Báo cáo khoa học: Cloning and functional characterization of Arabidopsis thaliana D-amino acid aminotransferase – D-aspartate behavior during germination pdf

Cloning and functional characterization of
Arabidopsis thaliana
D-amino acid aminotransferase –
D-aspartate behavior during germination
Miya Funakoshi
1,
*, Masae Sekine
1,
*, Masumi Katane
1
, Takemitsu Furuchi
1
, Masafumi Yohda
2
,
Takafumi Yoshikawa
1
and Hiroshi Homma
1
1 School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan
2 Tokyo University of Agriculture and Technology, Japan
All protein amino acids, with the exception of Gly,
have two optical isomers: the l-form and the d-form.
It has long been believed that only l-amino acids are
present in the mammalian body, and that d-amino
acids are unnatural or represent laboratory artefacts.
However, recent investigations have revealed that a
variety of d-amino acids are present in mammals in free
form or in proteins, and their biological functions are
being clarified [1]. Among the d-amino acids examined
in mammals, d-Ser and d-Asp are abundant [2–5].

d-Ser is present at high concentrations, especially in the
mammalian forebrain, throughout the lifespan of the
animal. This amino acid binds to the Gly-binding site
of the N-methyl-d-aspartate subtype of the Glu recep-
tor in the brain, and potentiates glutamatergic neuro-
transmission [2,3]. d-Ser is considered to be an intrinsic
coagonist of the N-methyl-d-aspartate receptor in the
mammalian brain. Serine racemase, which synthesizes
d-Ser from the l-isomer, has been cloned and
characterized [6]. Interestingly, it has been suggested
that d-Ser-degrading enzyme, d-amino acid oxidase
and its potential regulator G72 are associated with
schizophrenia [7]. In a recent study, an association was
suggested between this disease and PICK1, a protein
interactor of serine racemase [8]. These studies indicate
a possible role and involvement of d-Ser in the disease.
In addition to d-Ser, widespread and transient
occurrences of d-Asp have been reported in various
Keywords
A. thaliana;
D-amino acid aminotransferase;
D-alanine; D-aspartate; D-glutamate
Correspondence
H. Homma, School of Pharmaceutical
Sciences, Kitasato University, 5-9-1
Shirokane, Minato-ku, Tokyo 108-8641,
Japan
Fax: +81 3 5791 6381
Tel: +81 3 5791 6229
E-mail:

*These authors contributed equally to this
work
(Received 21 September 2007, revised 25
December 2007, accepted 8 January 2008)
doi:10.1111/j.1742-4658.2008.06279.x
The understanding of d-amino acid metabolism in higher plants lags far
behind that in mammals, for which the biological functions of these unique
amino acids have already been elucidated. In this article, we report on the
biochemical behavior of d-amino acids (particularly d-Asp) and relevant
metabolic enzymes in Arabidopsis thaliana. During germination and growth
of the plant, a transient increase in d-Asp levels was observed, suggesting
that d-Asp is synthesized in the plant. Administration of d-Asp suppressed
growth, although the inhibitory mechanism responsible for this remains to
be clarified. Exogenous d-Asp was efficiently incorporated and metabo-
lized, and was converted to other d-amino acids (d-Glu and d-Ala). We
then studied the related metabolic enzymes, and consequently cloned and
characterized A. thaliana d-amino acid aminotransferase, which is presum-
ably involved in the metabolism of d-Asp in the plant by catalyzing trans-
amination between d-amino acids. This is the first report of cDNA cloning
and functional characterization of a d-amino acid aminotransferase in
eukaryotes. The results presented here provide important information for
understanding the significance of d-amino acids in the metabolism of
higher plants.
Abbreviations
AspAT, aspartate aminotransferase; AT, aminotransferase; BCAT, branched chain amino acid aminotransferase;
D-AAT, D-amino acid
aminotransferase; GST, glutathione S-transferase; MS, Murashige and Skoog; PLP, pyridoxal 5¢-phosphate.
1188 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS
mammalian tissues. d-Asp appears to affect the func-
tions of neuroendocrine and endocrine tissues. d-Asp

suppresses melatonin release in the pineal gland
[9,10], stimulates prolactin secretion in the anterior
pituitary gland [11,12], modulates oxytocin and ⁄ or
vasopressin synthesis in the posterior pituitary gland
[13,14], and stimulates testosterone production in the
testis [15], by stimulating expression of the gene
encoding steroidogenic acute regulatory protein in
Leydig cells [16]. Recently, mutant mice with tar-
geted deletion of the gene for d-Asp oxidase were
reported [17,18]; this enzyme selectively catalyzes the
oxidative degradation of acidic d-amino acids. In
d-Asp oxidase-deficient mice, d-Asp levels are signifi-
cantly increased in numerous tissues. The mutant
mice displayed impaired sexual performance and
behavioral alterations, potentially reflecting dimin-
ished synthesis and levels of pituitary hormones.
Thus, the physiological functions of d-Asp have been
identified, but its precise synthetic pathway(s)
remains to be discovered.
In contrast to the depth of understanding of
d-amino acids in mammalian physiology, the impor-
tance of d-amino acids in the biological function of
higher plants remains unknown. To investigate the
physiological significance of d-amino acids and their
relevant metabolic enzymes in higher plants, we
selected Arabidopsis thaliana as a plant model [19].
We showed a transient increase in A. thaliana d-Asp
levels during germination and growth, suggesting that
d-Asp is synthesized in the plant. In addition, we
examined the metabolism of exogenously administered

d-Asp, and found that it was taken up and, in part,
metabolically converted to other d-amino acids
(d-Glu and d-Ala). Finally, we isolated a functional
d-amino acid aminotransferase (d-AAT) A. thaliana
clone, an enzyme potentially responsible for the
metabolism of d-Asp and concomitant appearance of
other d-amino acids. This is the first report, for
eukaryotes, of cDNA cloning and functional charac-
terization of d-AAT.
Results
Growth suppression of A. thaliana in Murashige
and Skoog (MS) medium containing
D-Asp
(MS +
D-Asp)
Germination and growth of A. thaliana was observed
in MS medium, MS medium containing l-Asp
(MS + l-Asp) and MS + d-Asp, as shown in
Fig. 1. In MS and MS + l-Asp media, significant
plant growth was observed (Fig. 1A,B). The elonga-
tion rate of main roots and hypocotyls appeared to
be greater in plants cultured in MS medium than in
those cultured in MS + l-Asp medium. It is interest-
ing to note that growth in MS + d-Asp medium
was significantly suppressed (Fig. 1C). Figure 2 shows
C
B
A
Fig. 1. Growth of A. thaliana in MS medium, MS + L-Asp medium
and MS +

D-Asp medium. After A. thaliana seeds were sown on
culture plates, seedlings were grown for 14 days, as described in
Experimental procedures. (A) MS medium. (B) MS medium contain-
ing 10 m
ML-Asp (MS + L-Asp). (C) MS medium containing 10 mM
D
-Asp (MS + D-Asp).
M. Funakoshi et al.
D-Amino acid aminotransferase in A. thaliana
FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1189
the inhibitory, dose-dependent effect of d-Asp on
cotyledon length. Elongation of the main root was
also diminished in MS + d-Asp medium, and the
underside of cotyledons appeared purple (Fig. 1C). It
is notable that growth in MS + d-Asp medium
appeared to be partially restored after approximately
14 days of culture.
Amino acid content in A. thaliana cultured in MS,
MS +
L-Asp and MS + D-Asp media
d-Asp and l-Asp content was determined in whole
plant homogenates after culturing in MS, MS +
l-Asp and MS + d-Asp media (Fig. 3). In homoge-
nate prepared from MS-cultured plants, d-Asp levels
transiently increased, with the highest level being
observed after 11 days of culture (Fig. 3A). The ratio
of d-Asp to l-Asp [D% = (D ⁄ D+L)· 100] was
0.43% and 0.35% at 4 days and 11 days of culture,
respectively (Fig. 3A). The high percentage of d-Asp
observed in the 4 day culture was attributed to a cor-

responding low l-Asp content for the same time
point (Fig. 3B). At 7 days of culture the l-Asp con-
tent was markedly increased, and it remained high
until 21 days of culture (Fig. 3B). d-Glu and l-Glu
content remained high and showed negligible change
during culture (Fig. 3B). It is interesting to note that
the level of d-Asp also transiently increased at early
stages of culture (approximately 14 days) in the gel-
rite medium to which only CaCl
2
and sucrose were
added (data not shown). In gelrite medium-cultured
plants, germination and main root elongation were
observed; however, growth was severely restricted
and no green seed leaves (cotyledons) formed.
Concentrations of d-Asp in these plants reached a
maximum value of approximately 0.4 nmolÆmg
)1
protein, which is similar to that of plants grown in
MS medium (Fig. 3A). As d-Asp was not supple-
mented in gelrite or MS medium during the culture,
these results suggest that d-Asp is actually synthesized
and retained in the plant, although its level is low.
d-Asp presumably plays some as yet unknown physi-
ological role(s) in the plant, especially in the early
stages of germination.
In MS + l-Asp medium cultures, l-Asp content
was significantly higher than that in MS medium cul-
tures (Fig. 3D), suggesting that l-Asp in the medium
was efficiently taken up by the plant. d-Asp content

was also high at the early stages of culture (4 days of
culture; Fig. 3C). This is presumably due to uptake
of d-Asp that was inevitably present in the l-Asp
preparation used to supplement the medium, as
d-Asp is supposed to be effectively taken up into the
plant as described below. In MS + d-Asp medium,
d-Asp levels were significantly high, because exoge-
nous d-Asp is taken up efficiently into the plant
(Fig. 3E). Interestingly, d -Asp levels in the plant
decreased considerably at advanced stages of culture
(14 days of culture; Fig. 3E), suggesting that d-Asp is
efficiently metabolized in the plant. This result is con-
sistent with the observation described above that
growth of plants cultured in MS + d-Asp medium
was partially (not fully) restored after 14 days of cul-
ture. It is postulated that d-Asp suppressed growth
(Figs 1 and 2), and that catabolism of d-Asp partially
restored growth after 14 days of culture. l-Asp, d-Glu
and l -Glu contents were shown to increase up to
21 days of culture (Fig. 3F).
D-Amino acid content in A. thaliana cultured in
MS +
D-Asp medium
Taken together, the results described above suggested
that d-Asp is endogenously synthesized and retained
in the plant, and that exogenous d-Asp is efficiently
taken up and metabolized in the plant. Therefore,
the contents of other d-amino acids metabolically
related to d-Asp, in particular d-Glu and d-Ala,
were determined. Plants cultured in MS medium, in

the absence of d-Asp, showed no detectable levels of
d-Glu or d-Ala (data not shown). In contrast, d-Glu
20151050
0
1
2
Length (mm)
3
4
5
D-Asp concentration (mM)
14 days
11 days
*
**
**
***
***
*
***
***
***
***
***
Fig. 2. Effect of D-Asp on cotyledon growth during culture of
A. thaliana. The lengths of cotyledons were determined in seed-
lings after 11 days and 14 days of culture on MS medium contain-
ing
D-Asp. As indicated on the abscissa, various concentrations
of

D-Asp were included in the MS medium. Data represent the
mean ± SD (n = 3–5). *P < 0.05, **P < 0.01, ***P < 0.001 (by
Student’s t-test).
D-Amino acid aminotransferase in A. thaliana M. Funakoshi et al.
1190 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS
and d-Ala were detected in plants cultured in
MS +d-Asp medium as early as 4 days of culture
(Fig. 4); they are presumably synthesized during
metabolism of exogenous d-Asp. The presence of
d-Glu and d-Ala is expected even in plants cultured
without d-Asp supplementation; however, they would
exist in quantities below the limit of detection, as
the concentration of endogenous d-Asp is very low
and those of other d-amino acids are even lower.
Plants cultured in MS + d-Asp medium showed
nearly constant levels of d-Glu, approximately
30 nmol Æ mg
)1
protein, up to 21 days of culture
(Fig. 4A). However, l-Glu levels markedly increased
after 14 days of culture (Fig. 4A), whereas d-Asp lev-
els decreased considerably (Fig. 3E). l-Asp levels were
shown to increase thereafter (Fig. 3F). Surprisingly,
levels of d-Ala were 3.6–4.6-fold higher than l-Ala lev-
els up to 9 days of culture (Fig. 4B). It is interesting
0
0.15
0.3
0.45
4 7 11 14 16 21

0.15
0.3
0.45
Days
4 7 11 14 16 21
Days
4 7 16 21
Days
47991621
Days
4 7 9 141621
Days
479141621
Days
D-Asp (nmol·mg protein
–1
)D-Asp (nmol·mg protein
–1
)D-Asp (nmol·mg protein
–1
)
L-Asp (nmol·mg protein
–1
)
D (= D /D+L) %D (= D /D+L) %
D (= D /D+L) %
L-Asp (nmol·mg protein
–1
)L-Asp (nmol·mg protein
–1

)
Glu (nmol·mg protein
–1
)Glu (nmol·mg protein
–1
)Glu (nmol·mg protein
–1
)
A
0
0
30
60
90
120
150
0
100
200
300
B
0.5
1.5
0
0.4
0.8
1.2
0
1
C

0
100
200
300
0
50
100
150
200
D
0
200
400
600
800
0
30
60
90
120
E
0
20
40
60
80
100
0
100
200

300
400
500
F
Fig. 3. Amino acid content in A. thaliana cultured in MS, MS + L-Asp and MS + D-Asp media. A. thaliana was cultured in MS medium, MS
medium containing 10 m
ML-Asp (MS + L-Asp), and MS medium containing 10 mMD-Asp (MS + D-Asp), and seedlings were collected at
various time points. Whole plant homogenates were prepared, and
D-Asp, L-Asp and Glu contents were determined as described in Experi-
mental procedures. (A, B) MS medium. (C, D) MS +
L-Asp medium. (E, F) MS + D-Asp medium. Two separate experiments were carried out
independently, where at least two determinations were performed for each time point, and essentially similar results were obtained. The
data shown in this figure represent the results obtained in an experiment.
M. Funakoshi et al.
D-Amino acid aminotransferase in A. thaliana
FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1191
that d-Ala levels markedly decreased at 14 days of
culture (Fig. 4B) in a manner similar to the decrease in
d-Asp levels, as depicted in Fig. 3E. These results indi-
cate that exogenous d-Asp is metabolized in the plant,
and that the appearance of d -Glu and d-Ala is corre-
lated with the metabolism of d-Asp.
Enzymes potentially responsible for d-Asp metabo-
lism in A. thaliana are amino acid aminotransferase
(AT), racemase and ⁄ or dehydrogenase. Among these
enzymes, AT catalyzes transamination of amino acids
into keto acids, which are in turn converted to other
amino acids. The concomitant changes in d-Glu, d-Ala
and d-Asp levels suggested involvement of AT(s).
Thus, we investigated various AT clones, i.e. several

clones of Asp ATs (AspATs), branched chain amino
acid ATs (BCATs) and a putative d-AAT. Their sub-
strate specificities, particularly for d-amino acids, were
characterized.
Gene cloning and functional characterization of
amino acid AT recombinant proteins from
A. thaliana
AspAT
Six A. thaliana AspAT clones (Atasp1–5 and prokary-
otic-type AspAT [20]) have been characterized to date.
However, to our knowledge, enantioselectivity for
amino acid substrates (i.e. comparison of activity for
d-Asp and l-Asp) has not yet been reported in detail.
In this work, we characterized three AT clones
(Atasp1, Atasp 3 and Atasp5) that were available as
full-length cDNA clones from RIKEN BioResource
Center, Tsukuba, Japan.
Recombinant AspAT 1, AspAT 3 and AspAT 5
were expressed in Escherichia coli cells and detected in
the crude extract by western blotting. Their apparent
molecular masses were in good agreement with those
calculated from their deduced amino acid sequences
(data not shown). These AT preparations, purified as
described in Experimental procedures, demonstrated
considerable activity when l-Asp and a-ketoglutarate
were used as an amino donor and an amino acceptor,
respectively. Kinetic parameters of enzyme activity
(K
m
values for l-Asp) were determined by Line-

weaver–Burk plots: AspAT 1, K
m
(l-Asp) 1.0 mm;
AspAT 3, K
m
(l-Asp) 2.5 mm; and AspAT 5, K
m
(l-Asp) 1.0 mm. These K
m
(l-Asp) values are compara-
ble with those previously reported (3.0 mm, AspAT 1;
1.4 mm, AspAT 2; and 2.9 mm, AspAT 5 [21]). How-
ever, none of the ATs exhibited activity for d-Asp or
d-Ala as amino donors.
BCAT
BCATs and d-AATs of bacterial origin show signifi-
cant similarity in their primary and tertiary structure,
and are classified as a subgroup of ATs [22] or as a
distinct fold-type family (type IV) of pyridoxal 5¢-phos-
phate (PLP)-dependent enzymes [23]. They are also
similar in stereospecificity for hydrogen transfer in
enzymatic transamination, which is a feature distinct
from other ATs [24]. Arabidopsis thaliana BCATs may
utilize d-amino acids as substrates; thus, we were inter-
ested in investigating their substrate specificity for
d-amino acids. Six A. thaliana BCAT clones have been
characterized so far, and other putative clones have
been predicted [25]. Among them, BCAT 2 and
BCAT 4 were investigated in this work.
Recombinant BCAT 2 and BCAT 4 were expressed

in E. coli cells and detected in the crude extract by
western blotting. Their apparent molecular masses
were in good agreement with those calculated from
150
100
50
0
500
400
300
200
100
0
Days
150
100
50
0
A
B
D
-Ala
L
-Ala
D
-Glu
L
-Glu
D-Glu (nmol·mg protein
–1

)
L-Glu (nmol·mg protein
–1
)
D,L-Ala (nmol·mg protein
–1
)
4 7 9 14 16 21
Days
4 7 9 14 16 21
Fig. 4. D- and L-Glu and D-Ala and L-Ala content in A. thaliana cul-
tured in MS +
D-Asp medium. A. thaliana was cultured in MS med-
ium containing 10 m
MD-Asp (MS + D-Asp), and seedlings were
collected at various time points. (A)
D-Glu and L-Glu and (B) D-Ala
and
L-Ala contents were determined as described in Experimental
procedures. Two separate experiments were carried out indepen-
dently, where at least two determinations were performed for each
time point, and essentially similar results were obtained. The data
shown represent the results obtained in an experiment.
D-Amino acid aminotransferase in A. thaliana M. Funakoshi et al.
1192 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS
their deduced amino acid sequences (data not shown).
These BCAT preparations, purified as described in
Experimental procedures, showed significant activity
for l-Leu, l-Ile and l-Val as amino donors and a-keto-
glutarate as an amino acceptor. Kinetic parameters of

the activities were determined to be as follows:
BCAT 2, K
m
(l-Leu) 0.71 mm; and BCAT 4, K
m
(l-Leu) 1.61 mm. However, these BCATs did not exhi-
bit activity for l-isomers of Asp and Ala. Furthermore,
these BCATs exhibited no activity for d-isomers of
Leu, Ile, or Val, or for d-isomers of Asp, Glu, or Ala.
D-AAT
An uncharacterized A. thaliana AT clone was identified
that showed sequence similarity to d-AATs of bacterial
origin [25]. The recombinant protein of this clone was
expressed in E. coli cells and detected in the crude
extract by western blotting (Fig. 5). Its apparent
molecular mass was in good agreement with that cal-
culated from its deduced amino acid sequence. The
d-AAT preparation, purified as described in Experi-
mental procedures, exhibited considerable AT activity
for d-Asp and d-Ala as amino donors with a-ketoglu-
tarate as an amino acceptor, and significant levels of
d-Glu were observed. d-AAT activity was not detected
for l-Asp, l-Ala, l-Leu, l-Ile, or l-Val. The reverse
transaminations were also observed, where an amino
group was transferred from d-Glu to pyruvate or oxa-
loacetate to produce d-Ala or d-Asp, respectively.
Kinetic parameters for these activities were determined,
and are shown in Table 1. In the transamination con-
version of amino acid to a-ketoglutarate, K
m

and V
max
values for d-Asp and d-Ala were 2.3 and 1 mm, and
2.5 and 5.0 lmolÆmin
)1
Æmg
)1
protein (Table 1); there-
fore, d-Ala is a more efficient d-AAT substrate than
d-Asp. In addition, the K
m
and V
max
values for d-Glu
in the transamination reaction to pyruvate to produce
d-Ala were 4 mm and 3.3 lmolÆmin
)1
Æmg
)1
protein,
respectively (Table 1). Therefore the affinity for d-Ala
is higher than that for d-Glu, and V
max
is higher for
d-Ala than for d-Glu, indicating that d-Glu predomi-
nates in the transamination between d-Ala and d-Glu.
When d-Ala (as an amino donor) and oxaloacetate or
a-ketoglutarate (as an amino acceptors) were used in
the enzyme assay, the production of d-Asp was
approximately 1.73% that of d-Glu, indicating that

a-ketoglutarate is a preferred amino acceptor as
compared to oxaloacetate.
The substrate specificity for d-amino acids as amino
donors was subsequently studied with a-ketoglutarate
as an amino acceptor. d-AAT shows the greatest sub-
strate affinity for d-Ala; however, other d-amino acids
can serve as amino donors, including d-Met, d-Tyr,
d-Phe, d-Gln, d-Trp and d-Asn (Fig. 6). This indicates
that A. thaliana d-AAT exhibits broad substrate speci-
ficity, which has been demonstrated in other character-
ized bacterial d-AATs [26–29]. When the enzyme assay
was performed in the absence of PLP, activity was
Fig. 5. Western blotting of recombinant A. thaliana D-AAT
expressed in E. coli cells. The expression of recombinant A. thali-
ana
D-AAT was examined by western blotting of the crude extract
of E. coli cells using anti-GST serum. The crude extracts (0.4 lg
each) were prepared from E. coli cells harboring empty plasmid (1)
and the
D-AAT expression plasmid (2). Details are as in Experimen-
tal procedures. Figures on the left side represent molecular masses
of marker proteins. The arrowhead indicates recombinant A. thali-
ana
D-AAT.
Table 1. Apparent kinetic parameters of the recombinant A. thaliana D-AAT. The A. thaliana D-AAT (15.6 lg of protein) was assayed as
described in Experimental procedures. Two separate determinations were carried out for each parameter, and similar values were calculated.
The data shown represent the results obtained in an experiment.
Aminotransfer reaction
K
m

(mM)
V
max
(lmolÆmin
)1
Æmg
)1
)D-Asp D-Ala a-Ketoglutarate D-Glu
D-Asp fi a-ketoglutarate 2.3 2.5
D-Ala fi a-ketoglutarate 1.0 27 5
D-Glu fi pyruvate 4.0 3.3
M. Funakoshi et al.
D-Amino acid aminotransferase in A. thaliana
FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1193
approximately 65% of that in the presence of PLP.
However, the addition of 1 mm hydroxylamine or
10 mm amino-oxyacetic acid completely abolished
activity, suggesting that A. thaliana d-AAT is a
PLP-dependent enzyme. A. thaliana d-AAT showed
23.8%, 26.2% and 19.6% sequence homology with
Bacillus sp. YM-1, Bacillus subtilis and Bacillus sphae-
ricus d-AATs, respectively. Figure 7 shows the amino
acid sequence alignment of d-AATs from A. thaliana
and Bacillus sp. YM-1, for which the three-dimen-
sional crystal structure has been determined [30,31]. It
is noteworthy that a chloroplast-targeting signal is
present in the A. thaliana d-AAT.
Discussion
D-Amino acids in A. thaliana
Free d-amino acids and conjugated forms of d-amino

acids have been detected in higher plants. In the 1960s,
N-malonyl-d-Trp was found in pea seedlings [32], and
other conjugated d-amino acids have since been
detected, such as N-malonyl-d-Ala and c-glutamyl-
d-Ala [33,34]. Free-form d-amino acids have also been
reported: d-Ala, d-Asp, d-Glu [35], and others [36,37].
These d-amino acids are presumably of endogenous
and exogenous origin. In A. thaliana, d-Asp levels
transiently increased during germination and growth
when the plant was cultured in the absence of d-Asp
Fig. 7. Alignment of the deduced amino acid sequences of A. thaliana D-AAT and Bacillus sp. YM-1 D-AAT. Amino acid residues identical in
the two sequences are shown as white letters on black background, and conserved amino acid residues with high and low similarity are indi-
cated by double dots and single dots, respectively. Amino acid residues plausibly involved in the binding of coenzyme (PLP) are conserved
or equivalent in these two sequences, and are indicated by closed triangles (conserved) or an open triangle (equivalent). Details are
described in the Discussion. A putative chloroplast-targeting signal sequence, predicted by
PSORT, is underlined.
0 20406080100
120
Relative activity (%)
D-Ala
D-Asp
D-Gln
D-Asn
D-Cys
D-Met
D-Thr
D-Ser
D-His
D-Arg
D-Lys

D-Leu
D-Val
D-Tyr
D-Phe
D-Trp
D-Pro
Fig. 6. D-Amino acid substrate specificity for transamination to
a-ketoglutarate by A. thaliana
D-AAT. Recombinant D-AAT (7 lgof
protein) was incubated with 1.5 m
M various D-amino acids, 50 mM
a-ketoglutarate, and 50 lM PLP, and the amounts of D-Glu pro-
duced were determined by HPLC as described in Experimental pro-
cedures. The data presented in this figure are average ± half range
from two separate experiments, and shown as values relative to
that of
D-Ala.
D-Amino acid aminotransferase in A. thaliana M. Funakoshi et al.
1194 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS
(Fig. 3A); therefore, it was presumably synthesized
within the plant. d -Amino acids in higher plants may
originate as a product of racemase activity. In pea
seedlings, trace analysis of double-isotopically labeled
d-Ala suggested a direct conversion of l-Ala to d-Ala
via a racemase reaction. In in vitro analysis, enzymatic
activity of racemase synthesizing d-Ala from l-Ala
was detected [38]. Other racemase activities acting on
Trp have also been reported [39,40]. Recently, alanine
racemase was purified from alfalfa seedlings [41], and a
clone encoding serine racemase has been isolated [42].

d-Asp may be synthesized by a racemase(s) in A. thali-
ana, although an Asp-specific racemase has not yet
been identified in higher plants.
It was reported that administration of radiolabeled
d-Ala (1-
14
C) in ryegrass root gave rise to labeled Val,
suggesting a metabolic conversion of d-amino acids
through transamination between d-amino acids; how-
ever, the configuration of the labeled Val was not
determined [43]. Different types of partially purified
and characterized d-AATs have been shown to trans-
fer amino groups from various d-amino acids to keto
acids in a process that forms other d-amino acids
[44,45]. d-Trp-specific ATs were partially purified
[46,47], and are presumed to be involved in the synthe-
sis of indole-3-acetic acid, a plant hormone (see
below). These ATs show stereospecificity and ⁄ or much
higher activity for d-enantiomers, and are apparently
PLP-dependent. In the current study, stereospecific
d-AAT from A. thaliana was cloned and characterized
for the first time. A. thaliana d-AAT is PLP-depen-
dent, which is consistent with previous reports, as
described above. As this enzyme appears to catalyze
transamination from various d-amino acids to oxalo-
acetate to produce d-Asp, albeit the activity is low,
endogenous d-Asp may be synthesized in A. thaliana
by the combination of a racemase(s) that produces
d-amino acid(s) other than d-Asp from l-isomer(s),
and d-AAT, which subsequently transfers an amino

group to oxaloacetate to synthesize d-Asp. This syn-
thetic pathway may produce endogenous d-Asp in
chloroplasts, where d -AAT is predicted to be localized
(Fig. 7). As shown in Fig. 3A, d-Asp levels increase
transiently during plant germination. Therefore, the
spatiotemporal localization of endogenous d-Asp and
d-AAT in A. thaliana warrants further investigation.
d-Amino acids are thought to be present in soil sys-
tem where higher plants grow, as a variety of bacteria
in the soil, symbiotic root bacteria and plants them-
selves [37,48] represent abundant sources of free and
conjugated forms of d-amino acids, including peptido-
glycan, from which free d-amino acids can be gener-
ated by hydrolysis. On the basis of our studies, we
posit that higher plants are capable of utilizing
d-amino acids from the soil. It was demonstrated that
exogenous d-Asp is efficiently taken up by A. thaliana
and metabolized, leading in part to the production of
other d-amino acids, namely d-Glu and d-Ala. Exoge-
nous d-amino acids in higher plants are presumably
subject to racemization, transamination and ⁄
or mal-
onylation, as well as deamination and decarboxylation
[43,49,50]. It was proposed that exogenous d-Trp is
metabolized to indolepyruvate by stereospecific d-Trp
AT (see above), and that this is followed by decarbox-
ylation and oxidation to form indole-3-acetic acid
[39,47]. The A. thaliana d-AAT studied in this report
demonstrates broad substrate specificity, with d-Glu
and d-Ala being high-affinity substrates. The various

d-amino acids found in most plants [37] may be
formed by the activity of homologous d-AATs that
metabolize exogenous (and endogenous) d-amino acids
to generate other d-amino acids. However, determina-
tion of the localization and physiological function of
these other d-amino acids requires further research.
As shown in Figs 1 and 2, culturing plants in med-
ium containing d-Asp suppressed the growth of A. tha-
liana. d-Trp has a growth-promoting effect on the
higher plants [39,47], and biological activities in plants
have been reported for several other d-amino acids,
including inhibition of salt uptake, growth inhibition
[51,52], chlorosis, promotion of abscission, and stimu-
lation of ethylene production [51,53]. However, the
details of these effects and their underlying mechanism
are not yet understood.
Plant
D-AAT
ATs constitute the AT superfamily, where AspAT
belongs to subgroup I and BCAT and d-AAT belong
to subgroup III [22]. The latter two ATs are classified
in the fold-type IV family of the PLP-dependent
enzyme superfamily [23]. BCAT and d-AAT are simi-
lar in their stereospecificity for hydrogen transfer of
the coenzyme [24]. AspATs from A. thaliana (Atasp1,
Atasp3 and Atasp5) are stereospecific for l-Asp, and
therefore AspATs are presumably not involved in the
metabolism of d-Asp in plants. A. thaliana BCAT
(Atbcat2 and Atbcat4) and d-AAT are apparently dis-
tinct in their substrate specificity. The BCATs studied

in this work act only on l-isomers of branched amino
acids, and not on d-amino acids, whereas d-AAT is
stereospecific for d-enantiomers, acting on a variety of
d-amino acids, including d-isomers of branched amino
acids (Fig. 6). The amino acid residues presumed to be
involved in substrate recognition and binding are not
conserved between BCAT from E. coli and d-AAT
M. Funakoshi et al. D-Amino acid aminotransferase in A. thaliana
FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1195
from Bacillus sp. YM-1, enzymes for which three-
dimensional crystal structures have been determined
[30,31,54]. Likewise, A. thaliana BCAT and d-AAT
probably differ in the structure of the active center,
although their overall spatial structures are similar and
the amino acid residues involved in coenzyme (PLP)
binding are conserved.
d-AATs of bacterial origin are stereospecific and
exhibit activities for a broad range of d-amino acids
[26–29]. The substrate preference of A. thaliana
d-AAT is quite similar to that of d-AAT from
B. sphaericus [27]; for these species, d-Met and d-Phe
are adequate d-AAT substrates, although this is not
so for other bacterial d-AATs. The amino acid
sequence alignment of d-AATs from YM-1 and
A. thaliana (Fig. 7) indicates that the critical residues
in the YM-1 enzyme are conserved or equivalent to
those in A. thaliana d-AAT, including the catalytic
site Lys146 (Lys222 in A. thaliana d-AAT) and other
residues putatively involved in PLP binding [30,31]:
Arg52 (Arg128 in A. thaliana d-AAT), Glu178

(Glu255), Thr206 (Thr284), and Thr242 (Ser325).
However, residues involved in substrate recognition
and binding are not conserved. Thus, Arg99, His101
and Tyr32 in YM-1 d-AAT, which comprise a trap
that binds the substrate a-carboxyl group [30,31], are
replaced by hydrophobic residues, Phe176, Leu178
and Phe109, respectively, in A. thaliana d-AAT. The
loop of Ser241-Thr-Thr-Ser244 in YM-1 d-AAT,
which defines the entrance for a substrate-locating
pocket [30,31], is Gly324-Ser-Gly-Ile327 in A. thaliana
d-AAT. The substrate preference of A. thaliana
d-AAT, which differs from that of YM-1 d-AAT,
may be due to these sequence differences. However,
the corresponding residues presumed to be involved
in substrate recognition in B. sphaericus d-AAT are
also not conserved in A. thaliana d-AAT. Therefore,
the amino acid residues in A. thaliana d-AAT
responsible for substrate recognition are not yet
clearly identified.
In conclusion, we observed a transient increase in
d-Asp levels during A. thaliana germination and
growth, suggesting that d-Asp is synthesized in the
plant. d-Asp administered to plants suppressed
growth, although the inhibitory mechanism remains
to be clarified. Exogenous
d-Asp was efficiently
incorporated and metabolized, and was in part con-
verted to other d-amino acids (d-Glu and d-Ala).
A. thaliana d-AAT, which is presumably involved in
the metabolism of d-Asp by catalyzing transamina-

tion between d-amino acids, was cloned and charac-
terized. This represents the first cDNA cloning and
functional characterization of a d-AAT of eukaryotic
origin. Further characterization of this d-AAT is
necessary. Investigation of its spatiotemporal expres-
sion and knockout phenotype will be important to
elucidate the underlying mechanism of d-AAT
enzyme activity.
Experimental procedures
Materials
A. thaliana seeds (Columbia, wild-type) were obtained from
H. Seki (RIKEN BioResource Center). The mixture of salt
ingredients used for MS medium, gelrite and 4-fluoro-
7-nitro-1,2,3-benzoxadiazole were purchased from Wako
Pure Chemical Ind. (Osaka, Japan). d-Amino acids and
l-amino acids were purchased from Sigma Chemical Co.
(St Louis, MO, USA), and other reagents and solvents were
of the highest grade commercially available.
The following A. thaliana cDNA clones were obtained
from RIKEN BioResource Center [55,56]: AspAT (Atasp1,
accession number AY059912; Atasp3, AY050765; Atasp5,
AY054660); putative d-AAT (AY099783); and BCAT (Atb-
cat2, AY370135; Atbcat4, AY052676).
Growth conditions of A. thaliana and the
preparation of its extracts
A. thaliana was grown in MS medium comprising 0.01%
myoinositol, 1 · 10
)4
mgÆmL
)1

thiamine hydrochloride,
5 · 10
)4
mgÆmL
)1
nicotinic acid, 5 · 10
)4
mgÆmL
)1
pyri-
doxine hydrochloride, 2 · 10
)4
mgÆmL
)1
glycine, 2.0%
sucrose, 0.3% gelrite, and a commercially available mix-
ture of salts for MS medium (Wako Pure Chemical Ind.).
The additives (10 mmd-Asp and ⁄ or 10 mml-Asp) were
filter-sterilized and added after autoclaving the medium.
A. thaliana seeds were sterilized in 70% ethanol, washed
and resuspended in sterilized water, sown on media plates,
and cold-treated for 1 day at 4 °C. The seedlings were
then grown at 21 °C under 24 h of continuous light
(3000 lux) for up to 23 days.
Seedlings were collected at various time points and
washed briefly and gently with NaCl⁄ P
i
. The buffer was
then wiped away, and the seedlings were immediately fro-
zen in liquid nitrogen. Two volumes of 100 m m potassium

phosphate buffer (pH 8.0), including protease inhibitors
(Roche Applied Science, Mannheim, Germany), were added
to the frozen sample in a mortar that had been chilled at
)20 °C, and the mixture was subsequently ground with a
pestle. The resultant homogenate was centrifuged at
20 600 g for 10 min at 4 °C. A portion of the supernatant
was stored at )80 °C prior to the analysis of amino acid
content. Protein concentrations were determined using a
protein assay reagent (BioRad Laboratories, Hercules, CA,
USA) and BSA as standard.
D-Amino acid aminotransferase in A. thaliana M. Funakoshi et al.
1196 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS
Determination of amino acid contents
d-Amino acid and l-amino acid contents in plant samples
were determined by HPLC as essentially described in our
previous reports [57,58]. To an aliquot (150 lL) of plant
homogenate prepared as described above, 50 lLofH
2
O
and 10 lL of 100% (w ⁄ v) trichloroacetic acid were added,
and the mixture was centrifuged at 4 °C for 10 min at
20 600 g to remove precipitated proteins. The supernatant
(130 lL) was then mixed with 50 lLof1m NaOH, 100 lL
of 200 mm borate buffer (pH 9.5), and 120 lLofH
2
O.
Subsequently, amino acids in the mixture (40 lL) were flu-
orescently derivatized by the addition of 30 lLof50mm
4-fluoro-7-nitro-1,2,3-benzoxadiazole in dry acetonitrile,
and this was followed by incubation at 60 °C for 5 min.

The reaction was terminated with 930 lL of 1% trifluoro-
acetic acid. The sample was filtered through a 0.45 lm filter
(Millex-LH; Millipore, Bedford, MA, USA) and applied to
a column-switching HPLC system for the determination of
d-Asp and l-Asp content, as previously described [58].
Analysis of d-Glu and l-Glu content was performed by
modifying the column-switching time of the system for glu-
tamate. d-Ala and l-Ala content was determined by HPLC
as described in our previous report [57].
Construction of A. thaliana amino acid AT
expression plasmids
Expression plasmids for AspAT, putative d-AAT and
BCAT were constructed as follows. AspAT cDNAs were
amplified by PCR using cDNA clones (as described above)
as templates and the following primers: Atasp1,5¢-GAGC
TCGATGGCTTTGGCGATGATGATCCG-3¢ and 5¢-CC
ATGGTTAAGATGACTTGGTGACTTCATG-3¢; Atasp3,
5¢-AGATCTATGAAAACTACTCATTTCTCTTCC-3¢ and
5¢-GGTACCTCAGACGGCTTTGGTGACAACAGC-3¢;and
Atasp5,5¢-GAGCTCGATGGCTTCTTTAATGTTATCT
CTC-3¢ and 5¢-CCATGGTCAGCTTACGTTATGGTAT
GAGTC-3¢. The SacI–NcoI fragment (for Atasp1 and
Atasp5) and BglI–KpnI fragment (for Atasp3) were sub-
cloned into pRSET-B (Invitrogen, Carsbad, CA, USA) to
generate N-terminal, His-tagged AspAT expression
plasmids.
Putative d-AAT and BCAT cDNAs were amplified by
PCR using cDNA templates (as described above) and
the following primers: putative d-AAT, 5¢-GTCGACCC
ATGGCAGGTTTGTCGCTGGAG-3¢ and 5¢-CTCGAG

TCAGTAAGGAACAAGAACACG-3¢; Atbcat2,5¢-GT
CGACAGATGATCAAAACA ATCACAT CTCTACGC -3¢
and 5¢-CTCGAGTCAGTTGATATCTGTGACCCATCC-
3¢; and Atbcat4,5¢-GAATTCATGGCTCCTTCTGCGCA
ACCTC-3¢ and 5¢-CTCGAGTCAGCCCTGGCGGTCA
ATCTCCAC-3¢. The SalI–XhoI fragment (for putative
d
-AAT and Atbcat2) and EcoRI–XhoI fragment (for
Atbcat4) were subcloned into pET-41a(+) (Novagen,
Madison, WI, USA) to generate N-terminal, glutathione
S-transferase (GST)-tagged, His-tagged and S-tagged AT
expression plasmids. In initial trials where the coding
regions of the putative d-AAT and BCAT were sub-
cloned into pRSET-B, the recombinant proteins were
nearly all recovered in the insoluble fraction. Therefore,
the coding regions of these ATs were subcloned into
another expression plasmid, pET-41a(+), instead of
pRSET-B. DNA sequences of the coding regions of these
expression plasmids were confirmed by sequencing, using
an ABI PRISM 310 DNA sequencer.
Expression and purification of recombinant
proteins
E. coli strain BL21(DE3)pLysS cells transformed with AT
expression plasmids were grown in LB medium under
optimized conditions. For AspAT, cells were grown at
37 °C in medium containing 100 lgÆmL
)1
ampicillin until
the attenuance (D
620 nm

) reached 0.5, and culturing was
then continued at 20 °C for an additional 20 h. For puta-
tive d-AAT and BCAT, cells were grown in medium
containing 25 lgÆmL
)1
kanamycin until the attenuance
(D
620 nm
) reached 0.5; isopropyl thio-b-d-galactoside was
then added to a final concentration of 0.01 mm, and cells
were cultured at 18 °C for another 20 h. After culturing,
cells were pelleted by centrifugation at 10 000 g for
10 min at 4 °C and resuspended in buffer (NaCl ⁄ P
i
,
pH 7.0, for AspAT; 20 mm Tris, pH 8.0, for putative
d-AAT and BCAT) that included protease inhibitors
(Roche Applied Science). The cell suspension was incu-
bated for 20 min at room temperature with gentle mixing
after addition of BugBuster Protein Extraction Reagent
(Novagen, ·10; 1 mL per gram wet cell paste). In the case
of putative d-AAT and BCAT, Lysonase Bioprocessing
Reagent (Novagen) was also included (3 lLÆmL
)1
). The
resulting lysates were centrifuged at 12 000 g at 4 °C for
20 min to pellet the insoluble cell debris and obtain the
crude extract fraction.
The crude extract fraction was subsequently applied to a
chelating column (HiTrap Chelating HP column; Amer-

sham Biosciences, Piscataway, NJ, USA), and the recombi-
nant AT was purified by affinity chromatography. For
AspAT purification, the column was equilibrated with
20 mm sodium dihydrogen phosphate buffer (pH 7.4),
0.5 m NaCl, and 10 mm imidazole. Following application
of the crude extract, the column was washed with the same
buffer, and the AspAT was eluted with the same buffer
containing 500 mm imidazole. The AspAT fraction was
used for enzyme assay after dialysis against 10 mm potas-
sium phosphate buffer (pH 8.0). For purification of puta-
tive d-AAT and BCAT, the column was equilibrated with
20 mm sodium dihydrogen phosphate buffer (pH 8.0),
0.5 m NaCl, and 50 mm imidazole, and the d-AAT and
BCAT fractions were eluted using the same buffer including
M. Funakoshi et al. D-Amino acid aminotransferase in A. thaliana
FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1197
300 mm imidazole. Other conditions were similar to those
used in the case of AspAT purification.
Enzyme assays
The standard reaction mixture (200 lL) for AspAT activity
included 100 mm potassium phosphate buffer (pH 8.0),
appropriate concentrations of d-Asp or l-Asp as an amino
donor (1–50 mm), 50 mm a-ketoglutarate as an amino
acceptor, 50 lm PLP, and the AspAT fraction. The mixture
was incubated at 37 °C for 5 or 10 min, and this was fol-
lowed by the addition of 10 lL of 100% trichloroacetic
acid to stop the reaction. After centrifugation at 4 °C for
10 min at 20 600 g, the supernatant (150 lL) was removed,
filtered through a 0.45 lm filter (Asahi Techno Glass Co.,
Tokyo, Japan), and applied to an automated HPLC system

to determine the amounts of d-Glu or l-Glu produced.
Details regarding the HPLC system have been described in
our previous report [59].
Branched chain amino acid aminotransferase activity was
assayed in a mixture containing 100 mm Tris (pH 8.0),
appropriate concentrations of d-amino acid or l-amino
acid as an amino donor, 5 mm a-ketoglutarate as an amino
acceptor, 50 lm PLP, and the BCAT fraction. The mixture
was incubated at 37 °C for 30 min. After the incubation,
the assay procedure was similar to that used in the AspAT
assay. The reaction mixture for putative d-AAT included
100 mm potassium phosphate (or Tris) buffer (pH 8.0),
appropriate concentrations of d-Asp or l-Asp as an amino
donor (0.4–12.5 mm), 50 mm a-ketoglutarate or 12.5 mm
pyruvate as an amino acceptor, 50 lm PLP, and the
d-AAT fraction. Other details for the assay were the same
as those for the AspAT and BCAT assay.
Miscellaneous methods
SDS ⁄ PAGE and western blotting with anti-His-tag
(His-probe; Santa Cruz Biotechnology, Santa Cruz, CA,
USA) and anti-GST serum (anti-GST mouse IgG
2a-j
; Naca-
lai Tesque, Kyoto, Japan) were carried out as described in
our previous reports [60]. The DNA sequence of A. thaliana
d-AAT was aligned with the d-AAT sequence of Bacillus sp.
YM-1 by using clustal w software [61]. The chloroplast
targeting signal was analyzed using the psort program [62].
Acknowledgements
The authors express their sincere appreciation to

Dr H. Seki (RIKEN BioResource Center, Tsukuba,
Japan) for his generous gift of A. thaliana seeds. This
work was supported in part by a Grant-in-Aid from
the Ministry of Education, Science, Sports and Culture
of Japan, a Grant-in-Aid for Scientific Research from
the Japan Society for the Promotion of Science, and a
Kitasato University Research Grant for Young
Researchers (M. Sekine and M. Katane).
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