Molecular and biochemical characterization of a serine racemase from Arabidopsis thaliana

Article abstract of DOI:10.1016/j.phytochem.2006.01.003

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5′-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, or Ni²⁺, at alkaline pH for both activities. The racemization process is highly specific toward l-serine, whereas l-alanine, l-arginine, and l-glutamine were poor substrates. The V_max/K_m values for racemase activity of l- and d-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for l- and d-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward l-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana.

Full text of DOI:10.1016/j.phytochem.2006.01.003

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 668–674
www.elsevier.com/locate/phytochem
Molecular and biochemical characterization of a serine
q
racemase from Arabidopsis thaliana
a
b
c
d
Yoshiyuki Fujitani , Nobuyoshi Nakajima , Koji Ishihara , Tadao Oikawa ,
e
a,
*
Kazutoshi Ito , Manabu Sugimoto
a
Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan
Collaborative Research Center, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan
b
c
Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan
Plant Bioengineering Research Laboratories, Sapporo Breweries Ltd., 37-1 Nitta-Kizaki-cho, Ohta, Gunma 370-0393, Japan
d
e
Received 23 August 2005; received in revised form 21 November 2005
Available online 17 February 2006
Abstract
A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana
0
and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5 -phosphate (PLP) are
conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyru-
2
+
2+
2+
2+
2+
vate. The enzyme is a homodimer and requires PLP and divalent cations, Ca , Mg , Mn , Fe , or Ni , at alkaline pH for both
activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor sub-
m
strates. The Vmax/K values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values
for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction
and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various
organs of A. thaliana.
ꢀ
2006 Elsevier Ltd. All rights reserved.
0
Keywords: Arabidopsis thaliana; Serine racemase; D-Amino acid; Pyridoxal 5 -phosphate
1
. Introduction
plants as well (Robinson, 1976; Hashimoto et al., 1992,
993; Bruckner and Weshauser, 2003). Recently, it has
1
D-Amino acids such as D-alanine and D-glutamate have
been reported that D-serine acts as an agonist at the glycine
site of a N-methyl-D-aspartate receptor and D-aspartate
serves as an agonist at its glutamate site in the mammalian
nervous system (Dunlop et al., 1986; Nagata et al., 1989,
been recognized as the components of peptidoglycans in
bacterial cell walls, although they have been isolated not
only from microorganisms but also from animals and
1994; Hashimoto et al., 1992, 1993, 1995), showing that
D-amino acids play an important role in eukaryotes.
D-Amino acids are synthesized by amino acid racemases
that catalyze the racemization of L- and D-amino acids,
and a number of the enzymes such as alanine, aspartate,
glutamate, serine, and phenylalanine racemase have been
isolated and their genes cloned from microorganisms
(Esaki et al., 2002). Serine racemase is a unique enzyme
0
Abbreviations: PLP, pyridoxal 5 -phosphate; PCR, polymerase chain
reaction; IPTG, isopropyl-b-D-thiogalactopyranoside; AtSR, Arabidopsis
thaliana serine racemase.
q
The sequence reported in this paper has been deposited in the DDBJ,
EMBL, and GenBank data banks under the Accession No. AB206823.
*
Corresponding author. Tel.: +81 86 424 1661; fax: +81 86 434 1249.
E-mail address: manabus@rib.okayama-u.ac.jp (M. Sugimoto).
0
031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.01.003

Y. Fujitani et al. / Phytochemistry 67 (2006) 668–674
669
discovered in eukaryotes, which requires pyridoxal
PLP-bound Lys62/Phe61/Gly241 which sandwiches the
PLP ring, Ser315 of which the side-chain is hydrogen-
bonded to the pyridium nitrogen of PLP, Asn89 which sta-
0
5
-phosphate (PLP) as a coenzyme (Uo et al., 1998; Wolos-
ker et al., 1999a). Interestingly, the amino acid sequence of
human and mouse brain serine racemases showed ca. 30%
identity to that of bacterial biosynthetic threonine dehydra-
tase (Wolosker et al., 1999b; De Miranda et al., 2000).
Moreover, mouse serine racemase catalyzes not only serine
racemization but also dehydration of serine to pyruvate in
the presence of divalent metal ions (De Miranda et al.,
0
bilizes the 3 oxygen of PLP by a hydrogen bond of E. coli
threonine dehydratase (Gallagher et al., 1998) correspond
to Lys59, Phe58, Gly238, Ser314, and Asn86 of AtSR,
respectively, and all of these residues were conserved in
human and mouse serine racemases (Wolosker et al.,
1999b; De Miranda et al., 2000). The glycine-rich group
which coordinates the phosphate group of PLP is consti-
tuted by a triglycine loop (Gly 186–188) in AtSR, whereas
it is a tetraglycine loop in human and mouse serine race-
mases (Fig. 1). Amino acid sequence alignment shows that
AtSR belongs to fold type II as for mammalian serine race-
mases and bacterial serine/threonine dehydratase, of which
the Schiff base lysine is near the N-terminus and the gly-
cine-rich loop is closer to the C-terminus; by contrast, bac-
terial and fungal alanine racemases belong to fold types III
and I, respectively (Grishin et al., 1995). These results indi-
cate that eukaryotic serine racemases represent a distinct
group in the overall amino acid racemase family.
2
002; Strisovsky et al., 2003; Foltyn et al., 2005). Thus,
mammalian serine racemases are distinct from other amino
acid racemases. In plants, a number of D-amino acids such
as D-alanine, D-aspartate, and D-glutamate were detected in
pea seedlings (Ogawa et al., 1977), barley grain, blossoms
of hops (Erbe and Bruckner, 2000), and tobacco leaves
(
Kullman et al., 1999); D-alanine was also identified as a
dipeptide such as in D-alanylglycine and D-alanyl-D-alanine
in wild rice (Manabe and Ohira, 1983; Manabe, 1985). The
genomic DNA of Arabidopsis thaliana formerly registered
on the database as a putative serine/threonine dehydratase
gene of Escherichia coli displays 46% amino acid sequence
identity to the human and rat serine racemases (Wolosker
et al., 1999b); however, there is no evidence whether the
homolog has serine racemase activity or some other amino
acid racemase activity.
In this paper, we cloned the cDNA encoding the homo-
log of mammalian serine racemases from A. thaliana and
characterized the gene product. The expressed protein is
a bifunctional PLP-dependent enzyme catalyzing racemiza-
tion of serine and dehydration of serine to pyruvate in the
same way as mammalian serine racemases.
2.2. Purification of recombinant A. thaliana serine racemase
E. coli cells harboring pAtSR produced an extra protein
whose expression level attained ca. 9% of the total protein
after 6 h of IPTG induction. Purification of the recombi-
nant AtSR with Ni-NTA column chromatography yielded
ꢀ95% pure preparation (Fig. 2). The molecular mass of
AtSR estimated by SDS–PAGE was ca. 35 kDa, which
matches to that of 35 kDa calculated from the amino acid
sequence, and ca. 60 kDa by gel filtration, suggesting that
AtSR is a homodimer protein.
2
. Results and discussion
The purified AtSR was incubated with L-serine and
CaCl that activates mammalian serine racemases (De Mir-
2
2
.1. Sequence analysis of A. thaliana serine racemase
anda et al., 2002; Strisovsky et al., 2003), and the produc-
tion of D-serine and pyruvate was determined. Specific
activities of racemization of L- to D-serine and dehydration
of L-serine to pyruvate were 4.1 and 86 nmol/min/mg,
respectively.
The putative open reading frame of the gene, predicted
from the genomic DNA sequence of A. thaliana, was a
homolog of mammalian serine racemase, and was ampli-
fied by RT-PCR. However, the resulting nucleotide
sequence and its deduced amino acid sequence were 45
bases and 15 amino acids shorter than the predicted
sequences, respectively. The deduced amino acid sequence
of A. thaliana serine racemase (AtSR) was compared with
that of human and mouse serine racemases (Wolosker
et al., 1999b; De Miranda et al., 2000), a homolog of mouse
serine racemase encoded in the YKL218c gene of Saccharo-
myces cerevisiae, and a catalytic N-terminal domain of
E. coli L-threonine dehydratase (Gallagher et al., 1998),
which belong to the fold type II of PLP enzymes (Grishin
et al., 1995). AtSR showed 46%, 45%, 39%, and 30%
identity with human and mouse serine racemases, the
homolog of S. cerevisiae, and the catalytic domain of
E. coli L-threonine dehydratase, respectively. Alignment
analysis of its amino acid sequence with the CLUSTAL
W algorithm (Tompson et al., 1994) showed that the
2.3. Enzymatic characterization of recombinant A. thaliana
serine racemase
The absorption spectrum of the purified AtSR showed
maxima at 280 and 415 nm. After dialysis against 500 vol-
umes of the dialyzing buffer containing 10 mM sodium
borohydride, the absorption at 415 nm disappeared and
an increase in absorbance at 330 nm was observed, with
loss of enzyme activity (Fig. 3A). Furthermore, the race-
mase activity of AtSR was inhibited completely by hydrox-
ylamine (Fig. 3B), indicating that AtSR depends on PLP
and is bound with it through a Schiff base (Grishin et al.,
1995).
The optimum pH for both L-serine racemization and
dehydration activities were examined in the reaction
mixture containing 1 mM CaCl . AtSR has maximum
2

670
Y. Fujitani et al. / Phytochemistry 67 (2006) 668–674
Fig. 1. Alignment of the deduced amino acid sequence of A. thaliana serine racemase with those of human and mouse serine racemases, S. cerevisiae
L-threo-3-hydroxyaspartate dehydratase, and E. coli biosynthetic L-threonine dehydratase. A. thaliana serine racemase (AtSR) corresponds to GenBank
Accession No. AB206823, human serine racemase (hSR) corresponds to AF169974, mouse serine racemase (mSR) corresponds to AF148321, and E. coli
biosynthetic L-threonine dehydratase (EcTD) corresponds to Swiss-Plot No. P04968. Gaps, indicated by dashes, are introduced in the sequences to
maximize the homology. Identical amino acid residues among serine racemases or all of four enzymes are represented by black boxes. An asterisk and
arrowheads indicate the Lys matching to the PLP-binding Lys and amino acid residues matching to those interacting with PLP in E. coli biosynthetic
L-threonine dehydratase, respectively.
2
+
2+
2+
activities for racemization and dehydration at pH 8.5 and
.5, and has high activities from pH 8.0 to 8.5 and from
pH 8.5 to 9.5, respectively.
The effects of divalent cations on L-serine racemization
and dehydration activities were analyzed by the addition
of 1 mM divalent ion to the reaction mixture at pH 8.5.
Racemase activity increased 3.6, 2.9, and 3.0 times with
racemase was activated by Ca , Mg , Mn , and ATP,
2
+
9
especially when Ca was bound to the enzyme directly
(Cook et al., 2002). Moreover, since there is a synergy
between Mg and ATP on activity (De Miranda et al.,
2002), we evaluated the optimum concentration of Ca
and the effect of ATP on AtSR activity. AtSR activity
increased with increasing amounts of Ca reaching maxi-
mum activity at 1 mM Ca . However, AtSR was not acti-
vated by 1 mM ATP and the enzyme activity in the
presence of 1 mM Mg and 1 mM ATP was no different
from that in the presence of 1 mM Mg .
2
+
2
+
2
+
2
+
2+
2+
2+
Ca , Mg , and Mn , respectively, when compared to
2
+
that in the presence of EDTA, whereas Fe had a more
2+
2+
limited effect to increase 1.4 times and both Ni
and
2
+
2+
2+
2+
2+
Zn inhibited activity. Addition of Ca , Mg , Mn
,
2
+
2+
Fe , and Ni increased 6.8, 7.5, 9.3, 2.8, and 2.7 times
The racemase activities converting various L- to D-amino
acids were examined in the reaction mixture containing
1 mM CaCl2 at pH 8.5. AtSR showed high substrate
2
+
dehydratase activity, respectively, and Zn had no effect
on activity (Fig. 4). It has been shown that mouse serine

Y. Fujitani et al. / Phytochemistry 67 (2006) 668–674
671
M
1
2
3
A
1
.0
7
5
3
5
0
7
2
5
0
.5
2
1
0
5
Fig. 2. Analysis of the expression of AtSR in E. coli by SDS–polyacryl-
amide gel. E. coli cells harboring pAtSR were harvested after IPTG
induction at 25 ꢁC for 6 h. Total E. coli lysate (lane 1), the soluble protein
solution (lane 2), and the purified AtSR with Ni-NTA column (lane 3)
were subjected to 15% SDS–PAGE with molecular mass marker (lane M)
followed by Coomassie Brilliant Blue R-250 staining.
0
250
300
350
400
450
500
Wavelength (nm)
B
specificity for L-serine, whereas L-alanine, L-arginine, and
L-glutamine were poor substrates for AtSR, having activi-
ties of ꢀ 13%, 7.0%, and 4.5% of that for L-serine,
respectively.
4
Alanine racemases from Salmonella typhimurium and
Schizosaccharomyces pombe could catalyze the racemiza-
tion of L-serine, but the activities on L-serine were 15%
and 3.7% of those on L-alanine, respectively (Esaki and
Walsh, 1986; Uo et al., 2001). An amino acid racemase
with low substrate specificity from Pseudomonas putida also
showed racemase activity with L-serine; however, the activ-
ity was much lower than those of basic and aliphatic
straight-chain amino acids such as L-arginine and L-gluta-
mine, respectively (Soda and Osumi, 1969). These results
show that the enzymatic properties of AtSR are apparently
different from those of microbial amino acid racemases,
except for the optimum pH at 8.0 to 8.5; bacterial alanine
racemases have maximum activity at alkaline pH (Esaki
et al., 2002).
3
2
1
2.4. Kinetic characterization of recombinant A. thaliana
serine racemase
0
0
0.001
0.01
0.1
1
The Km and Vmax values were estimated in the reaction
NH OH (mM)
2
mixture containing 1 mM CaCl , 10 lM PLP at pH 8.5.
2
Fig. 3. Absorption spectra of AtSR (A) and inhibition of AtSR by
hydroxylamine (B). A: The spectra of the purified AtSR (1 mg/ml) before
and after dialyzing against 500 volumes of 20 mM Tris–HCl (pH 7.5)
buffer containing 1 mM DTT, 10 lM PLP and 10 mM sodium borohy-
dride are shown as a solid line and a dashed line, respectively. B:
Racemase activity of the purified AtSR converting L- to D-serine was
assayed under the presence of different concentrations of hydroxylamine.
The results represent the average ± SE of three experiments.
The Km and Vmax values of racemase activity converting
L- to D-serine were 2.5 mM and 5.0 nmol/mg/min, and
those converting D- to L-serine were 0.77 mM and
1
.1 nmol/mg/min. The Vmax/Km values for the conversion
from L- to D-serine and from D- to L-serine were 2.0 and
.4 nmol/mg/min/mM. The Km and Vmax values of dehy-
dratase activity converting L-serine to pyruvate were
0 mM and 250 nmol/mg/min, and those eliminating D-ser-
1
2
ine to pyruvate were 5.0 mM and 26 nmol/mg/min. The
Vmax/Km values for L- and D-serine elimination were 13
and 5.3 nmol/mg/min/mM. The Km values for L-serine of
rat and mouse serine racemases are 9.8 and 30 mM, and
those for D-serine are 60 and 49 mM. The Vmax values
for L-serine of rat and mouse serine racemases are 83 and

672
Y. Fujitani et al. / Phytochemistry 67 (2006) 668–674
5
4
125
100
3
2
1
75
50
25
0
0
control EDTA MgCl2 CaCl2 MnCl2 FeCl2
NiCl2
ZnCl2
Fig. 4. Effect of divalent cations on racemase and dehydratase activities of AtSR. Racemase (black columns) and dehydratase (white columns) activities of
the purified AtSR were assayed under the presence of 1 mM metal chloride or 1 mM EDTA. The results represent the average ± SE of three experiments.
1
1
10 nmol/min/mg, and those for D-serine are 370 and
30 nmol/min/mg (Wolosker et al., 1999a; Strisovsky
et al., 2003). The K and V
values for both L- and D-ser-
m
max
ine of AtSR are thus one to two orders lower than those of
mammalian serine racemases, showing that AtSR has
higher affinity for L- and D-serine and lower activity than
mammalian serine racemases. When comparing the Vmax
/
K values for L- and D-serine of AtSR to those of mamma-
m
lian serine racemases, all enzymes indicate the same ratio of
the Vmax/Km for L- to D-serine, 1:0.7, which is consistent
with the theoretical value for the racemization reaction
kbp
2.0
1.5
(
Briggs and Haldane, 1925). On the dehydratase activity,
AtSR has higher affinity and activity than mouse serine
1
.0
racemase, of which the Km values for L- and D-serine are
7
5 and 75 mM and the Vmax values for L- and D-serine
0.5
are 77 and 4.3 nmol/min/mg (Strisovsky et al., 2003). The
Vmax values for L-serine of both enzymes are one order
higher than those for D-serine, showing that AtSR and
mouse serine racemase have the specificity of dehydration
toward L-serine. The ratios of the Vmax/Km for L- to D-ser-
ine of AtSR and rat serine racemase, 1:0.4 and 1:0.06, sug-
gest that AtSR has lower specificity to enantiomer than rat
serine racemase on the dehydratase activity.
Fig. 5. Expression profile of AtSR gene in various organs of A. thaliana.
Three microgram of each cDNA sample prepared from the shoots, roots,
rosette, and inflorescence were electrophoresed on 1.2% agarose gel and
transferred to a nylon membrane. The membrane was hybridized with a
fragment of AtSR cDNA.
2
.5. Localization of A. thaliana serine racemase
The expression level of the AtSR gene in the plant parts
3. Experimental
was analyzed by hybridization with the cDNA of AtSR as
a probe (Fig. 5). Our blot detected a single 1.1-kb band in
the shoots, roots, rosette, and inflorescence of A. thaliana.
A number of D-amino acids have been identified in plants,
however, plant biosynthetic mechanisms for D-amino acids
have not been found. We have thus elucidated that A. tha-
liana has a serine racemase and that its gene is expressed in
various organs, implying that plant cells require D-amino
acids or D-amino acid-containing peptides as the physiolog-
ically functional component and have a D-amino acid met-
abolic pathway.
3.1. Reagents
Ultrapure L-serine was purchased from Fluka and D-ser-
ine, L-alanine, L-arginine, and L-glutamine were from
Sigma. Pyridoxal 5 -phosphate (PLP), flavin adenine
dinucleotide (FAD), o-phenylenediamine (OPA), 2,4-dini-
trophenylhydrazine (2,4-DNP), pyruvate, and hydroxyl-
amine were obtained from Nacalai Tesque (Japan).
D-Amino acid oxidase from porcine kidney, L-amino acid
oxidase from Crotalus adamanteus, peroxidase from
0

Y. Fujitani et al. / Phytochemistry 67 (2006) 668–674
673
horseradish, and catalase from bovine liver were obtained
from Sigma. Other reagents were of analytical grade.
0.15 M NaCl and 10 lM PLP. The protein concentration
was quantified according to the method of Bradford
(1976) with bovine serum albumin as the standard.
3
.2. Plant material
3.5. Enzyme assays
Plants of Arabidopsis thaliana, ecotype Columbia, were
grown in a growth chamber in a light/dark cycle of 16 h/
Serine racemase activity was assayed according to the
8
h at 23 ꢁC.
method described by Cook et al. (2002) with some modifi-
cation. The activity in the direction from L- to D-serine was
determined with a reaction mixture (500 ll) containing
3
.3. Cloning of A. thaliana serine racemase gene
Based on the putative open reading frame of the A. tha-
20 mM Tris–HCl (pH 8.5), 10 lM PLP, 10 mM L-serine,
1 mM CaCl , 1 nM FAD, 50 lg/ml OPA, 3 U/ml D-amino
2
liana serine racemase gene (GenBank Accession Nos.
acid oxidase, 3 U/ml peroxide, and enzyme. The reaction
was incubated at 37 ꢁC for 1 h and absorbance at 411 nm
was measured to calculate the amount of D-serine. D-Serine
and L-amino acid oxidase instead of L-serine and D-serine
oxidase were used to determine the activity in the direction
from D- to L-serine. The calibration curve was made with 0
to 100 lM L- and D-serine showing a linear range.
Serine dehydratase activity was assayed according to the
method described by Uo et al. (2002). The reaction mixture
(500 ll) consisted of 20 mM Tris–HCl (pH 8.5), 10 lM
AL161532 and AL049500), two primers were designed as
0
follows: sense primer, 5 -CCCATATGGAAGCAAATA-
0
GAGAGAAGTA-3 , which creates a NdeI site (indicated
0
by an underline), and antisense primer, 5 -CCCTC-
0
GAGTTTTGAACTCTTAAATGAAT-3 , which creates
an XhoI site (indicated by an underline). The open reading
frame was amplified by reverse-transcription PCR (RT-
PCR) using the OneStep RT-PCR kit (Qiagen) with total
RNA from 2-week-old shoots of A. thaliana. The PCR
product of 1008 bp was cloned into a pGEM-T vector
PLP, 10 mM L- or D-serine, 1 mM CaCl , and the enzyme
2
(
Promega) and subjected to sequencing using an ABI
was incubated at 37 ꢁC for 30 min and stopped by addition
of 2 M HCl (0.5 ml) containing 0.03% (w/v) 2,4-DNP.
After incubation at 4 ꢁC for 5 min, 2 M NaOH (1 ml)
was added and absorbance at 520 nm was measured to cal-
culate the amount of pyruvate. The calibration curve was
made with 0–500 lM pyruvate showing a linear range.
L-Serine solution used in the enzyme assay was pre-trea-
ted with D-amino acid oxidase and catalase in order to
remove contaminating D-serine according to the method
described by De Miranda et al. (2002). L-Amino acid oxi-
dase was used to remove contaminating L-serine in D-serine
solution.
PRISM 310 DNA sequencer (Applied Biosystems). The
NdeI- and XhoI-digested fragment of the plasmid was cut
out and subcloned into a NdeI- and XhoI-digested
pET20b(+) vector (Novagen), in which the endogenous
stop codon of the serine racemase gene was substituted
by a polyhistidine tag gene. The resulting the plasmid,
pAtSR, was transformed into E. coli BL21(DE3) pLysS
cells.
3
.4. Expression and purification of serine racemase
E. coli cells harboring pAtSR were grown at 25 ꢁC in an
LB medium containing 50 lg/ml of ampicillin. When
OD600 became 0.5, isopropyl-b-D-thiogalactopyranoside
3.6. cDNA preparation and hybridization
(
IPTG) was added to the culture at a final concentration
cDNA was synthesized and amplified using the SMART
PCR cDNA Synthesis Kit (Clontech). Total RNA isolated
from shoots (2-week-old), roots, rosette, and inflorescence
(6–8-week-old) was reverse-transcribed at 42 ꢁC for 1 h
with the cDNA synthesis primer and the SMART II oligo-
nucleotide. The reaction mixture was amplified with the
polymerase chain reaction primer supplied in the kit
according to the manufacturer’s instruction. Three micro-
gram of cDNA was electrophoresed on 1.2% agarose gel
of 0.5 mM. After cultivation at 25 ꢁC for 6 h, cells were
harvested by centrifugation and frozen at ꢁ80 ꢁC at least
for 2 h. The frozen cell pellet was suspended in a Bug-
Busterꢂ HT protein extraction reagent (Novagen) accord-
ing to the manufacturer’s instruction and the soluble
protein solution was purified using a Ni-NTA column
(
7
Qiagen) equilibrated with 20 mM Tris–HCl buffer (pH
.9) containing 0.5 M NaCl and 5 mM imidazole. The col-
+
umn was washed with the same buffer followed by 60 mM
imidazole in the same buffer, and the absorbed protein was
eluted with 1 M imidazole in the same buffer. The enzyme
solution was collected, dialyzed against 20 mM Tris–HCl
buffer (pH 7.5) containing 1 mM DTT and 10 lM PLP,
and concentrated by Ultracent-30 (Tohso, Japan). The pur-
ity and the molecular mass of the enzyme were estimated
by SDS–PAGE and gel filtration with a Superdex 200
HR column (Amersham Biosciences) equilibrated with
and transferred onto Hybond-N nylon membrane (Amer-
sham Biosciences). The membrane was hybridized with a
fragment of the AtSR cDNA labeled with digoxigenin-
11-dUTP using a PCR DIG Labeling Kit (Boehringer
0
Mannheim) with primers 5 -TCAGGTATGGTGGTAA-
0
0
GGTTATATGGA-3 and 5 -TCCACTTGGTTCCACA-
0
GAGACCTTCA G-3 . Hybridization was performed at
42 ꢁC for 16 h in 50% formamide, 5· sodium saline citrate
(SSC), 0.25% non-fat milk powder, 0.1% sodium dodecyl
sulfate (SDS), 5· Denhardt’s reagent. The membrane was
5
0 mM sodium phosphate buffer (pH 7.0) containing

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Y. Fujitani et al. / Phytochemistry 67 (2006) 668–674
washed twice with 1· SSC and 0.1% SDS at room temper-
ature, and twice with 0.1· SSC and 0.1% SDS at 68 ꢁC.
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,
3
3–36.
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Acknowledgment
This research was supported in part by the Ohara Foun-
dation in Kurashiki.
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