Serine racemases from barley, Hordeum vulgare L., and other plant species represent a distinct eukaryotic group: Gene cloning and recombinant protein characterization

Article abstract of DOI:10.1016/j.phytochem.2007.03.040

Several d-amino acids have been identified in plants. However, the biosynthetic pathway to them is unclear. In this study, we cloned and sequenced a cDNA encoding a serine racemase from barley which contained an open reading frame encoding 337 amino acid residues. The deduced amino acid sequence showed significant identity to plant and mammalian serine racemases and contained conserved pyridoxal 5-phosphate (PLP)-binding lysine and PLP-interacting amino acid residues. The purified gene product catalyzed not only racemization of serine but also dehydration of serine to pyruvate. The enzyme requires PLP and divalent cations such as Ca²⁺, Mg²⁺, or Mn²⁺, but not ATP, whereas mammalian serine racemase activity is increased by ATP. In addition to the results regarding the effect of ATP on enzyme activity and the phylogenetic analysis of eukaryotic serine racemases, the antiserum against Arabidopsis serine racemase did not form a precipitate with barley and rice serine racemases. This suggests that plant serine racemases represent a distinct group in the eukaryotic serine racemase family and can be clustered into monocot and dicot types.

Full text of DOI:10.1016/j.phytochem.2007.03.040

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 1530–1536
www.elsevier.com/locate/phytochem
Serine racemases from barley, Hordeum vulgare L., and other
plant species represent a distinct eukaryotic group: Gene cloning
q
and recombinant protein characterization
a
a
b
a,
*
Yoshiyuki Fujitani , Terumi Horiuchi , Kazutoshi Ito , Manabu Sugimoto
a
Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan
Plant Bioengineering Research Laboratories, Sapporo Breweries Ltd., 37-1 Nitta-Kizaki-cho, Ohta, Gunma 370-0393, Japan
b
Received 20 September 2006; received in revised form 20 February 2007
Abstract
Several D-amino acids have been identified in plants. However, the biosynthetic pathway to them is unclear. In this study, we cloned
and sequenced a cDNA encoding a serine racemase from barley which contained an open reading frame encoding 337 amino acid res-
idues. The deduced amino acid sequence showed significant identity to plant and mammalian serine racemases and contained conserved
pyridoxal 5-phosphate (PLP)-binding lysine and PLP-interacting amino acid residues. The purified gene product catalyzed not only race-
2
+
2+
mization of serine but also dehydration of serine to pyruvate. The enzyme requires PLP and divalent cations such as Ca , Mg , or
2
+
Mn , but not ATP, whereas mammalian serine racemase activity is increased by ATP. In addition to the results regarding the effect
of ATP on enzyme activity and the phylogenetic analysis of eukaryotic serine racemases, the antiserum against Arabidopsis serine rac-
emase did not form a precipitate with barley and rice serine racemases. This suggests that plant serine racemases represent a distinct
group in the eukaryotic serine racemase family and can be clustered into monocot and dicot types.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Hordeum vulgare L.; Oryza sativa; Gramineae; Pyridoxal 5-phosphate; Serine racemase; Serine dehydratase; D-Amino acid
1
. Introduction
mization but also dehydration of 1a and 1b to pyruvate (2)
in the presence of divalent cations; this distinguishes it
from bacterial amino acid racemases (Cook et al., 2002;
De Miranda et al., 2002; Strisovsky et al., 2003; Foltyn
et al., 2005) (Fig. 1). In plants, several D-amino acids have
been discovered in pea seedlings (Ogawa et al., 1977), bar-
ley grain, hops blossoms (Erbe and Bruckner, 2000), and
tobacco leaves (Kullman et al., 1999). Recently, a serine
racemase gene was cloned from Arabidopsis thaliana and
expressed to produce a protein that has both racemase
and dehydratase activities (Fujitani et al., 2006). This gene
also has 46% and 45% identity to human and mouse serine
racemases, respectively, and the pyridoxal 5-phosphate
Serine racemase, which catalyzes racemization of L- and
D-amino acids, is found in mammals as a biosynthetic
enzyme for D-serine (1a), with the latter acting as an ago-
nist at the glycine site of the N-methyl-D-aspartate receptor
in the mammalian nervous system (Dunlop et al., 1986;
Nagata et al., 1989; Hashimoto et al., 1992, 1993, 1995;
Nagata et al., 1994). Mammalian serine racemase is an
unique enzyme that catalyzes not only serine (1a, 1b) race-
Abbreviations: PLP, pyridoxal 5-phosphate; PCR, polymerase chain
reaction; IPTG, isopropyl-b-D-thiogalactopyranoside.
(PLP)-binding domain is conserved as with mammalian
q
The sequence reported in this paper has been deposited in the DDBJ,
serine racemases. The PLP-dependent A. thaliana serine
EMBL, and GenBank data banks under the accession no. AB271213.
*
racemase also requires divalent cations but not ATP,
Corresponding author. Tel.: +81 86 424 1661; fax: +81 86 434 1249.
2
+
E-mail address: manabus@rib.okayama-u.ac.jp (M. Sugimoto).
whereas there is synergy between Mg and ATP in the
0
031-9422/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.03.040

Y. Fujitani et al. / Phytochemistry 68 (2007) 1530–1536
1531
to PLP, Phe61 and Gly241 sandwich the PLP ring, the
side-chain of Ser315 is hydrogen-bonded to the pyridium
nitrogen of PLP, and Asn89 stabilizes the 3 oxygen of
H
COOH
0
HO
PLP by hydrogen bonding. Additionally, in the PLP-bind-
ing domain of E. coli threonine dehydratase, a typical fold
type II PLP enzyme, these residues correspond to Lys67,
Phe66, Gly246, Ser322, and Asn94 of HvSR, respectively,
moreover, all of these residues were conserved in rice, A.
thaliana, human, and mouse serine racemases. The gly-
cine-rich loop, which coordinates the phosphate group of
PLP, is composed of a triglycine loop (Gly194–196 of
HvSR) in the three plant serine racemases known, whereas
it is a tetraglycine loop in human and mouse serine race-
mases. The phylogenetic tree presented shows that plant
serine racemases represent a distinct group from mamma-
lian serine racemases, and that the monocot serine race-
mases of barley and rice are distinct from the dicot serine
racemase of A. thaliana (Fig. 3).
NH2
D-Serine (1a)
H C
COOH
3
NH3
O
Pyruvate (2)
H
COOH
HO
NH2
L-Serine(1b)
Fig. 1. Reaction pathways of D-serine (1a) and L-serine (1b) by mamma-
lian serine racemase.
2.2. Purification and enzyme activity of recombinant barley
serine racemase
activity of mammalian serine racemases. These reports sug-
gest that this plant serine racemase is distinct from mam-
malian serine racemases. However, there has been no
report on the cloning and characterization of plant serine
racemases except for that in A. thaliana.
In this report, we describe the isolation and character-
ization of a cDNA encoding serine racemase from barley.
In addition, we obtained a rice serine racemase by cloning
a cDNA encoding a homolog of barley serine racemase and
compared the structure–function relationships of plant and
mammalian serine racemases.
SDS-PAGE analysis of E. coli cells harboring pHvSR
contained an extra protein with the expected molecular
mass of ca. 36 kDa, the expression level of which reached
ca. 10% of the total protein after 8 h of isopropyl-b-D-thio-
galactopyranoside (IPTG) induction. Purification of
recombinant HvSR by Ni-NTA column chromatography
yielded an ꢀ95% pure preparation (Fig. 4). The purified
HvSR was incubated with 5 lM PLP and 1 mM CaCl
2
which activates AtSR and mammalian serine racemases
(Cook et al., 2002; De Miranda et al., 2002; Strisovsky
et al., 2003), and the formation of 1a and 2 was determined.
The specific activities for racemization of L-serine (1b) to
1
a, and dehydration of 1b to 2, were 14 and 228 nmol/
min mg, respectively, and those for racemization of 1a to
b, and dehydration of 1a to 2, were 2.2 and 89 nmol/
2
. Results
1
2
.1. Sequence analysis of barley serine racemase
The resulting nucleotide sequence of the barley serine
min/mg, respectively.
The purified rice serine racemase expressed in E. coli
cells harboring pOsSR had a molecular mass ꢀ36 kDa,
as estimated by both the deduced amino acid sequence
and SDS-PAGE, respectively. It was also purified by Ni-
NTA column chromatography (Fig. 4), and displayed rac-
emase and dehydratase activities for 1b of 10 and
214 nmol/mg min, respectively, and those for 1a of 1.5
and 77 nmol/mg min, respectively.
racemase cDNA, except for the poly(A) sequence, is
380 bp in length and contains an open reading frame
1
encoding a polypeptide of 337 amino acid residues with a
calculated molecular mass of 35.7 kDa (HvSR,
AB271213). The deduced amino acid sequence of HvSR
showed 89% identity with that of a putative rice serine rac-
emase in data banks (OsSR, AL606647), and 68%, 46%,
4
5%, and 28% identity with a serine racemase from A. tha-
liana (AtSR, AB206823, Fujitani et al., 2006), as well as
human (hSR, AF169974, De Miranda et al., 2000) and
mouse (mSR, AF148321, Wolosker et al., 1999) serine
racemases, and the catalytic domain of L-threonine dehy-
dratase from Escherichia coli (EcTD, Swiss-Plot no.
P04969, Gallagher et al., 1998), respectively. The amino
acid sequence alignment (Fig. 2) shows that plant and
mammalian serine racemases belong to the fold type II
PLP enzymes. Specifically, it is known that Lys62 binds
2.3. Enzymatic characterization of recombinant barley serine
racemase
A maximum at 420 nm in the absorption spectrum of
the purified HvSR disappeared after dialysis against a buf-
fer containing 10 mM sodium borohydride with concomi-
tant loss of enzyme activity (Fig. 5). Additionally, the
enzyme activity of HvSR was inhibited completely by
0.1 mM hydroxylamine (data not shown), indicating that

1532
Y. Fujitani et al. / Phytochemistry 68 (2007) 1530–1536
Fig. 2. Alignment of the deduced amino acid sequences of barley and rice serine racemases with those of A. thaliana, human, and mouse serine racemases,
as well as E. coli biosynthetic L-threonine dehydratase. Barley, rice, A. thaliana, human, and mouse serine racemases (HvSR, OsSR, AtSR, hSR, and mSR)
correspond to GenBank accession nos. AB271213, AL606647, AB206823, AF169974, and AF148321, respectively, whereas the E. coli biosynthetic
L-threonine dehydratase (EcTD) corresponds to Swiss-Plot accession no. P04968. Gaps, indicated by dashes, are introduced in the sequences to maximize
the homology. Identical amino acid residues among either serine racemases or all six of the enzymes are represented by black boxes. The closed arrowhead,
open arrowheads, and asterisks indicate the PLP-binding Lys, amino acid residues that interact with PLP, and the glycine-rich loop, respectively.
The optimum pH and concentration of CaCl in the
racemization and dehydration of 1b were examined in a
2
AtSR
reaction mixture containing 10 lM PLP. HvSR has maxi-
mum activities for both racemization and the elimination
reaction at pH 8.5 in the presence of 1 mM CaCl2.
The racemase activities for converting various 1b to 1a
acids were examined in a reaction mixture containing
0
.142
0
.127
OsSR
0.060
hSR 0.048
.053
0.329
1 mM CaCl at pH 8.5. HvSR showed high substrate spec-
2
0
.064
ificity with regard to 1b, 14 nmol/mg min, whereas L-ala-
nine, L-glutamine, and L-arginine were poorer substrates:
the activities were 1.9, 0.74, and 1.7 nmol/mg min, respec-
tively. The Km and Vmax were estimated in a reaction mix-
0
HvSR
mSR
0
.1
ture containing 1 mM CaCl , 10 lM PLP at pH 8.5, with
2
Fig. 3. Phylogenetic analysis of eukaryotic serine racemases. The scale bar
represents 0.1 amino acid substitutions per site.
corresponding values for converting 1b to 1a being
2
9
.6 mM and 21 nmol/mg min, and for 1a to 1b of
.0 mM and 455 nmol/mg min, respectively. Additionally,
HvSR depends on PLP and is bound to PLP through a
Schiff base (Grishin et al., 1995).
the K and V
for dehydratase activity from 1b to 2 were
max
m
2.7 mM and 2.8 nmol/mg min, whereas those of 1a to pyru-
vate were 8.3 mM and 161 nmol/mg min, respectively.

Y. Fujitani et al. / Phytochemistry 68 (2007) 1530–1536
1533
M
1
2
3
M
4
5
kDa
7
5
3
5
0
7
2
5
2
0
Fig. 4. Analysis of the expression of serine racemase genes from barley and rice in E. coli cells by SDS-polyacrylamide gel. Total cell lysate (lane 1), soluble
protein (lane 2), purified enzyme following Ni-NTA column chromatography (CC) (lane 3) from E. coli cells harboring pHvSR, soluble protein (lane 4)
and purified enzyme following Ni-NTA CC (lane 5) from E. coli cells harboring pOsSR. Gels were obtained using 15% SDS-PAGE with molecular mass
marker series (lane M) for calibration, with detection by Coomassie Brilliant Blue R-250 staining.
1
1
5
0
3
00
1
.0
.5
0
2
1
00
00
5
0
0
0
EDTA ATP
ATP
MgCl
MgCl2 CaCl2 MnCl2 FeCl2 NiCl2
2
Fig. 6. Effects of divalent cations and ATP on racemase and dehydratase
activities of barley serine racemase. Racemase (black columns) and
dehydratase (white columns) activities of the purified recombinant enzyme
were assayed in the presence of 1 mM EDTA, 1 mM metal chloride, or
250
300
350
400
450
500
1
mM ATP. The results represent the average ± s.e. of three experiments.
Wavelength (nm)
Fig. 5. Absorption spectra of purified HvSR before and after NaBH
4
incubation. The spectra of the purified HvSR (1 mg/ml) before and after
dialyzing against 500 volumes of 20 mM Tris–HCl (pH 7.5) containing
2.5. Immunological properties
1
mM DTT, 10lM PLP, and 10 mM sodium borohydride are shown as
An immunological comparison among plant serine race-
mases was performed by Ouchterlony double diffusion
solid and dashed lines, respectively.
(Fig. 7). Anti-HvSR serum formed one line not only with
2
.4. Effects of divalent cations and ATP
purified recombinant HvSR but also with purified recombi-
nant OsSR and AtSR; however, the anti-AtSR serum also
formed a precipitate with AtSR and no line was detected
with HvSR and OsSR.
The effects of divalent cations and ATP on 1b racemiza-
tion and dehydration activities were analyzed by adding
mM divalent ion to a reaction mixture at pH 8.5
Fig. 6). Racemase activity was increased 2.9-, 3.8-, and
1
(
3
2
+
2+
2+
.8-fold by addition of Mg , Ca , and Mn , whereas
3. Discussion
2
+
2+
EDTA, Fe , or Ni inhibited this activity. The addition
of Mg , Ca , and Mn also increased dehydratase activ-
ity 13-, 11-, and 13-fold, respectively, whereas Fe showed
only a 2.8-fold increase and Ni inhibited the activity.
HvSR was not activated by 1 mM ATP, and the activity
with the combination of 1 mM Mg and 1 mM ATP was
slightly decreased compared to that in the presence of
1
2
+
2+
2+
We demonstrated that barley serine racemase, HvSR, is
a bifunctional PLP-dependent enzyme that catalyzes not
only racemization of 1a and 1b but also dehydration of
1a and 1b to 2. The enzyme activity of HvSR and A. thali-
ana serine racemase, AtSR, are enhanced by addition of
2
+
2
+
2
+
2
+
2+
2+
2+
divalent cations such as Ca , Mg , and Mn . Fe
inhibits racemase activity of HvSR, whereas that of AtSR
2
+
mM Mg .

1534
Y. Fujitani et al. / Phytochemistry 68 (2007) 1530–1536
Fig. 7. Ouchterlony double diffusion of the purified recombinant plant serine racemases. Ab1, anti-HvSR serum; Ab2, anti-AtSR serum; 1, OsSR; 2,
HvSR; 3, AtSR.
2
+
is slightly increased by addition of Fe (Fujitani et al.,
2
both divalent cations and ATP. In contrast to the eukary-
otic racemases, plant serine racemase activity was increased
by divalent cations but not by ATP. This suggests that the
interaction of the enzyme with ATP and the divalent cation
restricts the substrate specificity of eukaryotic racemases.
A novel mechanism of mammalian serine racemase has
been proposed, whereby dehydratase activity of serine rac-
emase regulates the intracellular 1a level. This effect occurs
especially in brain areas, since a D-amino acid oxidase,
which degrades D-amino acids in mammalian cells, is
restricted to the cerebellum and brainstem and a dehydra-
tase activity-deficient serine racemase mutant accumulates
1a (Foltyn et al., 2005). Several D-amino acids and D-amino
acid-containing peptides have been identified in plants.
However, both 1a and D-alanine inhibit plant growth at
low concentrations (Erikson et al., 2004), indicating that
the D-amino acid level should be strictly regulated in plant
cells. There is no previous report of a plant D-amino acid
oxidase, and the serine racemase is the only known enzyme
that degrades 1a in plants. The specific activities of
racemization and dehydration for 1b were as follows: 14
and 228 nmol/min/mg with regard to HvSR, 10 and
214 nmol/min/mg with regard to OsSR, and 4.1 and
86 nmol/min/mg with regard to AtSR (Fujitani et al.,
2006), respectively: the corresponding dehydratase activi-
ties were about 20-fold higher than racemase activity. This
is in contrast to the dehydration/racemization ratio of
about 0.7 for mouse serine racemase (De Miranda et al.,
2002). These results thus suggest that the main physiologi-
cal function of plant serine racemase might be to degrade,
rather than produce 1a.
2
+
006). While the combined effect of Mg and ATP on
mouse serine racemase leads to an increase in 1a produc-
2
+
tion, there was no synergy between Mg and ATP on
HvSR activity, as with AtSR. These results suggest that
the protein structure interacting with ATP might be differ-
ent between plant and mammalian serine racemases. The
amino acid residues that interact with PLP (Phe61,
Lys62, Asn89, Gly241, Ser315) are conserved in HvSR,
with other plant and mammalian serine racemases, which
belong to the fold type II group of PLP enzymes (Galla-
gher et al., 1998), except for the glycine-rich loop, which
consists of a triglycine and a tetraglycine in plant and
mammalian serine racemases, respectively. These results
show that plant serine racemase represents a distinct group
in the eukaryotic serine racemase family.
The amino acid sequence identities of HvSR with OsSR
and AtSR (89% and 68%, respectively) and the phyloge-
netic tree suggest that plant serine racemases can be clus-
tered into monocot and dicot types. To show the
structural differences between serine racemases of monocot
and dicot plants, we prepared anti-HvSR and anti-AtSR
sera, and then purified recombinant rice serine racemase
with both racemase and dehydratase activities: the latter
was obtained by expression of an open reading frame of
the putative rice serine racemase gene in DDBJ, EMBL,
and GenBank data banks. The specific cross-reactivity of
the anti-AtSR serum against AtSR by the Ouchterlony test
supports the possibility that there may be monocot and
dicot clusters in plant serine racemase based on a compar-
ison of the three plant enzymes examined so far.
Recently, an aspartate racemase gene was cloned from a
bivalve mollusk and expressed to characterize its enzyme
properties (Abe et al., 2006). The aspartate racemase is a
PLP-dependent enzyme and has both racemase and dehy-
dratase activities. The amino acid sequence shows 38%,
4. Experimental
4.1. Plant material and growth conditions
3
9%, 32%, 30%, and 36 % identities to human, mouse, bar-
ley, rice, and A. thaliana serine raceases, respectively, and
contains conserved PLP-interacting amino acid residues,
corresponding to Phe61, Lys62, Asn89, Gly241, and
Ser315 of E. coli threonine dehydratase. Interestingly, its
enzyme activity was decreased by ATP but not by EDTA,
whereas mammalian serine racemases was activated by
The seeds of barley, Hordeum vulgare L., cv. Haruna
nijo, were germinated and cultured in a hydroponic solu-
tion that consisted of 4 mM KNO , 1 mM NaNO ,
3
3
1 mM NaH PO , 1 mM MgSO , 1 mM CaCl , 45 lM Fe-
2
4
4
2
citrate, 18 lM H BO , 4.6 lM MnSO , 1.5 lM ZnSO ,
3
3
4
4
1.5 lM CuSO , and 1.5 lM Na MoO . The pH of the cul-
4
2
4

Y. Fujitani et al. / Phytochemistry 68 (2007) 1530–1536
1535
ture solution was adjusted to 5.5 with NaOH. After 1 week
4.4. Expression and purification of serine racemase
of cultivation, the roots were harvested, frozen in liquid N2,
and stored at ꢁ80 °C.
E. coli cells harboring pHvSR were grown at 25 °C in
Luria–Bertani (LB) medium containing 50 lg/ml of ampi-
cillin. When the OD600 became 0.5, isopropyl-b-D-thioga-
lactopyranoside (IPTG) was added to the culture at a
final concentration of 0.5 mM. After cultivation at 25 °C
for 6 h, the cells were harvested by centrifugation and fro-
zen at ꢁ80 °C for at least 2 h. In the case of E. coli cells har-
boring pOsSR, the culture of 0.5 OD was incubated at
4
.2. Rapid amplification of cDNA ends PCR (RACE PCR)
Total RNA was isolated from the storage roots using the
RNeasy Plant Mini Kit (Qiagen) and adaptor-ligated cDNA
was synthesized from 1 lg of total RNA using the SMART
RACE cDNA Amplification Kit (Clontech) according to
6
00
the manufacture’s instructions. The primers for 3- and
15 °C for 30 min and cultured at 15 °C for 8 h with
0
5
-RACE, 5 -GCCTTGAACTTTTGGAGCAAGTCCC-
0.5 mM IPTG. The frozen cell pellets were suspended in a
0
0
TM
TG-3 and 5 -TCCCTATTTTGCTGCTCTCATGCCAAG-
3
BugBuster
HT protein extraction reagent (Novagen)
0
, respectively, were synthesized based on the conserved
according to the manufacturer’s instructions. The resulting
recombinant protein was purified using an Ni-NTA CC
(Qiagen) initially equilibrated in 20 mM Tris–HCl buffer
(pH 7.9) containing 0.5 M NaCl and 5 mM imidazole (Buf-
fer A). The column was next washed with Buffer A, followed
by 60 mM imidazole in Buffer A, with the absorbed protein
finally eluted with 1 M imidazole in Buffer A. The enzyme
solution was collected, dialyzed against a 20 mM Tris–
HCl buffer (pH 7.5) containing 1 mM DTT and 10 lM
PLP, and concentrated by an Ultracent-30 (Tohso).
nucleotide sequences from truncated barley genes (accession
nos. BJ450541 and CA023779) and PCR was performed on
the cDNA with a synthesized primer and the universal pri-
mer mixture (‘‘UPM’’) supplied in the kit. Fragments of
about 280 and 800 bp amplified by 3- and 5-RACE PCR,
respectively, were cloned into a pGEM-T vector (Promega)
and subjected to nucleotide sequencing on both strands
using a BigDye terminator v1.1 cycle sequencing kit
(
Applied Biosystems) with a series of synthetic primers.
4
.3. Cloning of serine racemase gene
4.5. Enzyme and protein assays
Based on the putative open reading frame of the barley
The serine racemase and dehydratase activities were
assayed by calculating the hydroxyperoxide production
with amino acid oxidase–peroxidase–OPA and by calculat-
ing the pyruvate production with 2,4-dinitrophenylhydr-
azine, respectively, according to the method described
previously (Fujitani et al., 2006). The 1b solution used in
the enzyme assay was pre-treated with D-amino acid oxi-
dase and catalase to remove any contaminating 1a accord-
ing to De Miranda et al. (2002). L-Amino acid oxidase was
used to remove contaminating 1b in the 1a solution. Pro-
tein concentrations were quantified according to Bradford
(1976) with bovine serum albumin as the standard.
serine racemase gene predicted by the RACE PCR experi-
ment, primers were designed as follows: sense primer,
0
0
5
-GCCATATGGGAAGCAGAGATGACGATG-3 , which
0
creates an NdeI site (underlined), and antisense primer, 5 -
0
GACTCGAGTTTATACAAGGAATC-CCAT-3 , which
creates an XhoI site (underlined). The open reading frame
was amplified by reverse-transcription PCR (RT-PCR)
using the OneStep RT-PCR kit (Qiagen) with total RNA
from the barley roots. The PCR product of 1026 bp was
cloned into the pGEM-T vector and the NdeI- and XhoI-
digested fragment of the plasmid was cut out and sub-
cloned into an NdeI- and XhoI-digested pET20b(+) vector
(
Novagen), in which the endogenous stop codon of the ser-
4.6. Antiserum preparation
ine racemase gene was replaced by a polyhistidine tag gene.
The resulting plasmid, pHvSR, was transformed into
E. coli BL21(DE3) pLysS cells.
The purified recombinant serine racemase of barley or
A. thaliana was mixed with either Freund’s complete adju-
vant (for the first injection) or incomplete adjuvant (for fol-
lowing injections). The emulsified mixture containing
protein (1 mg) was used to immunize a male Japanese white
rabbit by subcutaneous injection followed by four subse-
quent injections, once every two weeks. Titration of the
antiserum was performed by standard enzyme-linked
immunosorbent assay (ELISA).
The open reading frame of the rice serine racemase gene
was amplified with total RNA from the rice, Oryza sativa,
cv. Nipponbare, and the primers were designed based on
the putative gene (AL606647), as follows: sense primer,
0
0
5
-CCCATATGGGGAGCAGAGGTGGAAGTGG-3 ,
which creates an NdeI site (underlined), and antisense pri-
0
mer, 5 -CCCTCGAGACGTTTATAGAGAGACTCCC-
0
3
, which creates an XhoI site (underlined) by OneStep
RT-PCR. The NdeI- and XhoI-digested fragment of the
032 bp PCR product was subcloned into an NdeI- and
4.7. Computer analysis
1
XhoI-digested pCold I vector (Takara), in which a polyhis-
tidine tag gene is fused upstream from the start codon. The
resulting plasmid, pOsSR, was transformed into E. coli
BL21 cells.
Analysis and translation of the nucleotide sequences
were performed using GENETYX-MAC (Software Devel-
opment). The homology searches, alignments, and phylo-
genetic analyses of the amino acid sequences were

1536
Y. Fujitani et al. / Phytochemistry 68 (2007) 1530–1536
performed using BLAST (Altschul et al., 1990; Karlin and
Altschul, 1990), CLUSTAL W (Tompson et al., 1994), and
TreeView (Page, 1996) programs, respectively.
Fujitani, Y., Nakajima, N., Ishihara, K., Oikawa, T., Ito, K., Sugimoto,
M., 2006. Molecular and biochemical characterization of a serine
racemase from Arabidopsis thaliana. Phytochemistry 67, 668–674.
Gallagher, D.T., Gilliland, G.L., Xiao, G., Zondlo, J., Fisher, K.E.,
Chinchilla, D., Eisenstein, E., 1998. Structure and control of pyridoxal
phosphate dependent allosteric threonine deaminase. Structure 6, 465–
Acknowledgement
4
75.
Grishin, N.V., Phillips, M.A., Goldsmith, E.J., 1995. Modeling of the
spatial structure of eukaryotic ornithine decarboxylases. Protein Sci. 4,
1291–1304.
This research was supported in part by the Ohara Foun-
dation in Kurashiki, Japan.
Hashimoto, A., Nishikawa, T., Hayashi, T., Fujii, N., Oka, T., Takahashi,
K., 1992. The presence of free D-serine in rat brain. FEBS Lett. 296,
References
3
3–36.
Hashimoto, A., Kumashiro, S., Nishikawa, T., Oka, T., Takahashi, K.,
Mito, T., Takashima, S., Doi, N., Mizutani, Y., Yamazaki, T.,
Kaneko, T., Ootomo, E., 1993. Embryonic development and postnatal
changes in free D-aspartate and D-serine in the human prefrontal
cortex. J. Neurochem. 61, 783–786.
Hashimoto, A., Oka, T., Nishikawa, T., 1995. Anatomical distribution
and postnatal changes in endogenous free D-aspartate and D-serine in
rat brain and periphery. Eur. J. Neurosci. 7, 1657–1663.
Karlin, S., Altschul, S.F., 1990. Methods for assessing the statistical
significance of molecular sequence features by using general scoring
schemes. Proc. Nalt. Acad. Sci. USA 87, 2264–2268.
Kullman, J.P., Chen, X., Armstrong, D.W., 1999. Evaluation of the
enantiomeric composition of amino acids in tobacco. Chirality 11,
669–673.
Nagata, Y., Konno, R., Yasumura, Y., Akino, T., 1989. Involvement of
D-amino acid oxidase in elimination of free D-amino acids in mice.
Biochem. J. 257, 291–292.
Nagata, Y., Horiike, K., Maeda, T., 1994. Distribution of free D-serine in
vertebrate brains. Brain Res. 634, 291–295.
Ogawa, T., Bando, N., Sasaoka, K., 1977. Occurrence of D-a-amino-n-
butyric acid in legume seedlings. Agric. Biol. Chem. 41, 1811–1812.
Page, R.D.M., 1996. TreeView: an application to display phylogenetic
trees on personal computers. Comput. Appl. Biosci. 12, 357–358.
Strisovsky, K., Jiraskova, J., Barinka, C., Majer, P., Rojas, C., Slusher,
B.S., Konvalinka, J., 2003. Mouse brain serine racemase catalyzes
specific elimination of L-serine to pyruvate. FEBS Lett. 535, 44–48.
Tompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Improving the
sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res. 22, 4673–4680.
Abe, K., Takahashi, S., Muroki, Y., Kera, Y., Yamada, R., 2006. Cloning
and expression of the pyridoxal 5-phosphate-dependent aspartate
racemase gene from the bivalve mollusk Scapharca broughtonii and
characterization of the recombinant enzyme. J. Biochem. 139, 235–244.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990.
Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein–
dye binding. Anal. Biochem. 72, 248–254.
Cook, S.P., Galve-Roperh, I., Martinez, d.P., Rodriguez-Crespo, I., 2002.
Direct calcium binding results in activation of brain serine racemase. J.
Biol. Chem. 277, 27782–27792.
De Miranda, J., Santoro, A., Engelender, S., Wolosker, H., 2000. Human
serine racemase: molecular cloning, genomic organization and func-
tional analysis. Gene 256, 183–188.
De Miranda, J., Panizzutti, R., Foltyn, V.N., Wolosker, H., 2002.
Cofactors of serine racemase that physiologically stimulate the
synthesis of the N-methyl-D-aspartate (NMDA) receptor coagonist
D-serine. Proc. Natl. Acad. Sci. USA 99, 14542–14547.
Dunlop, D.S., Neidle, A., McHale, D., Dunlop, D.M., Lajtha, A., 1986.
The presence of free D-aspartic acid in rodents and man. Biochem.
Biophys. Res. Commun. 141, 27–32.
Erbe, T., Bruckner, H., 2000. Chromatographic determination of amino
acid enantiomers in beers and raw materials used for their manufac-
ture. J. Chromatogr. A 881, 81–91.
Erikson, O., Hertzberg, M., Nasholm, T., 2004. A conditional marker
gene allowing both positive and negative selection in plants. Nat.
Biotechnol. 22, 455–458.
Foltyn, V.N., Bendikov, I., De Miranda, J., Panizzutti, R., Dumin, E.,
Shleper, M., Lu, P., Toney, M.D., Kartvelishvily, E., Wolosker, H.,
Wolosker, H., Blackshaw, S., Snyder, S.H., 1999. Serine racemase, a glial
enzyme synthesizing D-serine to regulate glutamate-N-methyl-D-aspar-
tate neurotransmission. Proc. Natl. Acad. Sci. USA 96, 13409–13414.
2005. Serine racemase modulates intracellular D-serine levels through
an a,b-elimination activity. J. Biol. Chem. 280, 1754–1763.