutant
enzymes were insensitive to the monovalent and divalent
metal ions (Fig. 5a, b). Neither Mg2? nor Ca2? showed any
inhibitory or stimulatory effects on the E207A and D213A
mutant enzymes in the Tris–HCl or borate–NaOH buffers.
These results suggest that both residues are required for the
divalent metal binding. E207 and D213 are also important
for Na?-dependent activation. The stimulatory effect of Na?
was completely abolished in the racemase and dehydrase
reactions catalyzed by the E207A mutant enzyme (Fig. 5a).
D213 mutation greatly weakened the Na? stimulatory
effect. Even with 100 mM NaCl, D213A-catalyzing L-serine
We examined the tryptophan fluorescence of DdSR to
investigate whether the metal ions and MgATP affect
enzyme structure. In the presence of Na? or Mg2?, no
changes were observed in the fluorescence emission spec-
trum at around 340 nm when the enzyme was excited at
295 nm. This suggests that these metal ions induced no
significant structural changes. On the other hand, MgATP
caused the fluorescent spectral change and probably
brought about the DdSR conformational change (Supple-
mentary Fig. 3).
The kinetic parameters of DdSR in L-serine dehydration
were obtained with Tris–HCl buffer in the presence of
these activators and summarized in Table 1. The Km and
kcat values for L-serine with no metal ions, 20 mM Na?,
and 1 mM Mg2? were 53.0 mM and 7.4 min-1, 42.3 mM
and 62.5 min-1, and 36.9 mM and 13.7 min-1, respec-
tively. Mg2? and Na? ions greatly increase the kcat values
without significantly changing the Km values. In contrast,
MgATP affected the Km value: in the presence of 1 mM
MgATP, the Km and kcat values in the L-serine dehydration
were determined to be 6.7 mM and 39.3 min-1, respec-
tively; in the presence of MgATP, the catalytic efficiency
123

1574
T. Ito et al.
Table 2 Effect of mutation of putative Mg2?-binding residues, E207
and D213
500 (A)
400
Wild type
E207A
D213A
Ratio Sa
300
Saa
Ratiob Sa
Ratio
200
Racemase activity
L-Serine to D-serine
D-Serine to L-serine
Dehydrase activity
100
0.77 100
0.56 100
0.09 11.4
0.09 15.7
0.16 21.4
0.37 66.5
0
L-Serine to pyruvate 1.26 100
D-Serine to pyruvate 0.36 100
0.13 10.2
0.47 37.0
0.15 42.5
160
(B)
0.03
8.6
140
120
100
80
The serine dehydrase activities were obtained by lactic dehydroge-
nase coupling assay method in the presence of 50 mM Borate–NaOH
(pH 8.5), 20 lM PLP, 0.3 mM NADH, 50 mM substrate, 10 U lactic
dehydrogenase, and 0.6–8.1 lM DdSR at 30°C. For the assay of
racemase activity, 3.1–16 lM DdSR was incubated at 30°C for
30 min in the presence of 50 mM Borate–NaOH (pH 8.5), 20 lM
PLP, and 50 mM substrate. After the incubation, the resultant amino
acids were determined by HPLC as described in ‘‘Materials and
methods’’. The values represented are normalized of three indepen-
dent experiments
60
40
20
0
a
Fig. 5 Effects of metal ions and MgATP on the E207A and D213A
mutant enzymes. The L-serine dehydratase activities of E207A (a) and
D213A (b) were determined in the presence of 20 mM NaCl, 0.1 mM
MgCl2, 0.1 mM CaCl2, 1 mM ATP, or 1 mM MgATP. The reaction
(100 ll) was performed in 50 mM Tris–HCl (pH 8.5) buffer
containing 20 lM PLP, 50 mM L-serine, each additive, and mutant
proteins (1.6 lM of E207A or 1.5 lM of D213A) at 30°C for 30 min.
The pyruvate formed was determined using the 2,4-DNP method.
Error bars indicate SD (n = 3)
The unit of Sa (specific activity) was lmol/min/mg
b
Ratio of the specific activity in each reaction catalyzed by the
mutant enzyme to that catalyzed by the wild-type enzyme
dehydration and racemization were enhanced only by
1.2- and 2.2-fold, respectively (data not shown).
On the other hand, fivefold activation by MgATP was
found in the E207A-catalyzing L-serine dehydrase activity.
The L-serine dehydrase activity of D213A was stimulated
1.5-fold by MgATP. ATP alone did not activate the mutant
enzymes, suggesting that the complexed MgATP is a
ligand of the enzyme. The kinetic parameters of E207A in
the presence or absence of MgATP are shown in Table 1.
Although E207A was substantially activated by MgATP,
the presence of MgATP caused a relatively small change in
the Km value of E207A mutant compared to the corre-
sponding value of the wild-type enzyme. The tryptophan
fluorescence of E207A was not significantly altered by the
addition of MgATP (data not shown). This implies that the
binding of Mg2? to the metal ion-binding site constituted
by E207 and D213 might be important for MgATP-medi-
ated dynamic structural rearrangement.
however, D. discoideum contains no genes that are
homologous to those of the NMDA receptors. D. discoid-
eum is one of the attractive targets for elucidating the
D-serine functions of low eukaryotic organisms.
Regulation of SR and D-serine in mammals has attracted
interest because of its physiological importance. Activity of
the mammalian SRs is regulated by various factors, such as
divalent cations, MgATP (De Miranda et al. 2002), S-nit-
rosylation (Mustafa et al. 2007), phosphorylation, and/or
translocation (Kim et al. 2005; Balan et al. 2009). In this
work, we studied the properties of DdSR, focusing on its
activation by metal ions and MgATP. DdSR is activated by
monovalent cations, such as Na? and Li? and divalent
cations, such as Mg2? and Ca2?, and MgATP. The metal
ions alone affect the kcat value of the enzyme without
drastic changes in the Km value. MgATP causes structural
change and affects the enzyme’s Km and kcat values. To the
best of our knowledge, DdSR is the first example of serine
racemase stimulated by Na?. Our results suggest that
monovalent and divalent cations share a binding site, which
is also the unique aspect of DdSR.
Discussion
D. discoideum contains a set of D-serine metabolizing
enzymes, DdSR, DdDSD, and DdDAO. We have cloned
and expressed all the genes of these enzymes in the E. coli
cells, purified the gene products, and confirmed their
activities. The existence of the set of D-serine metabolizing
enzymes implies that D. discoideum synthesizes and
metabolizes D-serine in vivo. As described above, D-serine
serves as a co-agonist of the NMDA receptor in mammals;
The Mg2?-binding site of DdSR is conserved in other
SRs. Thus, it is interesting whether other SRs that conserve
the similar metal ion-binding site are also activated by
monovalent cations. We have examined the Na?
123

Metal ion dependency of serine racemase from D. discoideum
1575
dependency of the mouse SR. The L-serine dehydrase
activity of the mouse SR was stimulated to 2.4- and 10-fold
by the addition of 1 mM Mg2? and 1 mM MgATP,
respectively (Supplementary Fig. 5). These results were
consistent with those reported previously (De Miranda
et al. 2002; Strisovsky et al. 2005). On the other hand,
addition of 100 mM NaCl enhanced the enzymatic activity
by only 1.2-fold (Supplementary Fig. 5). The mouse SR is
activated mainly by Mg2? and MgATP, but not by Na?.
The mechanism of the DdSR activation by monovalent
cations is interesting. Mg2? in S. pombe SR is octahedrally
coordinated with Glu208, Asp214, Gly212, and three water
molecules lined by Leu182, Gly183, Ala237, and Thr239.
The two carboxyl groups of acidic amino acids are
assumed to be one reason for the high affinity of Mg2?. We
constructed 3D structure models of DdSR by using a
Swiss-Model homology modeling server (http://www.
expasy.org/swissmod/SWISS-MODEL.html) (Fig. 6). In
the DdSR model structure, the Thr214 side chain, which is
located next to Asp213 and is unique for DdSR, intrudes
into the metal ion-binding site. The existence of Thr
seemingly expands the space slightly among the glycine-
rich region, Glu207, and Asp213 compared with that of the
Mg2?-binding site of the S. pombe SR. The widened space
probably enables the binding of larger cations.
2003). In the rat L-serine dehydratase, the K? is located
close to the tetra glycine loop and coordinates with the six
oxygen atoms of Gly168, Ala198, Leu223, Val225,
Glu194, and Ser200. These residues are well conserved in
both S. pombe SR and DdSR in all of the amino acids
except for Ser200, the rat L-serine dehydrase of which is
replaced by an aspartate residue (Asp214) in the S. pombe
SR (Goto et al. 2009; Yamada et al. 2003). DdSR have
unique threonine residues (Thr214) next to the corre-
sponding aspartate residue (Asp213). It might be possible
that in the monovalent cation binding of DdSR, Thr214,
not Asp213, participates in the binding. If this is the case,
we can explain why the D213A mutant enzyme was par-
tially activated by Na? but not by the divalent cations.
Although DdSR showed the highest kcat value in the
presence of Na?, Na? is probably not the principal regu-
lator of DdSR. We found the half-maximal activation
concentration of Mg2? is about 1800-fold lower than that
of Na?. In vivo, Mg2? is most likely the physiological
relevant cation of DdSR because its concentration in the
D. discoideum cells is reported to be about 3.5 mM (Padh
and Brenner 1984). The concentration is enough to fully
activate DdSR.
As shown in Fig. 3, the DdSR activity became higher at
higher pH. The other SRs, such as mouse and Arabidopsis
thaliana enzymes, show their maximum activity at around
pH 8–9. The physiological significance of the high pH
preference of DdSR is currently unclear because the
intracellular pH of D. discoideum is reported to be at
around pH 7.1 (Aerts et al. 1985). Because the alkaline pH
enhances both serine racemization and dehydration, the
solvent pH is assumed to affect the common catalytic steps
between serine racemization and dehydration. We believe
that the higher pH is at least beneficial for the formation of
the substrate-PLP Schiff base (external aldimine), as
The E. coli D-serine dehydratase and rat L-serine dehy-
dratase belonging to the fold-type II of PLP enzyme
probably bear similar primary and quaternary structures as
those of SRs and have been reported to be activated by K?
(Kojiro et al. 1989; Yamada et al. 2003). The Z scores
(strength of structural similarity) between the S. pombe SR
and the rat L-serine dehydratase were calculated to be 34.4
with 24% sequence identity. The Mg2? of yeast SR and the
K? of rat L-serine dehydratase locate with almost identical
geometry in each structure (Goto et al. 2009; Yamada et al.
Fig. 6 Model structure of
DdSR. Left The estimated
tertiary structure of DdSR with
putative metal ion-binding
residues. Right Superposition of
the putative metal ion-binding
sites of S. pombe SR and DdSR.
These structures were generated
with Swiss-Model homology
modeling server. The residues
of S. pombe SR and DdSR are
shown in gray and black,
respectively. The Mg2? and
oxygen atoms of water
molecules of S. pombe SR are
represented in gray and black
balls, respectively. The numbers
are for the residues of DdSR.
These two structural figures
were drawn with MolScript
(Kraulis 1991)
123

1576
T. Ito et al.
Saccharomyces cerevisiae. Anal Biochem 371:167–172. doi:
10.1016/j.ab.2007.07.030
alkaline conditions are probably more advantageous than
neutral conditions for the substrate nitrogen to react with
the carbonyl carbon of PLP upon the formation of the
external Schiff base.
Ito T, Hemmi H, Kataoka K, Mukai Y, Yoshimura T (2008) A novel
zinc-dependent D-serine dehydratase from Saccharomyces cere-
visiae. Biochem J 409:399–406. doi:10.1042/BJ20070642
Kim PM, Aizawa H, Kim PS, Huang AS, Wickramasinghe SR,
Kashani AH, Barrow RK, Huganir RL, Ghosh A, Snyder SH
(2005) Serine racemase: activation by glutamate neurotransmis-
sion via glutamate receptor interacting protein and mediation of
neuronal migration. Proc Natl Acad Sci USA 102:2105–2110.
doi:10.1021/bi051201o
To elucidate the physiological roles and reaction
mechanisms of DdSR, construction of the DdSR gene-
deletion mutant of D. discoideum and the X-ray crystal-
lography of the enzyme are now under way.
Kojiro CL, Marceau M, Shafer JA (1989) Effect of potassium ion on
the phosphorus-31 nuclear magnetic resonance spectrum of the
pyridoxal 50-phosphate cofactor of Escherichia coli D-serine
dehydratase. Arch Biochem Biophys 268:67–73. doi:10.1016/
0003-9861(89)90565-1
Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed
and schematic plots of protein structures. J Appl Crystallogr
24:946–950. doi:10.1107/S0021889891004399
Mustafa AK, Kumar M, Selvakumar B, Ho GP, Ehmsen JT, Barrow
RK, Amzel LM, Snyder SH (2007) Nitric oxide S-nitrosylates
serine racemase, mediating feedback inhibition of D-serine
formation. Proc Natl Acad Sci USA 104:2950–2955. doi:
10.1073/pnas.0611620104
Acknowledgments This work was supported in part by the Program
for Promotion of Basic and Applied Researches for Innovation in Bio-
oriented Industry (BRAIN) and the Grant-in-Aid for Scientific
Research 22380058 (to T. Y.), and the Grant-in-Aid for Research
Activity start-up 22880014 (to T. I.) from the Ministry of Education,
Culture, Sports, Science, and Technology. We thank Professor
Hideko Urushihara of the University of Tsukuba and Japan National
Bio Resource Project (NBRP-nenkin) for providing us the EST clone
of DdSR.
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