Exogenous d-Asp was efficiently incorporated and metabo-lized, and was converted to other d-amino acids d-Glu and d-Ala.. thaliana d-amino acid aminotransferase, which is presum-ably invo
Trang 1Arabidopsis thalianaD-amino acid aminotransferase –
Miya Funakoshi1,*, Masae Sekine1,*, Masumi Katane1, Takemitsu Furuchi1, Masafumi Yohda2, Takafumi Yoshikawa1and Hiroshi Homma1
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: hommah@pharm.kitasato-u.ac.jp
*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.
Trang 2mammalian 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 containingD-Asp
(MS +D-Asp)
Germination and growth of A thaliana was observed
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 M L -Asp (MS + L -Asp) (C) MS medium containing 10 m M
D -Asp (MS + D -Asp).
Trang 3the 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 CaCl2 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
20 15
10 5
0
0
1
2
3
4
5
D -Asp concentration (m M )
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).
Trang 4and 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 nmolÆmg)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 levlev-els 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
0.15 0.3 0.45
Days
Days
Days
Days
Days
Days
–1 )
–1 )
–1 )
–1 )
–1 )
–1 )
–1 )
–1 )
–1 )
A
30 60 90 120 150
0 100 200 300 B
0.5 1.5
0
0.4
0.8
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0 1 C
0 100 200 300
0 50 100 150
200 D
0
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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 M L -Asp (MS + L -Asp), and MS medium containing 10 m M D -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.
Trang 5that 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, Atasp3 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 (Km values for l-Asp) were determined by Line-weaver–Burk plots: AspAT 1, Km (l-Asp) 1.0 mm; AspAT 3, Km (l-Asp) 2.5 mm; and AspAT 5, Km (l-Asp) 1.0 mm These Km(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
–1 )
–1 )
–1 )
Days
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 M D -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.
Trang 6their 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, Km (l-Leu) 0.71 mm; and BCAT 4, Km
(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, Kmand Vmax values for d-Asp and d-Ala were 2.3 and 1 mm, and 2.5 and 5.0 lmolÆmin)1Æmg)1 protein (Table 1); there-fore, d-Ala is a more efficient d-AAT substrate than
d-Asp In addition, the Km and Vmax values for d-Glu
in the transamination reaction to pyruvate to produce
d-Ala were 4 mm and 3.3 lmolÆmin)1Æmg)1 protein, respectively (Table 1) Therefore the affinity for d-Ala
is higher than that for d-Glu, and Vmax 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 (m M )
V max (lmolÆmin)1Æmg)1)
Trang 7approximately 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.
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 lg of
protein) was incubated with 1.5 m M various D -amino acids, 50 m M
a-ketoglutarate, and 50 l M PLP, and the amounts of D -Glu
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.
Trang 8(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 (1-14C) 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 localizadetermina-tion and physiological funcdetermina-tion 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
PlantD-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
Trang 9from 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)4mgÆmL)1 thiamine hydrochloride,
5· 10)4mgÆmL)1 nicotinic acid, 5· 10)4mgÆmL)1 pyri-doxine hydrochloride, 2· 10)4mgÆmL)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 mm d-Asp and⁄ or 10 mm l-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 4C The seedlings were then grown at 21C 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⁄ Pi The buffer was then wiped away, and the seedlings were immediately fro-zen in liquid nitrogen Two volumes of 100 mm 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 4C 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
Trang 10Determination 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 lL of H2O
and 10 lL of 100% (w⁄ v) trichloroacetic acid were added,
and the mixture was centrifuged at 4C for 10 min at
20 600 g to remove precipitated proteins The supernatant
(130 lL) was then mixed with 50 lL of 1 m NaOH, 100 lL
of 200 mm borate buffer (pH 9.5), and 120 lL of H2O
Subsequently, amino acids in the mixture (40 lL) were
flu-orescently derivatized by the addition of 30 lL of 50 mm
4-fluoro-7-nitro-1,2,3-benzoxadiazole in dry acetonitrile,
and this was followed by incubation at 60C 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
CGACAGATGATCAAAACAATCACATCTCTACGC-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
37C in medium containing 100 lgÆmL)1 ampicillin until the attenuance (D620 nm) reached 0.5, and culturing was then continued at 20C 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
then added to a final concentration of 0.01 mm, and cells were cultured at 18C for another 20 h After culturing, cells were pelleted by centrifugation at 10 000 g for
10 min at 4C and resuspended in buffer (NaCl ⁄ Pi,
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 4C 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