Purification of three aminotransferases from Hydrogenobacter thermophilus TK-6 – novel types of alanine or glycine aminotransferase Enzymes and catalysis Masafumi Kameya, Hiroyuki Arai, Masaharu Ishii and Yasuo Igarashi Department of Biotechnology, The University of Tokyo, Japan Introduction Aminotransferase (EC 2.6.1) catalyses the conversion between amino acids and 2-oxo acids, transferring the amino group of the amino acid onto the 2-oxo acid. This enzyme is widespread, being present in almost all organisms, and plays a key role in the synthesis and degradation of amino acids. As the substrates ⁄ products of aminotransferase, namely 2-oxo acids and amino acids, are key metabolites in carbon and nitro- gen metabolism, this enzyme can be regarded as a physiologically important linkage within central meta- bolism. Furthermore, some aminotransferases have been reported to be coupled with further metabolic Keywords 2-oxo acid; amino acid; aminotransferase; Hydrogenobacter thermophilus; nitrogen anabolism Correspondence M. Ishii, Department of Biotechnology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Fax: +81 3 5841 5272 Tel: +81 3 5841 5143 E-mail: amishii@mail.ecc.u-tokyo.ac.jp (Received 6 January 2010, revised 27 January 2010, accepted 2 February 2010) doi:10.1111/j.1742-4658.2010.07604.x Aminotransferases catalyse synthetic and degradative reactions of amino acids, and serve as a key linkage between central carbon and nitrogen metabolism in most organisms. In this study, three aminotransferases (AT1, AT2 and AT3) were purified and characterized from Hydrogenobacter thermophilus, a hydrogen-oxidizing chemolithoautotrophic bacterium, which has been reported to possess unique features in its carbon and nitrogen anabolism. AT1, AT2 and AT3 exhibited glutamate:oxaloacetate amino- transferase, glutamate:pyruvate aminotransferase and alanine:glyoxylate aminotransferase activities, respectively. In addition, both AT1 and AT2 catalysed a glutamate:glyoxylate aminotransferase reaction. Interestingly, phylogenetic analysis showed that AT2 belongs to aminotransferase family IV, whereas known glutamate:pyruvate aminotransferases and gluta- mate:glyoxylate aminotransferases are members of family Ic. In contrast, AT3 was classified into family I, distant from eukaryotic alanine:glyoxylate aminotransferases which belong to family IV. Although Thermococcus litoralis alanine:glyoxylate aminotransferase is the sole known example of family I alanine:glyoxylate aminotransferases, it is indicated that this alanine:glyoxylate aminotransferase and AT3 are derived from distinct lin- eages within family I, because neither high sequence similarity nor putative substrate-binding residues are shared by these two enzymes. To our knowl- edge, this study is the first report of the primary structure of bacterial gluta- mate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase, and demonstrates the presence of novel types of aminotransferase phyloge- netically distinct from known eukaryotic and archaeal isozymes. Abbreviations AGT, alanine:glyoxylate aminotransferase; CFE, cell-free extract; GGT, glutamate:glyoxylate aminotransferase; GOT, glutamate:oxaloacetate aminotransferase; GPT, glutamate:pyruvate aminotransferase; 2-OG, 2-oxoglutarate; PLP, pyridoxal 5¢-phosphate; PSOT, phosphoserine: 2-oxoglutarate aminotransferase. 1876 FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS activities, e.g. enzymes involved in the malate shuttle, porphyrin synthesis [1], maintenance of intracellular redox status [2] or plant photorespiration [3]. A wide variety of substrates for aminotransferases have been reported, including branched-chain amino acids, aromatic amino acids, b-amino acids and their corresponding 2-oxo acids. To categorize diverse amin- otransferases, classifications based on the primary structure have been proposed. Such a classification divides aminotransferases into four families, numbered I–IV [4]. Family I is further divided into several subfamilies, such as Ia and Ic [5]. In this classification system, enzymes belonging to the same family or subfamily share common enzymatic characteristics to some extent. However, the substrate specificities of aminotransfe- rases are diverse, even within the same family or sub- family; therefore, at present, it is difficult to predict the specificities on the basis of the primary structures only. One reason for this difficulty is that the reaction mechanisms and structures of aminotransferases may be similar to each other, even if they react specifically with different substrates. Moreover, there are only a limited number of aminotransferases whose enzymatic properties and primary sequences have been deter- mined. For these reasons, the function of most puta- tive aminotransferase homologues found in the genome database remains to be ascertained. Some recent studies have revealed properties of several puta- tive aminotransferases by biochemical and enzymatic analyses [6–8], demonstrating the importance of a bio- chemical approach for the characterization of these enzymes. Hydrogenobacter thermophilus TK-6 is a thermo- philic, hydrogen-oxidizing, obligately chemolithoauto- trophic bacterium. The analysis of 16S rRNA sequences has shown that Hydrogenobacter species are located on the deepest branch in the domain Bacteria on the phylogenetic tree, together with other Aquificae species [9]. Reflecting this distinctive phylogenetic posi- tion, this bacterium shows many unique characteristics. One such characteristic is its carbon anabolism, where carbon dioxide is fixed via the reductive tricarboxylic acid cycle. Key enzymes in this cycle have been charac- terized and shown to have novel enzymatic features [10–13]. Furthermore, enzymatically peculiar character- istics have also been found in this bacterium’s nitrogen anabolism [14,15]. Although previous studies have demonstrated that H. thermophilus assimilates nitrogen in the form of ammonium to produce glutamate (Glu), it has not yet been clarified how Glu serves as the nitrogen donor for the synthesis of other nitrogenous compounds. The study of aminotransferases in this bacterium is of interest, firstly because of the need to characterize biochemically aminotransferases. The importance of this is emphasized by the belief that a novel amino- transferase would be found in this phylogenetically deep-rooted bacterium. Secondly, this study was expected to lead to further elucidation of the metabo- lism of H. thermophilus. Such elucidation would not be restricted to nitrogen metabolism, but would also include its unique central carbon metabolism. In this study, three aminotransferases were purified and characterized biochemically and presumed to contrib- ute to aspartate (Asp), alanine (Ala) and glycine (Gly) syntheses. Phylogenetic analysis of these enzymes showed a unique combination of substrate specificities and phylogenetic positions, providing novel insights into the aminotransferase classification. Results Aminotransferase activities in cell-free extract (CFE) Given that H. thermophilus operates a distinctive carbon pathway, the reductive tricarboxylic acid cycle, its central carbon metabolism is of interest. Therefore, we focused on amino acids with relatively simple carbon skeletons: Glu, Asp, Ala and Gly. Aminotransferase activities in the CFE were assayed combining Glu, Asp, Ala or Gly as the amino group donor and 2-oxoglutarate (2-OG), oxaloac- etate, pyruvate or glyoxylate as the amino group accep- tor. Consequently, the following four kinds of activity were detected: 0.96 UÆmg )1 glutamate:oxaloacetate aminotransferase (GOT; EC 2.6.1.1), 0.30 UÆmg )1 gluta- mate:pyruvate aminotransferase (GPT; EC 2.6.1.2), 0.30 UÆmg )1 glutamate:glyoxylate aminotransferase (GGT; EC 2.6.1.4) and 0.07 UÆmg )1 alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44). Although the GOT reaction was catalysed reversibly, the other reactions proceeded irreversibly as follows: GOT: Glu + oxaloacetate $ 2-OG + Asp GPT: Glu + pyruvate ! 2-OG + Ala GGT: Glu + glyoxylate ! 2-OG + Gly AGT: Ala + glyoxylate ! pyruvate + Gly Although GOT is a representative aminotransferase that has been studied extensively in many organisms [16–18], other aminotransferases have been less well studied, especially in bacteria. GPT has been purified and characterized in a few organisms, and only a M. Kameya et al. Three aminotransferases from H. thermophilus FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS 1877 limited number of GPT sequences have been deter- mined [2,6,19]. GGT and AGT have been subjected to considerably less research. GGT has been purified from a few organisms [20], and only those from Arabidopsis thaliana have been sequenced [3]. AGT has been sequenced and characterized in eukaryotes and archaea [21,22], but not in bacteria. Because of this background, the characterization of these aminotrans- ferase activities was expected to provide new insights into bacterial aminotransferases. Purification and phylogenetic analysis of aminotransferases Enzymes that exhibited GOT, GPT, GGT or AGT activity were subjected to purification, and three enzymes (AT1, AT2 and AT3) were purified from H. thermophilus CFE (Table 1). It was shown that GOT, GPT and AGT activities were derived from the single enzymes AT1, AT2 and AT3, respectively (Fig. 1). GGT activity was caused by AT1 and AT2, which exhibited 11 and 60 UÆ(mg purified protein) )1 of GGT activity, respectively. No other enzymes that exhibited GOT, GGT, GPT or AGT activity were detected throughout the purification, suggesting that the four kinds of activity in CFE were derived from only the three enzymes. Purified AT1, AT2 and AT3 gave single bands of 44, 42 and 45 kDa on SDS ⁄ PAGE, respectively (Fig. 2). The N-terminal amino acid sequences of AT1, AT2 and AT3 were determined to be MNLSKRVSHIKPAPT, MYQERLFTPG and MSEEWMFPKVKKL, respectively, and the full- length genes were identified in the H. thermophilus genome (AP011112). The molecular masses of AT1, AT2 and AT3 were calculated from their deduced pro- tein sequences to be 43.7, 41.9 and 45.6 kDa, respec- tively. These masses were consistent with those calculated from SDS ⁄ PAGE. The phylogenetic tree was constructed on the basis of the amino acid sequences (Fig. 3). GOT is known to be divided into two groups in subfamilies Ia and Ic, and AT1 belongs to aminotransferase subfamily Ic together with some other GOTs. Unexpectedly, AT2 is classified into family IV together with eukaryotic peroxisomal AGT, whereas other GPTs are members of family I. Interestingly, AT3 was located in family I, unlike eukaryotic AGT. There is only one report of a family I AGT, which was purified from Thermococ- cus litoralis [22]. The order of divergence of AT3 from enzymes in subfamily Ic is ambiguous in Fig. 3 Table 1. Purification of AT1, AT2 and AT3 from H. thermophilus. Enzyme Fraction Activity (U) a Protein (mg) Specific activity (UÆmg )1 ) a Purification (fold) Yield (%) AT1 CFE 636 660 0.96 1 100 Butyl-Toyopearl 245 13 19 20 39 DEAE-Toyopearl 74 1.3 59 61 12 MonoQ 61 0.26 239 248 10 AT2 CFE 275 927 0.30 1 100 Butyl-Toyopearl 65 24 2.7 9 24 DEAE-Toyopearl 31 3.0 10 35 11 Hydroxyapatite 15 0.3 51 171 5 MonoQ 10 0.13 79 266 4 AT3 CFE 129 1811 0.071 1 100 Butyl-Toyopearl 14 71 0.19 3 11 DEAE-Toyopearl 5.4 3.7 1.4 20 4 Hydroxyapatite 2.1 0.42 5.0 69 2 MonoQ 2.9 0.36 8.0 112 2 Phenyl Superose 1.2 0.063 19 270 1 a Representing GOT activity (in the direction of Asp synthesis) for AT1, GPT activity for AT2 and AGT activity for AT3. Asp Glu 2-OG OAA AT1 2-OG Pyr Ala AT2 2-OG Glyo Gly AT2 & AT1 PyrGlyo AT3 Fig. 1. Aminotransferase reactions catalysed by AT1, AT2 and AT3. Glyo, glyoxylate; OAA, oxaloacetate; Pyr, pyruvate. Three aminotransferases from H. thermophilus M. Kameya et al. 1878 FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS because of the low bootstrap values, although more detailed phylogenetic analysis indicated that AT3 is positioned separately from the known members of subfamily Ic (see below). Enzymatic properties Gel filtration estimated the molecular mass of AT1 to be 78 kDa, indicating that this enzyme forms a dimer of two identical subunits, as do many known amin- otransferases. The molecular masses of AT2 and AT3 were estimated to be 62 and 69 kDa, respectively. These values were 1.5-fold larger than each single peptide mass, indicating that these enzymes are mono- mers or homodimers. Considering that some thermo- philic enzymes have compact folding and their molecular masses are often underestimated by gel filtration [14], AT2 and AT3 might form a homodimer, although it cannot be excluded that they are mono- meric. The effects of pH on the aminotransferase activities of AT1, AT2 and AT3 were tested. AT1 exhibited the highest GOT activities in both directions over a broad pH range, 6.9–7.9 at 70 °C. AT2 and AT3 showed the highest GGT and AGT activities, respectively, at pH 7.9–8.4. These natural or slightly basic optimum pH values are common among known aminotransfe- rases. Some aminotransferases are known to be acti- vated by the addition of pyridoxal 5¢-phosphate (PLP), the catalytic cofactor of aminotransferase, to the reac- tion mixture [2]. The addition of PLP did not affect the activities of AT1, AT2 or AT3, suggesting that PLP binds tightly to these enzymes or extrinsic PLP cannot reactivate the apoenzymes. AT1 catalyses the GOT reaction reversibly and the GGT reaction only in the direction of Gly synthesis. AT2 catalyses the GPT reaction in the direction of Ala synthesis, and shows only trace activity (< 5% of that in the forward direction) in the reverse direction. This enzyme also irreversibly catalyses the GGT reaction in the direction of Gly synthesis, as well as AT1. Many known GPTs catalyse the GPT reaction reversibly and lack GGT activity. GPTs from A. thaliana share these properties with AT2 [3], although these GPTs belong to subfamily Ic distant from AT2, which is a member of family IV (Fig. 3). AT3 specifically catalyses the AGT reaction irreversibly in the direction of Gly synthesis. The irreversibility of GGT and AGT is a common feature among known GGTs and AGTs [20,22,23]. Although some eukaryotic AGTs have been reported to exhibit serine:pyruvate aminotransferase activity [21], AT3 did not show this activity, suggesting a high substrate specificity for Ala and glyoxylate compared with these AGTs. Some members of family IV are known as phospho- serine:2-oxoglutarate aminotransferases (PSOT; EC 2.6.1.52), which catalyse the conversion of phos- phoserine and 2-OG to phosphohydroxypyruvate and Glu [7,24,25]. AT2, which belongs to family IV, exhib- ited PSOT activity at 16 UÆmg )1 , corresponding to about one-quarter of its GGT activity. It is noteworthy that, although AT2 has a higher similarity to known AGTs than to known PSOTs, it does not have AGT activity but shows PSOT activity (Fig. 3). Kinetic characterization The kinetic parameters of AT1, AT2 and AT3 were determined for the reactions that followed typical Michaelis–Menten kinetics (Table 2). AT1 exhibited higher V max values in GOT reactions than in the GGT reaction. K m values for Glu, Asp and 2-OG in the GOT reaction were comparable with those of other reported GOTs [16,26]. With regard to GGT activity, both AT1 and AT2 showed K m values as low as those of known GGTs [3,20]. Although the GGT specific activity of AT1 was less than one-fifth of that of AT2, both specific activities were higher than those of reported GGTs (such as 5.71 UÆmg )1 from A. thaliana and 3.25 UÆmg )1 from Rhodopseudomonas palustris). These data indicate that, not only AT2, but also AT1 has GGT catalytic efficiency comparable with or 12 3 4 (kDa) 97 66 45 31 22 14 Fig. 2. SDS ⁄ PAGE (13%) of purified AT1, AT2 and AT3. Lane 1, purified AT1; lane 2, purified AT2; lane 3, purified AT3; lane 4, molecular mass markers. M. Kameya et al. Three aminotransferases from H. thermophilus FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS 1879 higher than that of known enzymes. AT2 also showed GPT activity, but its K m value for pyruvate was too high to determine accurately. Further investigations are required to verify the extent to which AT2 contrib- utes to the GPT reaction in vivo. K m values of AT3 were estimated to be equivalent to those of known AGTs. All determined K m values, except for that of AT2 for pyruvate, were less than or equivalent to those of known aminotransferases. These results indicate that AT1, AT2 and AT3 are adequately efficient to serve as GOT or GGT, GGT or PSOT, and AGT, respectively. Discussion In this study, GOT, GGT, GPT and AGT activities were detected in H. thermophilus, and three aminotransferases were identified. These activities are believed to enable this bacterium to synthesize Asp, Ala and Gly by transferring the amino group of Glu as the nitrogen source. These enzymes were completely purified and characterized and, as such, this report represents, to our knowledge, the first description of the characterization of bacterial GGT and AGT at an enzymatic and gene level. Comparison of the amino acid sequences with known enzymes showed the phylogenetic position of each aminotransferase. AT2 showed high similarity to eukaryotic AGT in family IV, whereas AT2 possessed GGT, GPT and PSOT activities instead of AGT activity. Most GGTs have been reported to lack GPT activity, with the exception of the GGT from A. thaliana [3]. In addition, GPTs have been identified in several organisms, such as Corynebacterium glutami- cum, Pyrococcus furiosus and mammals [2,6,19], and all are classified into subfamily Ic rather than into family IV. Therefore, it is obvious that AT2 is phylo- genetically distinct from known GGTs and GPTs. AT2 also possessed PSOT activity, which is found in some enzymes belonging to family IV. A study of the struc- ture of the Escherichia coli PSOT identified several conserved residues that bind to the substrates [25]. His41, Arg42, His328 and Arg329 in the E. coli PSOT are involved in the interaction with the negatively charged phosphate group of the phosphoserine. These residues are conserved not in AGTs, but are found in all PSOTs (Fig. S1, see Supporting information). Inter- estingly, AT2 harbours two of these four conserved residues (His29 and Arg30 in AT2). It may be that these partially conserved residues endow AT2 with PSOT activity, which is uncommon among known AGTs of family IV. AT3 also occupies an unusual phylogenetic position in family I, considering that this enzyme exhibited Fig. 3. Phylogenetic tree of aminotransfe- rases on the basis of the amino acid sequences. The numbers at the nodes are bootstrap confidence values expressed as percentages of 1000 bootstrap replicates. The order of the divergence was presumed to be reliable only when the bootstrap values were above 50. The tree was con- structed using the neighbor-joining method and showed the same overall topology as that constructed by the maximum likelihood method. Plus signs indicate the activities proven experimentally. The accession num- bers of each enzyme are shown in paren- theses. Enzymes from the following organisms were used: Arabidopsis thaliana [3,21,26], Bacillus circulans [24], Bacillus sp. YM-2 [17], Corynebacterium glutamicum [6], Escherichia coli [25], Entamoeba histolytica [7], human [35], H. thermophilus, Pyrococcus furiosus [2,36], rat [19,37,38], Saccharomyces cerevisiae [39], Sulfolobus solfataricus [40], T. litoralis [22] and Thermus thermophilus [18]. Three aminotransferases from H. thermophilus M. Kameya et al. 1880 FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS AGT activity. An AGT belonging to family I has only been found in T. litoralis [22]. This AGT has several characteristics similar to those of AT3, such as compa- rable specific activity (29 UÆmg )1 ) and strict substrate specificity. However, AT3 seems to be phylogenetically distant from the T. litoralis AGT, because of the low similarity between them: AT3 shows 26% identity to AGT, which is lower than the identity between AT3 and Thermus thermophilus GOT (31%). Furthermore, AT3 lacks several residues that are presumed to affect the substrate specificity of the T. litoralis AGT, e.g. Thr108 in the T. litoralis AGT is supposed to serve to the specificity for Ala [27], but this residue is replaced by Lys105 in AT3 (Fig. S2, see Supporting informa- tion). In addition, Leu19, which is located near the substrate in T. litoralis AGT, is replaced by Phe18 in AT3. These phylogenetic and structural differences suggest that AT3 has a substrate recognition mecha- nism distinct from that presumed in the T. litoralis AGT. The high similarity between the T. litoralis AGT and kynurenine aminotransferase II [28,29] has noted [27], and they also share similarity with a-aminoadipate aminotransferase [30] and aromatic aminotransferase [31]. These enzymes form a cluster in the phylogenetic tree, but AT3 is clearly located outside of the cluster (Fig. 4). This position also supports the phylogenetic dissimilarity between AT3 and T. litoralis AGT. Instead of these enzymes, AT3-like genes are found in genomes of Aquificales and c-ord-proteobacteria (a few of the homologues are depicted in Fig. 4). None of these homologues has been subjected to biochemical studies, and their enzymatic properties and functions are of interest. It has been shown that H. thermophilus has GGT activity and that this activity is derived from two enzymes, AT1 and AT2, with specific activities signifi- cantly higher than those of known GGTs. GGT activi- ties derived from AT1 and AT2 in the CFE are calculated to be 0.044 and 0.23 UÆmg )1 , respectively, from the specific activities and purification factors of each enzyme. These values indicate that most of the GGT activity can be attributed to AT2. Although functional analyses for aminotransferase in vivo are necessary to clarify their physiological roles, it can be speculated that AT2 plays a major role in the GGT reaction to synthesize Gly, and AT1 mainly serves in the GOT reaction. Although no bacterial GGT gene has been identified, GGT purification has been reported from two species, Rhodopseudomonas palustris and Lacto- bacillus plantarum [20,23]. AT1 and AT2 homo- logue genes are found in the genomes of both species (NP_949667 and NP_946142 in R. palustris; NP_785312 and NP_784469 in L. plantarum), and it is possible that the reported GGT activities were derived from these gene products. Further biochemical research is needed to clarify the distribution of these types of homologue with GGT activity. One of the noteworthy findings in this study is that AT2 and AT3 showed novel substrate specificities from the viewpoint of the well-established aminotransferase classification (Fig. 3), suggesting that the substrate specificity of aminotransferases is broader than previ- ously known. The enzymatic data obtained are expected to be of use in predicting the function of putative aminotransferase homologues that are found in the genome database. It remains unclear whether similar aminotransferases are distributed among a broad range of organisms or whether these enzymes evolved after the divergence from other bacteria early in evolution. Further biochemical study is needed to solve this question. Another intriguing question con- cerns glyoxylate metabolism in H. thermophilus. Although all three aminotransferases purified in this work use glyoxylate as their substrate, no enzymatic activities for the glyoxylate cycle were detected (not shown), and no genes encoding these enzymes are found in the genome. Glycolate oxidase (EC 1.1.3.15), which catalyses the conversion of glycolate into glyoxylate, may be one of the candidates for physio- logical glyoxylate synthesis. Several genes in the H. thermophilus genome share similarity with those of Table 2. Kinetic parameters of AT1, AT2 and AT3 (ND, not deter- mined). Enzyme Reaction Substrate K m (mM) Apparent V max (UÆmg )1 ) AT1 GOT Glu 20 ± 2 280 ± 10 Oxaloacetate a 0.38 ± 0.05 240 ± 10 Asp 2.3 ± 0.3 110 ± 10 2-OG 0.92 ± 0.04 110 ± 10 GGT Glu 1.5 ± 0.2 11 ± 0 Glyoxylate 4.3 ± 0.8 13 ± 1 AT2 GGT Glu 1.2 ± 0.1 64 ± 2 Glyoxylate 6.5 ± 1.8 70 ± 8 GPT Glu ND ND Pyruvate >50 >50 PSOT Phosphoserine 0.66 ± 0.07 17 ± 0 2-OG 1.9 ± 0.3 18 ± 1 AT3 AGT Ala 8.1 ± 0.1 23 ± 1 Glyoxylate 0.90 ± 0.08 24 ± 1 a The estimate of the K m value for oxaloacetate may be higher than the true value because of the instability of oxaloacetate at the assay temperature. M. Kameya et al. Three aminotransferases from H. thermophilus FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS 1881 glycolate oxidase. However, it remains unclear whether these genes actually encode glycolate oxidase and, fur- thermore, no genes have been found to explain how glycolate can be synthesized in this bacterium. More- over, elucidation of an unidentified carbon metabolism is needed to explain glyoxylate and Gly biosyntheses in this bacterium. Studies to clarify these pathways are in progress, and these may elucidate a novel central carbon metabolism in this bacterium. Materials and methods Bacterial strain and growth conditions Hydrogenobacter thermophilus TK-6 (IAM 12695, DSM 6534) was cultivated in an inorganic medium at 70 °C under a gas phase of 75% H 2 , 10% O 2 and 15% CO 2 ,as described previously [32]. Ammonium sulfate in the med- ium and CO 2 in the gas phase were the sole nitrogen and carbon sources, respectively. Aminotransferase assay Reaction mixtures contained 50 mm NaPO 4 (pH 8.0), 5 mm amino acid, 5 mm 2-oxo acid and the enzyme solution. If necessary, 100 lm PLP was added. For GOT, GGT, GPT, AGT and PSOT assays, substrate concentrations were mod- ified as follows: 100 mm Glu and 10 mm oxaloacetate or 10 mm Asp and 10 mm 2-OG for GOT, 20 mm Glu and 20 mm glyoxylate for GGT, 20 mm Glu and 30 mm pyru- vate for GPT, 40 mm Ala and 5 mm glyoxylate for AGT, and 10 mm phosphoserine and 10 mm 2-OG for PSOT. For the AT1 assay, the pH in the reaction mixture was changed to 7.2. The reaction mixtures were incubated at 70 °C, the optimum growth temperature of this bacterium. Amino- transferase activities were determined by measuring the production of the amino acid or the 2-oxo acid. To measure amino acid production, the reaction mix- tures were subjected to phenylthiocarbamyl derivatization, and the derivatized samples were analysed with a reverse- phase column (Inertsil ODS-3, 4.6 mm · 25 cm; GL Science, Tokyo, Japan) to determine the amino acid production [14]. One unit of activity was defined as the activity producing 1 lmol of an amino acid or a 2-oxo acid per minute. To measure 2-oxo acid production, 150 lL of the reac- tion mixtures were incubated at 70 °C and the reaction was stopped by the addition of 16 lL of 50% trichloroacetate. Denatured proteins were removed by centrifugation and the supernatants were neutralized with 74 lLof2m Tris ⁄ HCl (pH 8.0). The concentration of 2-OG was determined in reaction mixtures containing 50 mm NaPO 4 (pH 7.2), 0.2 mm NADH, 10 mm NH 4 Cl and 3 UÆmL )1 glutamate dehydrogenase from beef liver (Oriental Yeast, Tokyo, Japan) by measuring the absorbance change at 340 nm. Pyruvate concentration was determined in a reaction buffer containing 1 UÆmL )1 lactate dehydrogenase from rabbit Fig. 4. Phylogenetic tree of AT3, T. litoralis AGT homologues and subfamily Ic aminotransferases. The numbers at the nodes are bootstrap confidence values expressed as percentages of 1000 bootstrap replicates. The order of the divergence was presumed to be reliable only when the bootstrap values were above 50. The trees were constructed using the neighbor-joining method and showed the same overall topology as the trees constructed by the maximum likelihood method. In addition to the sequences in Fig. 3, those from the following organ- isms were used: Desulfovibrio vulgaris (YP_010112), Halorhodospira halophila (YP_001001722), human (NP_872603), Hydrogenivirga sp. 128-5-R1-1 (ZP_02176974), Hydrogenobaculum sp. Y04AAS1 (YP_002121232), Nitrococcus mobilis (ZP_01127658), Pyrococcus horikoshii (1X0M_A), Sulfurihydrogenibium sp. YO3AOP1 (YP_001931603) and Thermus thermophilus (BAC76939). AAAAT, a-aminoadipate aminotrans- ferase; KAT-II, kynurenine aminotransferase II. Three aminotransferases from H. thermophilus M. Kameya et al. 1882 FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS muscle (Roche, Basel, Switzerland) instead of NH 4 Cl and glutamate dehydrogenase. For the kinetic assay of GOT activity in the direction of Glu synthesis, a coupling method was applied using thermostable malate dehydrogenase from Thermus flavus (Sigma, St Louis, MO, USA). The reaction mixture con- tained 50 mm NaPO 4 (pH 7.2), 10 mm Asp, 5 mm 2-oxoglutarate, 0.2 mm NADH, 1 UÆmL )1 malate dehydro- genase and the enzyme solution. The mixture was incubated at 70 °C, and the absorbance was monitored at 340 nm to estimate the decrease in NADH. Enzyme purification AT1 was purified from 10 g of wet cells. Active fractions were selected according to GOT and GPT activities. The cells were washed with 20 mm Tris ⁄ HCl buffer (pH 8.0) and disrupted by sonication. Cell debris was removed by centri- fugation at 100 000 g for 1 h. The supernatant, which was designated CFE, was applied to a DE52 open column (25 mm · 15 cm; Whatman, Brentford, Middlesex, UK) equilibrated with 20 mm Tris ⁄ HCl buffer (pH 8.0) contain- ing 1 mm MgCl 2 . After the elution of bound proteins with buffer containing 1 m NaCl, ammonium sulfate was added to the fractions obtained to 30% saturation, and the samples were applied to a Butyl-Toyopearl column (22 mm · 15 cm; Tosoh, Tokyo, Japan) equilibrated with 20 mm Tris ⁄ HCl buffer (pH 8.0) containing 1 mm MgCl 2 and ammonium sul- fate at 30% saturation. This and subsequent chromatogra- phy steps were performed using an A ¨ KTA purifier system (GE Healthcare, Piscataway, NJ, USA). Proteins were eluted with a gradient of ammonium sulfate from 30% to 0% over 230 mL at a flow rate of 4 mLÆmin )1 . The active fractions were dialysed against 20 mm Tris ⁄ HCl buffer (pH 8.0) con- taining 1 mm MgCl 2 , and were applied to a DEAE-Toyo- pearl column (22 mm · 15 cm; Tosoh) equilibrated with 20 mm Tris ⁄ HCl buffer (pH 8.0) containing 1 mm MgCl 2 . Proteins were eluted with a gradient of NaCl from 0 to 1 m over 380 mL at a flow rate of 4 mLÆmin )1 . The active frac- tions were dialysed against 20 mm Tris ⁄ HCl buffer (pH 8.0) containing 1 mm MgCl 2 , and were applied to a MonoQ HR 5 ⁄ 5 column (bed volume, 1 mL; GE Healthcare) equilibrated with 20 mm Tris ⁄ HCl buffer (pH 8.0) containing 1 mm MgCl 2 . Proteins were eluted with a gradient of NaCl from 0 to 1 m over 40 mL at a flow rate of 0.5 mLÆmin )1 . The active fractions were designated purified AT1, and stored at )80 °C until use. AT2 was purified from 20 g of wet cells. Active fractions were selected according to GGT and GPT activities. CFE was prepared from the cells and applied to the DE52 column, Butyl-Toyopearl column and DEAE-Toyopearl column, as described above. The active fractions were applied to a CHT Ceramic Hydroxyapatite column (16 mm · 11 cm; Bio-Rad, Hercules, CA, USA) equilibrated with 1 mm KPO 4 buffer (pH 7.0). Proteins were eluted with a gradient of KPO 4 buffer from 1 to 400 mm over 90 mL at a flow rate of 3 mLÆ min )1 . The active fractions were dialysed against 20 mm Tris ⁄ HCl buffer (pH 8.0) containing 1 mm MgCl 2 , and were applied to the MonoQ column in the same way as AT1. The active fractions were designated purified AT2, and stored at )80 °C until use. AT3 was purified from 40 g of wet cells. Active fractions were selected according to GGT activity. CFE was pre- pared from the cells and applied to the DE52 column, Butyl-Toyopearl column, DEAE-Toyopearl column, CHT Ceramic Hydroxyapatite column and MonoQ column in the same way as AT2. Ammonium sulfate was added to the fractions obtained to 30% saturation, and the samples were applied to a Phenyl Superose column (bed volume, 1 mL; GE Healthcare) equilibrated with 20 mm Tris ⁄ HCl buffer (pH 8.0) containing 1 mm MgCl 2 and ammonium sulfate at 30% saturation. Proteins were eluted with a gradient of ammonium sulfate from 30% to 0% over 15 mL at a flow rate of 0.5 mLÆmin )1 . The active fractions were designated purified AT3, and stored at –80 °C until use. N-terminal amino acid sequencing The N-terminal amino acid sequences of purified amin- otransferases were determined by Procise 492HT (Applied Biosystems, Foster City, CA, USA) from a blotted mem- brane [0.2 lm Sequi-Blot poly(vinylidene) difluoride; Bio- Rad]. Protein assay Protein concentrations were measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). A calibration curve was plotted using bovine serum albumin as a standard protein. Gel filtration For the estimation of the molecular mass, gel filtration was performed using a Superose 6 HR 10 ⁄ 30 column (GE Healthcare) or a Shim-pack Diol-300 column (Shimadzu, Kyoto, Japan) equilibrated with 20 mm Tris ⁄ HCl (pH 8.0) buffer containing 1 mm MgCl 2 and 150 mm NaCl at flow rate of 0.5 or 1 mLÆmin )1 , respectively. Gel Filtration Stan- dard (Bio-Rad) was used as a molecular maker for calibra- tion. Each measurement of standards or samples was performed in triplicate. Phylogenetic tree construction Amino acid sequences were aligned using the muscle program [33]. After gap regions had been removed, phylo- genetic trees were constructed by the neighbor-joining method or the maximum likelihood method using phylip 3.67 [34]. M. Kameya et al. Three aminotransferases from H. thermophilus FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS 1883 Nucleotide sequence accession numbers Nucleotide sequences of AT1, AT2 and AT3 have been deposited in the DDBJ ⁄ EMBL ⁄ GenBank nucleotide sequence database under accession numbers AB536750, AB536751 and AB536752, respectively. Acknowledgement This work was supported by a Grant-in-Aid for JSPS Fellows (20-6284). References 1 Ilag LL, Jahn D, Eggertsson G & So ¨ ll D (1991) The Escherichia coli hemL gene encodes glutamate 1-semial- dehyde aminotransferase. J Bacteriol 173, 3408–3413. 2 Ward DE, Kengen SW, van Der Oost J & de Vos WM (2000) Purification and characterization of the alanine aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus and its role in alanine production. J Bacteriol 182, 2559–2566. 3 Liepman AH & Olsen LJ (2003) Alanine aminotransfer- ase homologs catalyze the glutamate:glyoxylate amino- transferase reaction in peroxisomes of Arabidopsis. Plant Physiol 131, 215–227. 4 Mehta PK, Hale TI & Christen P (1993) Aminotransfe- rases: demonstration of homology and division into evolutionary subgroups. Eur J Biochem 214, 549–561. 5 Jensen RA & Gu W (1996) Evolutionary recruitment of biochemically specialized subdivisions of Family I within the protein superfamily of aminotransferases. J Bacteriol 178, 2161–2171. 6 Marienhagen J, Kennerknecht N, Sahm H & Eggeling L (2005) Functional analysis of all aminotransferase proteins inferred from the genome sequence of Coryne- bacterium glutamicum. J Bacteriol 187, 7639–7646. 7 Ali V & Nozaki T (2006) Biochemical and functional characterization of phosphoserine aminotransferase from Entamoeba histolytica, which possesses both phos- phorylated and non-phosphorylated serine metabolic pathways. Mol Biochem Parasitol 145, 71–83. 8 Muratore KE, Srouji JR, Chow MA & Kirsch JF (2008) Recombinant expression of twelve evolutionarily diverse subfamily Ia aminotransferases. Protein Expr Purif 57, 34–44. 9 Pitulle C, Yang Y, Marchiani M, Moore ER, Siefert JL, Aragno M, Jurtshuk P Jr & Fox GE (1994) Phylo- genetic position of the genus Hydrogenobacter. Int J Syst Bacteriol 44, 620–626. 10 Miura A, Kameya M, Arai H, Ishii M & Igarashi Y (2008) A soluble NADH-dependent fumarate reductase in the reductive tricarboxylic acid cycle of Hydrogeno- bacter thermophilus TK-6. J Bacteriol 190, 7170–7177. 11 Aoshima M & Igarashi Y (2008) Nondecarboxylating and decarboxylating isocitrate dehydrogenases: oxalo- succinate reductase as an ancestral form of isocitrate dehydrogenase. J Bacteriol 190, 2050–2055. 12 Yamamoto M, Ikeda T, Arai H, Ishii M & Igarashi Y (2010) Carboxylation reaction catalyzed by 2-oxogluta- rate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus. Extremophiles 14, 79–85. 13 Ikeda T, Yamamoto M, Arai H, Ohmori D, Ishii M & Igarashi Y (2010) Enzymatic and electron paramagnetic resonance studies of anabolic pyruvate synthesis by pyruvate: ferredoxin oxidoreductase from Hydrogenob- acter thermophilus. FEBS J 277, 501–510. 14 Kameya M, Ikeda T, Nakamura M, Arai H, Ishii M & Igarashi Y (2007) A novel ferredoxin-dependent gluta- mate synthase from the hydrogen-oxidizing chemoauto- trophic bacterium Hydrogenobacter thermophilus TK-6. J Bacteriol 189 , 2805–2812. 15 Kameya M, Arai H, Ishii M & Igarashi Y (2006) Purifi- cation and properties of glutamine synthetase from Hydrogenobacter thermophilus TK-6. J Biosci Bioeng 102, 311–315. 16 Yagi T, Kagamiyama H, Nozaki M & Soda K (1985) Glutamate-aspartate transaminase from microorgan- isms. Methods Enzymol 113, 83–89. 17 Sung MH, Tanizawa K, Tanaka H, Kuramitsu S, Kagamiyama H & Soda K (1990) Purification and characterization of thermostable aspartate aminotrans- ferase from a thermophilic Bacillus species. J Bacteriol 172, 1345–1351. 18 Okamoto A, Kato R, Masui R, Yamagishi A, Oshima T & Kuramitsu S (1996) An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8. J Biochem 119, 135–144. 19 Ishiguro M, Suzuki M, Takio K, Matsuzawa T & Titani K (1991) Complete amino acid sequence of rat liver cytosolic alanine aminotransferase. Biochemistry 30, 6048–6053. 20 Yamaguchi H, Ohtani M, Amachi S, Shinoyama H & Fujii T (2003) Some properties of glycine aminotrans- ferase purified from Rhodopseudomonas palustris No. 7 concerning extracellular porphyrin production. Biosci Biotechnol Biochem 67, 783–789. 21 Liepman AH & Olsen LJ (2001) Peroxisomal ala- nine:glyoxylate aminotransferase (AGT1) is a photore- spiratory enzyme with multiple substrates in Arabidopsis thaliana. Plant J 25, 487–498. 22 Sakuraba H, Kawakami R, Takahashi H & Ohshima T (2004) Novel archaeal alanine:glyoxylate aminotransfer- ase from Thermococcus litoralis. J Bacteriol 186, 5513– 5518. 23 Galas E & Florianowicz T (1975) l-Glutamate-glyoxy- late aminotransferase in Lactobacillus plantarum. Acta Microbiol Pol B 7, 243–252. Three aminotransferases from H. thermophilus M. Kameya et al. 1884 FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS 24 Battchikova N, Himanen JP, Ahjolahti M & Korpela T (1996) Phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus: purification, gene cloning and sequencing. Biochim Biophys Acta 1295, 187–194. 25 Hester G, Stark W, Moser M, Kallen J, Markovic- Housley Z & Jansonius JN (1999) Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A ˚ resolution: comparison of the unligated enzyme and a complex with a-methyl-l-glutamate. J Mol Biol 286, 829–850. 26 de la Torre F, De Santis L, Sua ´ rez MF, Crespillo R & Ca ´ novas FM (2006) Identification and functional analy- sis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism. Plant J 46, 414–425. 27 Sakuraba H, Yoneda K, Takeuchi K, Tsuge H, Katu- numa N & Ohshima T (2008) Structure of an archaeal alanine:glyoxylate aminotransferase. Acta Crystallogr D: Biol Crystallogr 64, 696–699. 28 Chon H, Matsumura H, Koga Y, Takano K & Kanaya S (2005) Crystal structure of a human kynurenine ami- notransferase II homologue from Pyrococcus horikoshii OT3 at 2.20 A ˚ resolution. Proteins 61, 685–688. 29 Chon H, Matsumura H, Shimizu S, Maeda N, Koga Y, Takano K & Kanaya S (2005) Overproduction and pre- liminary crystallographic study of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3. Acta Crystallogr Sect F: Struct Biol Cryst Commun 61, 319–322. 30 Miyazaki T, Miyazaki J, Yamane H & Nishiyama M (2004) alpha-Aminoadipate aminotransferase from an extremely thermophilic bacterium, Thermus thermophi- lus. Microbiology 150, 2327–2334. 31 Andreotti G, Cubellis MV, Nitti G, Sannia G, Mai X, Adams MW & Marino G (1995) An extremely thermostable aromatic aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus. Biochim Biophys Acta 1247, 90–96. 32 Shiba H, Kawasumi T, Igarashi Y, Kodama T & Minoda Y (1982) The deficient carbohydrate metabolic pathways and the incomplete tricarboxylic-acid cycle in an obligately autotrophic hydrogen-oxidizing bacterium. Agric Biol Chem 46, 2341–2345. 33 Edgar RC (2004) MUSCLE: multiple sequence align- ment with high accuracy and high throughput. Nucleic Acids Res 32, 1792–1797. 34 Felsenstein J (2005) PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Wash- ington, Seattle, WA. 35 Purdue PE, Lumb MJ, Fox M, Griffo G, Hamon- Benais C, Povey S & Danpure CJ (1991) Characteri- zation and chromosomal mapping of a genomic clone encoding human alanine:glyoxylate aminotransferase. Genomics 10, 34–42. 36 Ward DE, de VosWM & van der Oost J (2002) Molecu- lar analysis of the role of two aromatic aminotransfe- rases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus. Archaea 1, 133–141. 37 Horio Y, Tanaka T, Taketoshi M, Nagashima F, Tanase S, Morino Y & Wada H (1988) Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli. J Biochem 103, 797–804. 38 Oda T, Miyajima H, Suzuki Y & Ichiyama A (1987) Nucleotide sequence of the cDNA encoding the precur- sor for mitochondrial serine:pyruvate aminotransferase of rat liver. Eur J Biochem 168, 537–542. 39 Morin PJ, Subramanian GS & Gilmore TD (1992) AAT1, a gene encoding a mitochondrial aspartate ami- notransferase in Saccharomyces cerevisiae. Biochim Bio- phys Acta 1171, 211–214. 40 Marino G, Nitti G, Arnone MI, Sannia G, Gambacorta A & De Rosa M (1988) Purification and characteriza- tion of aspartate aminotransferase from the thermoacid- ophilic archaebacterium Sulfolobus solfataricus. J Biol Chem 263, 12305–12309. Supporting information The following supplementary material is available: Fig. S1. Multiple sequence alignment of AT2, PSOT and AGT. Fig. S2. Multiple sequence alignment of AT3, the T. litoralis AGT and family I aminotransferases. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. M. Kameya et al. Three aminotransferases from H. thermophilus FEBS Journal 277 (2010) 1876–1885 ª 2010 The Authors Journal compilation ª 2010 FEBS 1885 . Purification of three aminotransferases from Hydrogenobacter thermophilus TK-6 – novel types of alanine or glycine aminotransferase Enzymes and catalysis Masafumi Kameya, Hiroyuki Arai, Masaharu Ishii and. the first report of the primary structure of bacterial gluta- mate:glyoxylate aminotransferase and alanine: glyoxylate aminotransferase, and demonstrates the presence of novel types of aminotransferase. aminotransferases. Purification and phylogenetic analysis of aminotransferases Enzymes that exhibited GOT, GPT, GGT or AGT activity were subjected to purification, and three enzymes (AT1, AT2 and