Cloning and functional characterization of Arabidopsis thaliana D-amino acid aminotransferase – D-aspartate behavior during germination Miya Funakoshi1,*, Masae Sekine1,*, Masumi Katane1, Takemitsu Furuchi1, Masafumi Yohda2, Takafumi Yoshikawa1 and Hiroshi Homma1 School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan Tokyo University of Agriculture and Technology, Japan 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 5791 6381 Tel: +81 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 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 metabolized, 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 presumably involved in the metabolism of d-Asp in the plant by catalyzing transamination 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 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 neurotransmission [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 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 D-Amino M Funakoshi et al mammalian tissues d-Asp appears to affect the functions 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 targeted 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 significantly increased in numerous tissues The mutant mice displayed impaired sexual performance and behavioral alterations, potentially reflecting diminished 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 importance 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 characterization of d-AAT acid aminotransferase in A thaliana 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 interesting to note that growth in MS + d-Asp medium was significantly suppressed (Fig 1C) Figure shows A B C 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 In MS and MS + l-Asp media, significant plant growth was observed (Fig 1A,B) The elonga- Fig 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 containing 10 mM L-Asp (MS + L-Asp) (C) MS medium containing 10 mM D-Asp (MS + D-Asp) FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1189 D-Amino acid aminotransferase in A thaliana M Funakoshi et al 14 days 11 days Length (mm) * ** * ** *** *** *** *** *** *** *** 0 D-Asp 10 15 concentration (mM) 20 Fig Effect of D-Asp on cotyledon growth during culture of A thaliana The lengths of cotyledons were determined in seedlings after 11 days and 14 days of culture on MS medium containing 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) 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 homogenate 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 days and 11 days of culture, respectively (Fig 3A) The high percentage of d-Asp observed in the day culture was attributed to a corresponding low l-Asp content for the same time point (Fig 3B) At days of culture the l-Asp content 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 1190 stages of culture (approximately 14 days) in the gelrite 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 nmolỈmg)1 protein, which is similar to that of plants grown in MS medium (Fig 3A) As d-Asp was not supplemented 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 physiological 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 cultures (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 exogenous 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 consistent with the observation described above that growth of plants cultured in MS + d-Asp medium was partially (not fully) restored after 14 days of culture It is postulated that d-Asp suppressed growth (Figs 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 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS D-Amino M Funakoshi et al A B 11 14 16 21 (nmol·mg protein–1) 300 120 90 200 60 100 30 11 Days 21 D 0.4 D-Asp 0.5 150 200 100 100 50 16 21 Days F 90 400 60 200 30 14 16 21 L-Asp 600 (nmol·mg protein–1) 120 D (= D /D+L) % 800 16 21 Days E 100 500 80 400 60 300 40 200 20 100 Days 14 16 21 Glu (nmol·mg protein–1) 200 L-Asp D (= D /D+L) % 0.8 (nmol·mg protein–1) 300 1.5 Glu (nmol·mg protein–1) 1.2 (nmol·mg protein–1) 16 Days C (nmol·mg protein–1) 14 Glu (nmol·mg protein–1) L-Asp D (= D /D+L) % 0.15 0.15 D-Asp (nmol·mg protein–1) 0.3 0.3 150 0.45 0.45 D-Asp acid aminotransferase in A thaliana Days Fig 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 mM L-Asp (MS + L-Asp), and MS medium containing 10 mM 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 Experimental 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 and d-Ala were detected in plants cultured in MS +d-Asp medium as early as 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 nmolỈmg)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 levels 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 levels up to days of culture (Fig 4B) It is interesting FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1191 acid aminotransferase in A thaliana M Funakoshi et al L-Glu 400 100 300 200 50 100 14 16 21 (nmol·mg protein–1) 500 D-Glu D-Glu (nmol·mg protein–1) A 150 L-Glu D-Amino Days D-Ala L-Ala 100 D,L-Ala (nmol·mg protein–1) B 150 50 14 16 21 Days Fig D- and L-Glu and D-Ala and L-Ala content in A thaliana cultured in MS + D-Asp medium A thaliana was cultured in MS medium containing 10 mM 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 independently, 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 Gene cloning and functional characterization of amino acid AT recombinant proteins from A thaliana AspAT Six A thaliana AspAT clones (Atasp1–5 and prokaryotic-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 and AspAT 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 Lineweaver–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 comparable with those previously reported (3.0 mm, AspAT 1; 1.4 mm, AspAT 2; and 2.9 mm, AspAT [21]) However, none of the ATs exhibited activity for d-Asp or d-Ala as amino donors BCAT 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 indicate that exogenous d-Asp is metabolized in the plant, and that the appearance of d-Glu and d-Ala is correlated with the metabolism of d-Asp Enzymes potentially responsible for d-Asp metabolism 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 substrate specificities, particularly for d-amino acids, were characterized 1192 BCATs and d-AATs of bacterial origin show significant 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¢-phosphate (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 interested 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 and BCAT were investigated in this work Recombinant BCAT and BCAT 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 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS D-Amino M Funakoshi et al Fig Western blotting of recombinant A thaliana D-AAT expressed in E coli cells The expression of recombinant A thaliana 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 Experimental procedures Figures on the left side represent molecular masses of marker proteins The arrowhead indicates recombinant A thaliana D-AAT 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-ketoglutarate 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 exhibit 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 acid aminotransferase in A thaliana 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 calculated from its deduced amino acid sequence The d-AAT preparation, purified as described in Experimental procedures, exhibited considerable AT activity for d-Asp and d-Ala as amino donors with a-ketoglutarate 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 oxaloacetate to produce d-Ala or d-Asp, respectively Kinetic parameters for these activities were determined, and are shown in Table In the transamination conversion of amino acid to a-ketoglutarate, Km and Vmax values for d-Asp and d-Ala were 2.3 and mm, and 2.5 and 5.0 lmolỈmin)1Ỉmg)1 protein (Table 1); therefore, 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 mm and 3.3 lmolỈmin)1Ỉmg)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 predominates 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 substrate 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 specificity, which has been demonstrated in other characterized bacterial d-AATs [26–29] When the enzyme assay was performed in the absence of PLP, activity was Table 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 Km (mM) Aminotransfer reaction D-Asp fi a-ketoglutarate fi a-ketoglutarate D-Glu fi pyruvate D-Glu Vmax (lmolỈmin)1Ỉmg)1) 4.0 2.5 3.3 2.3 D-Asp D-Ala D-Ala a-Ketoglutarate 1.0 27 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1193 D-Amino acid aminotransferase in A thaliana M Funakoshi et al 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 sphaericus d-AATs, respectively Figure shows the amino acid sequence alignment of d-AATs from A thaliana and Bacillus sp YM-1, for which the three-dimensional crystal structure has been determined [30,31] It is noteworthy that a chloroplast-targeting signal is present in the A thaliana d-AAT 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 20 40 60 80 100 120 Relative activity (%) Fig 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 mM various D-amino acids, 50 mM a-ketoglutarate, and 50 lM PLP, and the amounts of D-Glu produced were determined by HPLC as described in Experimental procedures The data presented in this figure are average ± half range from two separate experiments, and shown as values relative to that of D-Ala approximately 65% of that in the presence of PLP However, the addition of mm hydroxylamine or 10 mm amino-oxyacetic acid completely abolished activity, suggesting that A thaliana d-AAT is a 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-glutamyld-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 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 indicated 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 1194 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS M Funakoshi et al (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 thaliana, 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; however, the configuration of the labeled Val was not determined [43] Different types of partially purified and characterized d-AATs have been shown to transfer 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 synthesis 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-dependent, which is consistent with previous reports, as described above As this enzyme appears to catalyze transamination from various d-amino acids to oxaloacetate 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 synthetic 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 system where higher plants grow, as a variety of bacteria in the soil, symbiotic root bacteria and plants themselves [37,48] represent abundant sources of free and conjugated forms of d-amino acids, including peptidoglycan, from which free d-amino acids can be generated by hydrolysis On the basis of our studies, we D-Amino acid aminotransferase in A thaliana 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 Exogenous d-amino acids in higher plants are presumably subject to racemization, transamination and ⁄ or malonylation, 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 decarboxylation 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, determination of the localization and physiological function of these other d-amino acids requires further research As shown in Figs and 2, culturing plants in medium containing d-Asp suppressed the growth of A thaliana 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 stimulation 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 similar 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 distinct 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 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1195 D-Amino acid aminotransferase in A thaliana M Funakoshi et al from Bacillus sp YM-1, enzymes for which threedimensional 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 converted 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 transamination between d-amino acids, was cloned and characterized This represents the first cDNA cloning and functional characterization of a d-AAT of eukaryotic 1196 origin Further characterization of this d-AAT is necessary Investigation of its spatiotemporal expression 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-fluoro7-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 (Atbcat2, 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, · 10)4 mgỈmL)1 thiamine hydrochloride, · 10)4 mgỈmL)1 nicotinic acid, · 10)4 mgỈmL)1 pyridoxine hydrochloride, · 10)4 mgỈmL)1 glycine, 2.0% sucrose, 0.3% gelrite, and a commercially available mixture 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 day at °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 ⁄ Pi The buffer was then wiped away, and the seedlings were immediately frozen 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 at °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 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS M Funakoshi et al 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 lL of H2O and 10 lL of 100% (w ⁄ v) trichloroacetic acid were added, and the mixture was centrifuged at °C for 10 at 20 600 g to remove precipitated proteins The supernatant (130 lL) was then mixed with 50 lL of 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 fluorescently 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 60 °C for The reaction was terminated with 930 lL of 1% trifluoroacetic 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 glutamate 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 subcloned 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¢-CTCGAGTCAGTTGATATCTGTGACCCATCC3¢; 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, D-Amino acid aminotransferase in A thaliana 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 subcloned 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 lgỈmL)1 ampicillin until the attenuance (D620 nm) reached 0.5, and culturing was then continued at 20 °C for an additional 20 h For putative d-AAT and BCAT, cells were grown in medium containing 25 lgỈmL)1 kanamycin until the attenuance (D620 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 at °C 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 incubated for 20 at room temperature with gentle mixing after addition of BugBuster Protein Extraction Reagent (Novagen, ·10; mL per gram wet cell paste) In the case of putative d-AAT and BCAT, Lysonase Bioprocessing Reagent (Novagen) was also included (3 lLỈmL)1) The resulting lysates were centrifuged at 12 000 g at °C for 20 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; Amersham Biosciences, Piscataway, NJ, USA), and the recombinant 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 potassium phosphate buffer (pH 8.0) For purification of putative 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 FEBS Journal 275 (2008) 1188–1200 ª 2008 The Authors Journal compilation ª 2008 FEBS 1197 D-Amino acid aminotransferase in A thaliana M Funakoshi et al 300 mm imidazole Other conditions were similar to those used in the case of AspAT purification Kitasato University Research Grant for Researchers (M Sekine and M Katane) Enzyme assays References 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 or 10 min, and this was followed by the addition of 10 lL of 100% trichloroacetic acid to stop the reaction After centrifugation at °C for 10 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, mm a-ketoglutarate as an amino acceptor, 50 lm PLP, and the BCAT fraction The mixture was incubated at 37 °C for 30 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 Konno R, Bruckner H, D’Aniello A, Fisher G, Fujii N & ă Homma H (2007) D-Amino Acids: a New Frontier in Amino Acid and Protein Research, Practical Methods and Protocols Nova Science Publishers, Inc., New York, NY Nishikawa T (2005) Metabolism and functional roles of endogenous d-serine in mammalian brains Biol Pharm Bull 28, 1561–1565 Mustafa AK, Kim PM & Snyder SH (2004) d-Serine as a putative glial neurotransmitter Neuron Glia Biol 1, 275–281 Homma H (2007) Biochemistry of d-aspartate in mammalian cells Amino Acids 32, 3–11 D’Aniello A (2007) d-Aspartic acid: an endogenous amino acid with an important neuroendocrine role Brain Res Rev 53, 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