Occurrence of D-serine in rice and characterization of rice serine racemase

Article abstract of DOI:10.1016/j.phytochem.2009.01.003

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg²⁺ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg²⁺, and this structural change probably regulates the two enzymatic activities.

Full text of DOI:10.1016/j.phytochem.2009.01.003

Phytochemistry 70 (2009) 380–387
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Occurrence of D-serine in rice and characterization of rice serine racemase
*
Yoshitaka Gogami, Katsuyoshi Ito, Yuji Kamitani, Yuki Matsushima, Tadao Oikawa
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials, and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu 564-8680, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 March 2008
Received in revised form 29 December 2008
Available online 25 February 2009
Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the
D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative
amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine
racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza
sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product
(SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of
34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D-
and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and
1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the
racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhib-
ited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the ser-
ine dehydratase activity. Kinetic analysis showed that Mg2+ increases the catalytic efficiency of the serine
racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching
analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition
of Mg2+, and this structural change probably regulates the two enzymatic activities.
Keywords:
Unpolished rice
Oryza sativa L.
Gramineae
D-serine
D-amino acid
Serine racemase
Serine dehydratase
Pyridoxal 50-phosphate
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) (Ono
et al., 2006). During the course of this study, the D-amino acid con-
D-amino acids were once considered as unnatural and unusual
compounds that were not essential amino acids for humans.
Accordingly, studies of D-amino acids and their metabolism have
focused intensively on microbial amino acid racemases and D-ami-
no acid transaminases (Inagaki et al., 1986; Uo et al., 2001; Yorifuji
et al., 1971), since D-alanine and D-glutamate are known to be two
essential components of the peptide glycan in the microbial cell
wall (Osborn, 1969). But, with development of improved analytical
and detection techniques, D-amino acids have recently been found
in a much broader range of living organisms (Hashimoto and Oka,
1997), and our understanding of them has gradually changed. In
particular, D-serine (1a) (Fig. 1) has recently been demonstrated
to act as a neuromodulator in humans (De Miranda et al., 2002),
and it is currently undergoing clinical testing in patients as a treat-
ment for schizophrenia (Tsai et al., 1998). Furthermore, several
kinds of D-amino acids have been discovered in plant seedlings,
with the first occurrence of an amino acid racemase in plants being
tent of various vegetables and fruits (Gougami et al., 2006) was
analyzed. We estimated that germinated, unpolished rice (Oryza
sativa L.) contains a substantial amount of D-serine (1a) and the ra-
tio of the D-enantiomer (1a) to the L-enantiomer (1b) was higher
for serine than for other amino acids. To attempt to discern the bio-
synthetic pathway to D-serine 1a in O. sativa L., we searched for a
putative metabolic gene encoding a protein affording D-serine (1a)
formation in the rice genome from the International Rice Genome
Sequencing Project (IRGSP) in 2004. Only one candidate, a putative
serine/threonine racemase (serr) was found. Moreover, the cloning
of the serr into Escherichia coli was reported at the Annual Meeting
of the Vitamin Society of Japan in 2006 (Ito et al., 2006), and later
by Fujitani et al. (2007). In the current study, we first describe our
initial finding, as well as demonstrate both the occurrence of D-
serine (1a) in germinated seeds of O. sativa L. and the enzymolog-
ical characterization of the rice serine racemase.
2. Results and discussion
Abbreviations: CAPS, N-cyclohexyl-3-aminopropanesulfonic acid; CHES, 2-(N-
cyclohexylamino)-ethanesulfonic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazi-
neethanesulfonic acid; NAC, N-acetyl-L-cysteine; OPA, o-phtalaldehyde; PIPES,
piperazine-1,4-bis(2-ethanesulfonic acid); PLP, pyridoxal 50-phosphate; SerR, serine
racemase form Oryza sativa L.
2.1. Detection of D-serine (1a) in germinated unpolished rice
Germinated unpolished rice was found to contain a high amount
of D-serine (1a) relative to the total amount of L-serine (1b)
(Table 1). The fraction of D-serine (D/D + L%) reached approximately
* Corresponding author. Tel.: +81 6 6368 0812; fax: +81 6 6388 8609.
E-mail address: oikawa@ipcku.kansai-u.ac.jp (T. Oikawa).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.01.003

Y. Gogami et al. / Phytochemistry 70 (2009) 380–387
381
the serine racemase from O. sativa L. (SerR, E.C.5.1.1.18) was simi-
lar to that of H. vulgare L. (identity, 88.5%; accession No.
BAF63026), Arabidopsis thaliana (identity, 64.4%; accession No.
NP192901), Schizosaccharomyces pombe (identity, 40.8%; accession
No. NP587715), Homo sapiens (identity, 46.7%; accession No.
NP068766), and Mus musculus (identity, 46.3%; accession No.
NP038789) (See Fig. 2). The amino acid residues in the binding mo-
tifs for the cofactor, PLP (Lys68, Ser323, Asn95, Gly195, Gly196, and
Gly197), and for Mg2+ (Glu219, Ala223, and Asp225) were highly
conserved among these organisms. SerR was expressed in insolu-
ble fractions of the cell-free extract of E. coli harboring pET-21b/
serr, but, by decreasing the cultivation temperature from 37 to
15 °C, the enzyme was gradually localized in the soluble fractions.
There were twenty-three rare codons (AGA (4), AGG (1), CGG (1),
ATA (9), CTA (1), and CCC (1), and GGA (6)) contained in serr, which
is probably one of the reasons for the low yield of SerR.
a
b
COOH
COOH
C H
CH2
H
C
NH2
H2N
CH2
OH
L-Serine (1b)
OH
D-Serine (1a)
COOH
COOH
COOH
C
O
H C NH2
CH2
C
H
CH2
OH
H2N
NH
+
3
CH3
2.3. Purification of serine racemase from O. sativa L.
OH
D-Serine (1a)
Pyruvate (2)
L-Serine (1b)
SerR was purified to homogeneity using a Ni–NTA column
according to the purification protocol of Qiagen (Fig. 3). About
3.5 mg of purified enzyme was obtained from one liter of culture
(Table 2). The atomic absorption analysis indicated that Ni did
not contaminate the purified enzyme (data not shown). During
Fig. 1. Reaction pathways of D-serine (1a) and L-serine (1b) by serine racemase
from Oryza sativa L. (a) Racemase reaction; (b) Dehydratase reaction.
purification of the enzyme, 20 lM PLP and 0.01% 2-mercap-
10% six days after germination. The D-serine (1a) content did not
increase with the germination period from 0 to 6 days, and it
was detected even before germination (0 day). In previous studies,
D-alanine, D-aspartate, and D-glutamate were detected in both
free and conjugated forms from pea seedlings (Pisum sativum)
(Ogawa et al., 1977), barley grains (Hordeum vulgare L.) (Erbe
and Brückner, 2000), and hops blossoms (Humulus lupulus L.).
D-alanine and D-alanyl-D-alanine were also found in wild rice
(Oryza australiensis Domin) (Manabe, 1985), but there has been
no report of the occurrence of D-serine (1a) in any plant including
germinated, unpolished rice.
toethanol were present in the buffer. The enzyme can be stored
in a 20 mM sodium phosphate buffer (pH 8) at 4 °C for 5 days
without loss of activity, but in a 20 mM sodium or potassium
phosphate buffer (pH 7), the enzyme was unstable and became
inactivated, forming aggregates. About 30% of the initial activity
was lost after the enzyme was stored in a 20 mM sodium phos-
phate buffer (pH 7) for 3 days. The purified enzyme migrated as a
single band in SDS–PAGE (Fig. 3a) with an apparent molecular
mass of 34.5 kDa. The molecular mass of the native enzyme
determined by gel filtration with Superdex 200 Hiload was
87.0 kDa, suggesting that the enzyme was
a homodimer
(Fig. 3b), like the serine racemases from A. thaliana (Fujitani
2.2. Cloning of a putative serine/threonine racemase gene, serr, in the
genomic DNA of Oryza sativa L.
´
et al., 2006) and Mus musculus (Strísovsky et al., 2003). The FPLC
elution profile of mouse brain serine racemase showed that the
enzyme exists in solution in equilibrium between dimeric and
tetrameric forms (Cook et al., 2002). The elution profile of SerR
by gel filtration indicated that the monomeric and tetrameric
forms were completely absent, and that the subunit association
properties of SerR were therefore quite different from those of
mouse brain racemase.
The project to sequence the full-length genome of rice (O. sativa
L. ssp. Japonica cv. Nipponbare) was completed in the early 2000s
(Sasaki et al., 2002). Three research institutes – the National Insti-
tute of Agrobiological Sciences (NIAS), the Foundation for Advance-
ment of International Science (FAIS), and the Institute of Physical
and Chemical Research (RIKEN) – collaborated under the coordina-
tion of the Bio-oriented Technology Research Advancement Insti-
tute (BRAIN) on this project. Using the Knowledge-based Oryza
Molecular Biological Encyclopedia (KOME, http://cdna01.d-
na.affrc.go.jp/cDNA/), we tried to identify putative candidate genes
related to D-serine (1a) metabolism in O. sativa L. One putative ser-
ine/threonine racemase gene (serr, clone No. 001-110-B03) was de-
tected on chromosome 4 of the genomic DNA of O. sativa L. ssp.
Japonica cv. Nipponbare, which encodes an open reading frame
of 1,020 bp, having 50.1% GC content. The primary structure of
2.4. Enzyme reactions and substrate specificity of SerR
We found that SerR catalyzes D- and L-serine (1a and 1b) dehy-
dratase reactions in addition to a serine racemase reaction. This
catalytic feature is shared with the serine racemases from M. mus-
´
culus (Strísovsky et al., 2003), S. pombe (Yoshimura and Goto,
2008), and Bombyx mori (Uo et al., 1998). Fig. 4 shows the time
course for the reaction products of SerR in the presence of D-serine
(1a) (Fig. 4a) or L-serine (1b) (Fig. 4b) as a substrate. D-Serine (1a)
and L-serine (1b) were converted to, respectively, L-serine (1b) and
pyruvate or D-serine (1a) and pyruvate. The specific activities for
the L-serine and D-serine dehydratase activities were determined
to be 2.73 and 1.42 nkatal/mg, respectively. SerR is highly specific
for serine (1), whereas other amino acids, such as L-alanine, L-argi-
nine, L-threonine, L-glutamate, and L-aspartate, did not serve as
substrates. By contrast, the serine racemases from A. thaliana and
H. vulgare L. act on L-alanine, L-arginine, and L-glutamine in addi-
tion to serine. Although these three enzymes are all derived from
plants, the substrate specificities are characteristically quite differ-
ent from each other.
Table 1
D- and L-serine (1a and 1b) concentration in germinated unpolished rice.
Germination (day)
D-ser (
lmol/g)
L-ser (lmol/g)
1
2
3
4
5
6
0.37 ꢀ 10ꢁ2
0.76 ꢀ 10ꢁ2
0.67 ꢀ 10ꢁ2
0.59 ꢀ 10ꢁ2
0.64 ꢀ 10ꢁ2
0.67 ꢀ 10ꢁ2
2.7 ꢀ 10ꢁ2
1.4 ꢀ 10ꢁ2
0.87 ꢀ 10ꢁ2
0.74 ꢀ 10ꢁ2
2.2 ꢀ 10ꢁ2
0.63 ꢀ 10ꢁ2

382
Y. Gogami et al. / Phytochemistry 70 (2009) 380–387
Fig. 2. Alignment of primary structures of various serine racemases O. sativa L., Hordeum vulgare L., A. thaliana, S. pombe, H. sapiens, and M. musculus represent the serine
racemase amino acid sequences from Oryza sativa L. (accession No. AB425957), H. vulgare L. (accession No. BAF63026), Arabidopsis thaliana (accession No. NP192901),
Schizosaccharomyces pombe (accession No. NP587715), Homo sapiens (accession No. NP068766), and Mus musculus (accession No. NP038789). The shaded area represents the
amino acid residues conserved between all serine racemases. d, probable amino acid residues composing a magnesium binding site; s, tryptophan residues in SerR; ꢂ, amino
acid residues composing a PLP binding site.
a
b
116.0 kDa
0.9
0.5
66.2 kDa
45.0 kDa
35.0 kDa
25.0 kDa
18.4 kDa
14.4 kDa
0
104
105
106
Molecular mass (Da)
1
2
3
4
Fig. 3. Subunit and quaternary structures of the serine racemase from Oryza sativa L. (a) SDS–PAGE Electrophoresis was conducted with a Ready gel cell 108 BR (Bio-Rad
Japan, Tokyo) equipped with a Power pac 300 (Bio-Rad). The concentration of acrylamide was 12%T. Lane 1: protein marker: b-galactosidase from Escherichia coli, 116.0 kDa;
bovine serum albumin from bovine plasma, 66.2 kDa; ovalbumin from chicken egg white, 45.0 kDa; lactate dehydrogenase from porcine muscle, 35.0 kDa; restriction
endonuclease Bsp 981 from E. coli, 25.0 kDa; b-galactoglobulin from bovine milk, 18.4 kDa; lysozyme from chicken egg white, 14.4 kDa. Lane 2: a crude extract of E. coli BL21
(DE3) cells harboring pET-21b/serr (protein: 8 lg). Lane 3: purified SerR (protein: 8 lg). Lane 4: protein marker. b. Gel filtration. Kav values were calculated according to the
equation of Kav = Ve ꢁ Vo/Vt ꢁ Vo, where Ve is the elution volume for the protein, Vo is the column void volume, i.e., an elution volume for Blue Dextran 2000, and Vt is the total
bead volume. The mobile phase used was 10 mM potassium phosphate buffer containing 300 mM NaCl. The flow rate was 0.3 ml/min. The column and protein marker used
are described in Section 3.11.

Y. Gogami et al. / Phytochemistry 70 (2009) 380–387
383
Table 2
Purification of a putative serine racemase from Oryza sativa L.
Step
Total activity* (nkatal)
Total protein (mg)
Specific activity (nkatal/mg)
Yield (%)
Purification (fold)
Crude extract
Ni–NTA agarose
52.0
13.0
396
6.00
1.31 ꢀ 10ꢁ1
100
25.0
1.00
16.9
2.17
*
Activity is expressed as serine dehydratase activity.
a
b
100
100
80
60
40
20
80
60
40
20
0
0
0
1
2
3
4
5
0
1
2
3
4
5
Reaction time (h)
Reaction time (h)
Fig. 4. Time course of the reaction products of serine racemase from Oryza sativa L. (a) D-Serine (1a) was used as substrate. (b) L-Serine (1b) was used as substrate. The
preparation of a reaction mixture and the measurement of D- and L-serine (1a and 1b) and pyruvate were conducted according to the method described in Section 3.6. An
aliquot (500 ll) was taken from the reaction mixture after 0, 1, 2, 3, 4, and 5 h. Black bar: D-serine, white bar: L-serine, striped bar: pyruvate.
2.5. Effects of temperature and pH
2.6. Effects of inhibitors
The optimum temperature for the serine racemase and dehy-
dratase activities of SerR was determined to be 35 °C, which agrees
well with the optimum germination temperature of O. sativa L.
(30–35 °C). A similar optimum temperature (37 °C) was found for
the serine racemase from Rattus norvegicus (Wolosker et al.,
1999a). SerR was stable up to about 40 °C and became inactivated
at 50 °C. The activation energies for the serine racemase and serine
dehydratase reactions catalyzed by SerR were calculated to be
61.8 kJ and 36.2 kJ, respectively. The optimum pH for the serine
racemase reaction was 9.5, while that for the serine dehydratase
was pH 9.0. These values are slightly higher than those of the en-
zymes from mammals such as R. norvegicus (pH 8–9) (Wolosker
Since the salicylaldehyde method that is used for the determi-
nation of the serine dehydratase activity interfered with the inhib-
itors used, we examined only the effects of various inhibitors on
serine racemase activity. Like the serine racemase from R. norvegi-
cus (Wolosker et al., 1999a,b), SerR was completely inhibited by
1 mM of semicarbazide, hydroxylamine, aminooxyacetate, sodium
borate, or phenylhydrazine, respectively. SerR showed maximum
absorption at 417 nm, and this disappeared after the enzyme solu-
tion was treated with hydroxylamine to form an apoenzyme
(Fig. 5). These results suggest that SerR probably contains PLP as
a coenzyme.
´
et al., 1999a) and Mus musculus (pH 8–9.5) (Strísovsky et al.,
2.7. Effect of metal ions and ATP
2003). SerR was most stable at pH 8.5 and was almost inactivated
at pH 6 and 11.
ATP, Mg2+, and Ca2+ are known to affect both serine racemase
and dehydratase activities of serine racemases from mammalian
brain (Foltyn et al., 2005), M. musculus (Cook et al., 2002; Neidle
and Dunlop, 2002), and A. thaliana. In contrast to the mammalian
brain and M. musculus enzymes, the serine racemase activity of
SerR characteristically decreased upon addition of ATP, but the ser-
ine dehydratase activity of SerR increased (for [ATP] > 0.1 mM), as
it does for those enzymes (Fig. 6a). ADP had no effect on either
activity. Various metal ions affected the serine racemase activity
of SerR in many of the same ways that they affect the activities
of other serine racemases: Mg2+ and Ca2+ increased activity, and
Zn2+ and Cu2+ decreased it (Fig. 6b). However, the serine dehydra-
tase activity of SerR decreased with increasing the Mg2+ concentra-
tion (Fig. 6c). The serine racemase activity decreased in the
presence of both ATP and Mg2+ (Fig. 6d). These results suggest that
ATP, Mg2+, and Ca2+ affect serine racemase and dehydratase activ-
ities of SerR differently from how they affect those of other serine
racemases.
0.7
0.25
0.6
0.2
0.15
0.5
0.4
0.3
0.1
0.05
0.2
0.1
0
0
270
290 300
350
400
450
Wavelength (nm)
Fig. 5. Absorption spectra of serine racemase from Oryza sativa L. Solid line: the
holo SerR (1 mg/ml in a 10 mM sodium phosphate buffer (pH 8.0) containing
10 mM PLP and 0.02% 2-mercaptoethanol); Narrow line: the reduced form of SerR
2.8. Kinetic parameters
To clarify the effects of Mg2+ on serine racemase and dehydra-
tase activities of SerR, we determined the kcat, Km, and kcat/Km val-
ues for both activities in the presence or absence of Mg2+ (1 mM)
using L-serine (1b) as a substrate (Table 3). Mg2+ decreased the
(1 mg/ml in
mercaptoethanol after reduction with NaBH4); Dashed line: the apo SerR (1 mg/ml
in 10 mM sodium phosphate buffer (pH 8.0) containing 0.02% 2-mercap-
toethanol). The spectra were taken with a Jasco V550 spectrophotometer (Jasco,
Tokyo) at room temperature (about 25 °C).
a 10 mM sodium phosphate buffer (pH 8.0) containing 0.02% 2-
a

384
Y. Gogami et al. / Phytochemistry 70 (2009) 380–387
3.8 sꢁ1 for the racemase activity) and Km (3.8 mM for the dehydra-
tase activity; 4.0 mM for the racemase activity) values of the serine
a
160
140
120
100
80
´
racemase from M. musculus (Strísovsky et al., 2003), those of SerR
were one order of magnitude lower and higher, respectively. The
mammalian brain enzyme has a kcat/Km value for the L-serine
dehydratase activity (0.22 sꢁ1) about 10 times higher than that of
SerR (Foltyn et al., 2005), and a higher Km value (1.9 mM), suggest-
ing that SerR has lower affinity to L-serine (Baumgart et al., 2007).
60
40
20
0
0
0.001
0.01
0.1
1
ATP conc. (mM)
2.9. Quenching of SerR tryptophan fluorescence by acrylamide
b
c
140
120
100
80
We estimated the positions of the four tryptophan residues
(Trp136, Trp257, Trp312, and Trp333) of SerR using the Swiss-
PDB viewer and the three-dimensional structure of the serine rac-
emase from S. pombe (PDB 1V71) (Fig. 7a and b). Trp257 probably
exists next to a Mg2+-binding site in the active site, and the other
tryptophan residues may be distributed on the surface of the en-
zyme. There are no tryptophan residues in the serine racemase
from S. pombe, and those in SerR may serve as probes for a fluores-
cence-quenching analysis (Fig. 2). To examine the regulation
mechanism of Mg2+, we tried to analyze the fluorescence quench-
ing of tryptophan residues in SerR by acrylamide in the presence or
absence of Mg2+ (1 mM) (Fig. 8a and b). Fig. 8c shows the Stern-
60
40
20
0
Mg2+ Ca2+ Co2+ Cu2+ Fe2+ Al3+ Ni2+ Zn2+
No metal
added
160
140
120
100
80
Volmer plots for SerR, and the Stern-Volmer constants (Ksv
)
60
determined are summarized in Table 4. The Ksv constants in the
presence of Mg2+ were lower than those in the absence of Mg2+
regardless of whether or not D- or L-serine (1a and 1b) were added
as substrates. These results suggest that the accessibility of the
SerR tryptophan residues to the solvent is decreased by the addi-
tion of Mg2+. Accordingly, the structure of SerR is distorted by
the addition of Mg2+, and this structural change probably regulates
the two activities (serine racemase and dehydratase). Lys57 and
Ser82 in the serine racemase of S. pombe are thought to be involved
40
20
0
0
0.001
0.01
0.1
1
Mg2+ conc. (mM)
d
120
100
80
60
40
20
0
in abstracting the a-proton and forming a hydrogen bond to the b-
OH in L- and D-serine (1a and 2b), respectively (Yoshimura and
Goto, 2008), but the regulation mechanism for both reactions is
still unknown. Lys68 and Ser93 in the primary structure of SerR
probably correspond to Lys57 and Ser82 of S. pombe, respectively
(Fig. 2).
0
0.5
1
2
Mg2+ , ATP conc. (mM)
Fig. 6. Effects of metal ions and ATP on serine racemase activity. (a) Effect of ATP;
(b) effects of various metal ions; (c) effect of Mg2+; (d) effects of Mg2+ and ATP.
Reactions were carried out according to the methods described in Section 3.6. Black
bar: serine racemase activity of SerR; white bar: L-serine dehydratase activity of
SerR.
3. Experimental
3.1. Materials
Unpolished rice (Oryza sativa. L. cv. Koshihikari) was purchased
from JA, Japan. o-Phtalaldehyde (OPA), N-acetyl-L-cysteine (NAC),
Table 3
Kinetic parameters.
D- and L-serine (1a and 1b), and pyridoxal 50-phosphate (PLP) were
purchased from Wako (Osaka, Japan), whereas Ni–NTA Agarose
was purchased from Qiagen (Hilden Germany). The rice full-length
cDNA clone (clone name: 001-110-B03) was obtained from the
Rice Genome Resource Center, National Institute of Agrobiological
Science, Tsukuba, Ibaraki, Japan. E. coli BL21 (DE3), E. coli NovaBlue,
pT7 Blue-2 T-vector, and pET-21b were purchased from EMD Bio-
sciences, Inc. (San Diego, CA, USA). Blend Taq polymerase was pur-
chased from Toyobo, Osaka. NdeI and XhoI were purchased from
Roche Diagnostics (Basel, Switzerland). All other reagents were
the best grade commercially available unless otherwise stated.
kcat (sꢁ1
)
Km for L-ser (mM)
kcat/Km (Sꢁ1 ꢃ mMꢁ1
)
For racemase activity
None
3.6 ꢀ 10ꢁ1
3.1 ꢀ 10ꢁ1
18
10
2.0 ꢀ 10ꢁ2
3.1 ꢀ 10ꢁ2
+Mg2+ (1 mM)
For dehydratase activity
None
6.1 ꢀ 10ꢁ1
28
18
2.2 ꢀ 10ꢁ2
2.0 ꢀ 10ꢁ2
+Mg2+(1 mM)
3.6 ꢀ 10ꢁ1
Km value but did not affect the overall kcat value of the serine rac-
emase activity, and, subsequently, the kcat/Km value increased
about 1.6 times. Regarding the serine dehydratase activity, both
the Km and kcat values decreased; therefore, the kcat/Km value de-
creased slightly. These results suggest that Mg2+ characteristically
increases the catalytic efficiency of the serine racemase activity
and decreases that of the serine dehydratase activity (Table 3).
As compared with the kcat (4.0 sꢁ1 for the dehydratase activity;
3.2. Germination of unpolished rice
Unpolished rice (20 g) was sterilized with EtOH–H2O (7:3, v/v)
and 5% NaOCl containing 0.01% Triton X-100 and washed five times
with sterilized H2O. The washed, unpolished rice, was put into a
sterilized Erlenmeyer flask (500 ml) and statically incubated at
30 °C for 22 h.

Y. Gogami et al. / Phytochemistry 70 (2009) 380–387
385
Fig. 7. Proposed three-dimensional subunit structure of serine racemase from Oryza sativa L. (a) Subunit structure: The three-dimensional subunit structure of SerR (yellow
ribbon) was superimposed on that of serine racemase from Schizosaccharomyces pombe (yellowish-green ribbon). Mg2+ is shown as a red sphere. (b) Active site of SerR:
Try257, PLP, and Mg2+ are shown as a blue ribbon with purple sticks, pink sticks, and a red sphere, respectively. The structures were estimated by the method described in
Section 3.11.
Table 4
Stem-Volmer constants.
Substrate
None
Mg2+ (mM)
Ksv
0
+1
0
+1
0
+1
3.12
2.96
3.26
3.07
3.10
2.89
L-Ser (1b)
D-Ser (1a)
homogenate was put into a centrifuge tube (50 ml, Sumitomo
Bakelite Co., Ltd., Tokyo, Japan) and stored in a cold room (about
4 °C). After 20 h, the homogenate was centrifuged at 26,100ꢀg
for 30 min at 4 °C. The amino acids in the supernatant solution
were derivatized with OPA and NAC according to the method of
Aswad (1984). The HPLC analyses of D- and L-amino acids were
carried out under the same conditions as described previously
(Ono et al., 2006).
3.4. Cloning of the putative serine/threonine racemase gene in the rice
genome
The oligonucleotides serac1 (50-TCATATGGGGAGCAGAGGTG-30)
and serac2 (50-CTCGAGACGTTTATAGAGAGACTCCC-30), based on
the 50- and 30- sequences of the insert in the O. sativa cDNA clone
(O. sativa L. ssp. Japonica cv. Nipponbare, 001-110-B03), were used
as PCR primers (0.2 pmol) to amplify the serr gene from the cDNA
clone (1 ng). The PCR involved 25 cycles of denaturation at 95 °C
for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for
1 min. PCR amplification was carried out with Blend Taq polymer-
ase in a Gene Amp PCR system 9700 (PE Applied Biosystems, Pis-
cataway, NJ, USA). The resulting 1,020-bp serr fragment with
NdeI and XhoI restriction sites was sequenced with a DNA
sequencing system, SQ5500 (Hitachi, Tokyo, Japan), ligated into
Fig. 8. Quenching of SerR tryptophan fluorescence by acrylamide. (a) Fluorescence
spectra in the absence of Mg2+. (b) Fluorescence spectra in the presence of Mg2+. (c)
Stern-Volmer plot. The experiment was carried out according to the method
described in Section 3.10.
the pT7 Blue-2 T-vector (50 ng/ll) and transformed into E. coli
NovaBlue. After cultivation in an LB medium containing ampicillin,
the plasmid was extracted with the alkaline mini-prep method and
precipitated with polyethylene glycol 6,000. The purified DNA ob-
tained was digested with NdeI and XhoI and ligated into an NdeI-
and XhoI-digested pET-21b vector to form pET-21b/serr, which was
transformed into E. coli BL21 (DE3).
3.3. Extraction of D-amino acids in germinated, unpolished rice
The germinated, unpolished rice (3 g) was frozen in liquid N2
and ground in a mortar with EtOH–H2O (7:3, v/v, 27 ml). The

386
Y. Gogami et al. / Phytochemistry 70 (2009) 380–387
3.5. Purification of the serine racemase homologue of O. sativa L.
pHs at 4 °C for 5 h. The substrate specificities of the serine race-
mase and serine dehydratase activities of SerR were determined
under the standard assay conditions in the presence of various
10 mM substrates. The effects of various enzyme inhibitors on SerR
were determined as follows: the enzyme solution was incubated
with 1 mM of reagents such as semicarbazide, hydroxylamine,
aminooxyacetate, sodium borate, or phenylhydrazine for 30 min
at pH 9.0 on ice, and the residual activities were measured under
the standard assay conditions.
After the E. coli BL21 (DE3) cells harboring pET-21b/serr were
selected on an LB agar medium containing 100
the clone was grown in 1 l of an LB medium containing 100
l
g/ml ampicillin,
g/ml
l
ampicillin at 37 °C until the absorption at 600 nm reached 0.6.
The cells were collected by centrifugation (14,000ꢀg, 20 min),
suspended in 24 ml of
a 10 mM sodium phosphate buffer,
pH 8.0, and stored at ꢁ20 °C. The frozen cells were restored with
a 10 mM sodium phosphate buffer (pH 8.0) containing 10 M PLP,
l
10 mM imidazole, and 300 mM NaCl on ice and disrupted by
ultrasonication (model UD-201, Tomy Seiko Co., Tokyo; output
6, duty cycle, 30; 2 minꢀ5 times). The suspension was centrifuged
(26,100ꢀg, 20 min) and ultracentrifuged (100,000ꢀg, 1 h), and the
supernatant was dialyzed against a 10 mM sodium phosphate
buffer (pH 8.0) containing 10 mM PLP, 10 mM imidazole, and
300 mM NaCl. The crude extract obtained was applied to a
column of Ni–NTA (u1 ꢀ 2 cm), which was equilibrated with a
10 mM sodium phosphate buffer containing 10 mM imidazole
and 300 mM NaCl and washed (first with 100 ml of the same
buffer, then with 200 ml of a 10 mM sodium phosphate buffer
containing 20 mM imidazole and 300 mM NaCl, and finally with
60 ml of a 10 mM sodium phosphate buffer containing 50 mM
imidazole and 300 mM NaCl). The flow rate was 0.5 ml/min. The
adsorbed protein was eluted with 50 ml of a 10 mM sodium phos-
phate buffer containing 250 mM imidazole and 300 mM NaCl. The
elution was collected with Bio-collector (ATTO, Tokyo, 2 ml/tube),
and the protein concentration was monitored spectrophotometri-
cally at 280 nm.
3.8. Effect of metal ions, ADP, and ATP
The effects of metal ions, ADP, or ATP on the serine racemase
and serine dehydratase activities of SerR were examined in the
presence of 1 mM AlCl2, CaCl2, CoCl2, CuCl2, FeCl2, MgCl2, NaCl,
NiCl2, ZnCl2, ADP, or ATP under the standard assay conditions
(pH 9.0, 30 °C). The effects of the concentrations of MgCl2 (0,
0.001, 0.01, 0.1, and 1.0 mM), ATP (0, 0.001, 0.01, 0.1, and
1.0 mM), or ATP (0, 0.5, 1, and 2 mM) in the presence of MgCl2
(1 mM) were also examined.
3.9. Kinetic analysis
Kinetic parameters (kcat, Km, and kcat/Km) were determined for
both serine racemase and serine dehydration reactions of SerR. L-
serine was used as a substrate, and kcat, Km, and kcat/Km values were
calculated in the presence or absence of Mg2+ according to Linewe-
aver-Burk double-reciprocal plots.
3.10. Quenching of SerR tryptophan fluorescence by acrylamide
3.6. Assay methods
A fluorescence-quenching experiment was conducted by addi-
tion of various concentrations (0, 0.05, 0.10, 0.15, 0.20, and
0.25 M) of acrylamide dissolved in a 10 mM CHES–NaOH buffer,
pH 9.0 (0.25 ml), to the enzyme solution (protein conc.: 0.5 mg/ml,
0.1 ml) in the presence or absence of the substrate (L- or D-serines
(1a or 1b)) dissolved in the same buffer (0.65 ml). The fluorescence
intensity was measured with the Hitachi 650-60 fluorescence
spectrophotometer (Hitachi, Ibaragi) equipped with a data proces-
sor under a 5 nm slit width. The excitation wavelength used for the
tryptophan residue was 295 nm, and fluorescence was measured at
330 nm at room temperature (about 20 °C). The Stern-Volmer
quenching constant (Ksv) was determined according to the Stern-
Volmer equation
The serine racemase and serine dehydratase activities were
measured with the following assay mixture: a 500 mM CHES–
NaOH buffer, pH 9.0 (100
1b) (50 l), 1 mM PLP (5
and deionized water (245
l
l
l
l), 100 mM L- or D-serines (1a and
l), a 1 mg/ml crude extract (100 l),
l), for a total volume of 500 l. The as-
l
l
l
say mixture, except for the crude extract, was put into a microtube
(1.5 ml, Greiner, Heidelberg, Germany) and incubated at 30 °C. The
enzymatic reaction was started by the addition of the crude ex-
tract. After the reactions were stopped by heat treatment (100 °C,
5 min), any D- or L-serines (1a and 1b) produced were measured
by HPLC (for serine racemase activity), and any pyruvate produced
was measured using the salicylaldehyde method (for serine dehy-
dratase activity) (Saier and Jenkins, 1967). One unit of the enzyme
Fo=F ¼ 1 þ Ksv½Qꢄ;
was defined as the amount of enzyme that produces 1 lmol of D-
where Fo is the fluorescence intensity in the absence of a quencher
(i.e., acrylamide), F is the fluorescence intensity at the molar
quencher concentration [Q]. Ksv is obtained experimentally from
the slope of the plot of Fo/F versus [Q].
or L-serine (1a and 1b) from racemization, or pyruvate from dehy-
dration, per min at 30 °C.
3.7. Basic enzymatic properties
3.11. Other methods
Serine racemase and serine dehydratase activities of SerR were
examined at various reaction temperatures: 10, 20, 30, 35, 40, 50,
60, 70, 80, and 90 °C. The activation energies for the serine race-
mase and serine dehydratase reactions were calculated from
Arrhenius plots. The thermal stability of SerR was estimated by
both the serine racemase and the serine dehydratase activities
after the enzyme was treated at various temperatures (10, 20, 30,
35, 40, 50, 60, 70, 80, and 90 °C) for 1 h. The effects of pH on the
serine racemase and serine dehydratase activities of SerR were
measured under the standard assay conditions at various pHs (a
PIPES–NaOH buffer for pH 6.0–7.0; a HEPES buffer for pH 7.0–8.5;
a CHES–NaOH buffer for pH 8.5–10.0; and a CAPS–NaOH buffer
for pH 10.0–11.0), and the pH stability of the enzyme was esti-
mated by both the serine racemase and the serine dehydratase
activities after the enzyme was treated in buffers with various
Metal analysis was conducted using a polarized Zeeman atomic
absorption spectrophotometer Z-8200 (Hitachi High-Technologies
Co., Tokyo). Ni was monitored at 232.0 nm, with measurements re-
peated at least twice. These protein concentrations were measured
using the Bradford method, with bovine serum albumin (Wako
Chemical Co., Osaka; product No. 011-07493) as a standard. The
molecular mass of SerR was estimated by gel filtration with a col-
umn (1.6 by 70 cm) of Superdex 200 Hiload (16/60) (GE Healthcare
UK Ltd., Amersham Place, England), and with thyroglobulin
(669 k), catalase (232 k), albumin (69 k), and chymotrypsinogen
A (25 k) as standards. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis was carried out by the method of Laemmli
(Laemmli, 1970). Gels were stained with Coomassie Blue R-250.

Y. Gogami et al. / Phytochemistry 70 (2009) 380–387
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3.12. Nucleotide sequence accession number
The DNA sequence of the gene encoding serine racemase from
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In this study, we clarified the enzymological properties of SerR
in rice. The function of SerR is probably either catabolic (the degra-
dation of D- or L-serine (1a and 1b) to pyruvate and ammonia) or
anabolic (the production of D-serine (1a) from L-serine (1b)) but
this still remains unknown. We are currently studying the three-
dimensional structure of SerR by X-ray crystallography to clarify
the reaction and regulation mechanisms of the bifunctional en-
zyme, as well as the physiological functions of SerR in germinated,
unpolished rice.
Sasaki, T., Matsumoto, T., Yamamoto, K., Sakata, K., Baba, T., Katayose, Y., Wu, J.,
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H., Arita, K., Hamada, M., Harada, C., Hijishita, S., Honda, M., Ichikawa, Y.,
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Hahn, J.H., Kim, H.I., Eun, M.Y., Yano, M., Jiang, J., Gojobori, T., 2002. The genome
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Acknowledgements
We thank Dr. Kenji Soda, Professor Emeritus of Kyoto Univer-
sity, Japan and Dr. Toshihiko Ikeuchi, Professor of Kansai Univer-
sity, Japan, for their helpful advices and encouragements. This
work was supported in part by the Strategic Project to Support
the Formation of Research Bases at Private Universities.
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