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J Bacteriol. Aug 2004; 186(16): 5513–5518.
PMCID: PMC490878

Novel Archaeal Alanine:Glyoxylate Aminotransferase from Thermococcus litoralis


A novel alanine:glyoxylate aminotransferase was found in a hyperthermophilic archaeon, Thermococcus litoralis. The amino acid sequence of the enzyme did not show a similarity to any alanine:glyoxylate aminotransferases reported so far. Homologues of the enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced.

Alanine:glyoxylate aminotransferase (AGT; EC catalyzes the transfer of the α-amino group of l-alanine to glyoxylate, forming glycine and pyruvate. The enzyme is widely distributed in eucarya (16, 26, 33). One of the functions of AGT is the detoxification of glyoxylate. In humans, primary hyperoxaluria type 1 is characterized as an autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal AGT enzyme (6, 17). With respect to bacteria, aminotransferases that catalyze transamination between l-alanine and glyoxylate have been found in only a few organisms (9, 12). These bacterial enzymes have not yet been well characterized, and their physiological role remains obscure. On the other hand, serine:glyoxylate aminotransferase (SGT; EC has been found in methylotrophic bacteria such as Hyphomicrobium methylovorum (8, 10). Although H. methylovorum SGT does not catalyze transamination between l-alanine and glyoxylate, the enzyme shows 27% amino acid sequence identity to human AGT (8). The enzyme is reported to play important roles in the serine pathway for the assimilation of one-carbon compounds: it is involved in the formation of an acceptor (glycine) for a one-carbon unit and in the conversion of l-serine to hydroxypyruvate (2). The presence of AGT has not yet been described for Archaea, the third domain of living organisms.

Thermococcus litoralis is a typical marine hyperthermophilic archaeon that can grow at temperatures near the boiling temperature of water (15). T. litoralis is known to anaerobically utilize maltose and cellobiose as carbon and energy sources in a modified Embden-Meyerhof pathway, similar to many other strains of the Thermococcales order (27, 30, 31). This organism also utilizes peptides and pyruvate for growth. This suggests the presence of various amino acid and organic acid metabolic pathways in the hyperthermophiles. However, studies of these metabolic pathways and their related enzymes are very limited. Recently, members of our laboratory found the presence of a novel glyoxylate reductase in T. litoralis (22). This was the first biochemical evidence of an enzyme involved in glyoxylate metabolism in hyperthermophilic archaea (22). During the course of screening for enzymes associated with glyoxylate metabolism, we found a high level of AGT activity in the crude extract of T. litoralis. The presence of both glyoxylate reductase and AGT in T. litoralis indicates that glyoxylate metabolism may be present in the organism. To obtain insight into this metabolic pathway, we purified AGT from T. litoralis and characterized it. In addition, we cloned and sequenced the gene encoding the enzyme.

T. litoralis DSM5473 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). T. litoralis was grown as previously described (21). AGT activity was assayed by the method of Rowsell et al. (24). Pyruvate formed by the AGT reaction was assayed by spectrophotometric NADH determination using lactate dehydrogenase (EC The assay mixture contained, unless specified otherwise, 20 mM l-alanine, 5 mM glyoxylate, 20 μM pyridoxal 5′-phosphate, 100 mM potassium phosphate buffer (pH 7.5), and the enzyme preparation in a total volume of 0.4 ml. After incubation for an appropriate time at 37°C, the reaction was stopped with 50 μl of trichloroacetate (50% [wt/vol]), and the mixture was centrifuged (12,000 × g for 10 min). A 400-μl portion of the supernatant was neutralized by the addition of 500 μl of Tris-HCl (2 M) and was used for the spectrophotometric assay of pyruvate. The protein concentration was measured by the method of Bradford (3), with bovine serum albumin as the standard.

A typical result for the purification of AGT from a crude extract of T. litoralis is summarized in Table Table1.1. About 20 g (wet weight) of cells was used as a starting material for the purification. Unless otherwise indicated, the operations were performed at room temperature (~25°C). The cells were suspended in buffer A (20 mM potassium phosphate buffer [pH 7.5] containing 20% glycerol, 1 mM EDTA, and 0.1 mM dithiothreitol) supplemented with 1 mM phenylmethylsulfonyl fluoride and disrupted by incubation for 90 min at 37°C in the presence of lysozyme (1 mg/ml) and DNase (0.1 mg/ml) (4). The cell debris was removed by centrifugation at 15,000 × g for 40 min, and the supernatant solution was used as a crude extract. The crude extract was placed on a DEAE-Toyopearl (Tosoh, Tokyo, Japan) column equilibrated with buffer A. AGT was eluted with a linear gradient of 0 to 0.5 M NaCl in buffer A. The active fractions were pooled and the enzyme was dialyzed against buffer A. Solid (NH4)2SO4 was added to the enzyme solution up to 1.3 M. The enzyme solution was loaded onto a phenyl-Toyopearl (Tosoh) column that was previously equilibrated with buffer A supplemented with 1.3 M (NH4)2SO4. The enzyme was eluted with a linear gradient of 1.3 to 0 M (NH4)2SO4 in buffer A. The active fractions were collected and dialyzed against buffer A. Preparative polyacrylamide gel electrophoresis was performed with a 7.5% acrylamide gel according to a previously described method (20). The protein was extracted from the gel by centrifugation at 20,000 × g for 10 min, and the extraction was repeated twice. The supernatant solution exhibiting enzyme activity was concentrated by ultrafiltration. Solid (NH4)2SO4 was added to the enzyme solution up to 1.3 M. The solution was loaded onto a butyl-Toyopearl (Tosoh) column that was previously equilibrated with buffer A supplemented with 1.3 M (NH4)2SO4. The enzyme was eluted with a linear gradient of 1.3 to 0 M (NH4)2SO4 in buffer A. The active fractions were collected and dialyzed against buffer B (20 mM potassium phosphate buffer [pH 6.5] containing 20% glycerol, 1 mM EDTA, and 0.1 mM dithiothreitol). The enzyme solution was loaded onto a red-Sepharose CL-4B (19) column equilibrated with buffer B. The enzyme was eluted without adsorption to the affinity resin. The active fractions were collected and dialyzed against buffer B. The enzyme solution, concentrated by ultrafiltration, was subjected to fast-performance liquid chromatography on an Uno Q (Bio-Rad) column equilibrated with buffer B. The enzyme was eluted with a linear gradient of 0 to 0.5 M NaCl in buffer B. The active fractions were pooled, dialyzed against buffer A, and used as the final preparation of AGT. The final preparation was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (13) (Fig. (Fig.1).1). The molecular mass of the native enzyme was estimated to be about 170 kDa by native gradient PAGE (32; also data not shown), and the molecular mass of the subunit was about 42 kDa by SDS-PAGE (Fig. (Fig.1),1), so the enzyme must exist as a homotetramer. The subunit structure of bacterial AGT has not yet been reported. In eucaryotes, peroxisomal AGTs are usually homodimers, with a subunit size of 38 to 45 kDa, whereas mitochondrial AGTs have a homotetrameric structure, with a subunit size of 50 to 56 kDa (16, 23). H. methylovorum SGT, which shows 27% amino acid identity to human peroxisomal AGT, has been reported to have a homotetrameric structure with a subunit size of 40 kDa (10). In this regard, T. litoralis AGT is similar to bacterial SGT.

FIG. 1.
SDS-PAGE of purified AGT from T. litoralis. Lane 1, marker proteins (New England BioLabs) with molecular masses indicated (in kilodaltons); lane 2, purified AGT from T. litoralis crude extract.
Purification of AGT from T. litoralis

The N-terminal amino acid sequence of the enzyme was analyzed with a Shimadzu model PPSQ-10 protein sequencer as described previously (22) and was determined to be MDYTKYLAGRANWIKG. The optimum pH of AGT activity was about pH 7.5. The enzyme was stable up to 80°C during incubation for 10 min, but the remaining activity after incubation at 90°C was <50%. The activity of AGT increased with increases in temperature from 37 to 90°C. The assay could not be performed above 90°C because of the instability of glyoxylate. The effects of some chemicals on enzyme activity were examined. Enzyme assays were performed with standard reaction mixtures containing a 1 or 10 mM concentration of the tested compound. A significant effect on activity was not observed with 10 mM EDTA and 10 mM p-chloromercuribenzoic acid, but the enzyme was significantly inhibited by 10 mM l-penicillamine (86% inhibition) and 10 mM hydroxylamine (100% inhibition). In general, the pyridoxal 5′-phosphate enzyme is known to be sensitive to some carbonyl reagents, such as penicillamine and hydroxylamine. Saccharomyces cerevisiae AGT and H. methylovorum SGT are significantly inhibited (70 to 80% inhibition) by 0.5 to 1 mM hydroxylamine (10, 33). However, T. litoralis AGT appears to be less sensitive to the reagent because the enzyme was not inhibited by 1 mM l-penicillamine and only slightly inhibited by 1 mM hydroxylamine (27% inhibition).

The substrate specificity of the enzyme was then examined. When glyoxylate was used as an amino acceptor, the reactivities of l-alanine, l-serine, l-valine, l-leucine, l-isoleucine, l-threonine, l-asparagine, l-glutamine, l-aspartate, l-glutamate, l-histidine, l-methionine, l-phenylalanine, l-tyrosine, l-tryptophan, l-proline, l-ornithine, d-alanine, β-alanine, d-serine, and 4-aminobutyrate as amino donors were tested by measuring the formation of glycine from glyoxylate by use of an amino acid analyzer (Beckman system 6300E). When l-alanine was used as an amino donor, the reactivities of hydroxypyruvate, p-hydroxyphenylpyruvate, and oxaloacetate as amino acceptors were examined by measuring the formation of l-serine, l-tyrosine, and l-aspartate, respectively. Transamination between l-serine and pyruvate was examined by measuring the formation of l-alanine. The assay mixture comprised 20 mM amino acid, 5 mM 2-oxo acid, 20 μM pyridoxal 5′-phosphate, 100 mM potassium phosphate buffer (pH 7.5), and the enzyme preparation in a total volume of 0.4 ml. l-Tyrosine was used at a concentration of 6 mM due to its insolubility. Transamination between l-alanine and 2-oxoglutarate was analyzed by enzymatic measurement of the pyruvate formed, as for AGT, but 2-oxoglutarate was used instead of glyoxylate in the standard assay mixture. When glyoxylate was used as the amino group acceptor, T. litoralis AGT showed a strict specificity for l-alanine as the amino group donor. l-Valine, l-leucine, l-isoleucine, l-threonine, l-asparagine, l-glutamine, l-aspartate, l-glutamate, l-histidine, l-methionine, l-phenylalanine, l-tyrosine, l-tryptophan, l-proline, l-ornithine, d-alanine, β-alanine, d-serine, and 4-aminobutyrate were inert. When l-serine was used as the substrate instead of l-alanine, only a trace amount of glycine from glyoxylate could be detected. When l-alanine was used as the amino group donor, the enzyme also showed a strict specificity for glyoxylate as the amino group acceptor. Hydroxypyruvate, p-hydroxyphenylpyruvate, 2-oxoglutarate, and oxaloacetate were inert as amino group acceptors. The enzyme did not catalyze transamination between l-serine and pyruvate. In general, AGT shows a high transamination activity between l-serine and glyoxylate (or pyruvate) (18). Therefore, the strict specificity for l-alanine as an amino group donor and for glyoxylate as an amino group acceptor is one of the remarkable characteristics of T. litoralis AGT. Typical Michaelis-Menten kinetics were observed for both substrates. The Km values for l-alanine and glyoxylate were determined to be 0.92 and 0.32 mM, respectively, from Lineweaver-Burk plots (5). Reverse transamination of the enzyme with 20 mM glycine and 5 mM pyruvate was examined by measuring the formation of l-alanine from pyruvate by use of an amino acid analyzer. A negative result was obtained; the enzyme was found to catalyze an irreversible transamination between l-alanine and glyoxylate, similar to AGTs from other sources (16, 33).

For screening of the AGT gene, an oligonucleotide mixture probe (a mixture of 128 sequences [5′-ATGGAYTAYACNAARTAYYT-3′]) was synthesized based on the N-terminal amino acid sequence (underlined) of the enzyme as determined in this study (MDYTKYLAGRANWIKG). The probe was labeled with [γ-32P]ATP by the use of T4 polynucleotide kinase and Megalabel (Takara Biochemicals, Kyoto, Japan), purified through a ProbeQuant G-50 Micro column (Amersham Bioscience, Tokyo, Japan), and used as a specific probe for colony and Southern hybridizations. To obtain a clone containing the AGT gene, we prepared T. litoralis chromosomal DNA as previously described (22), digested it with several restriction enzymes, and then separated the fragments by 0.8% agarose gel electrophoresis. The separated DNA fragments in the agarose gel were subjected to Southern blotting with a 32P-labeled probe. An approximately 4-kb XbaI fragment that gave a positive signal by Southern hybridization was extracted from the gel. The fragment was inserted into the XbaI site of plasmid pUC18, and then Escherichia coli JM109 cells were transformed. Transformants were selected on a Luria-Bertani plate (28) containing ampicillin (0.003%). The colonies were transferred and fixed on Hybond N+ nylon membranes (Amersham Bioscience). Prehybridization and hybridization with the 32P-labeled probe were performed according to the manufacturer's instructions. Positive clones were detected with a BAS-1500 system (Fuji Film, Tokyo, Japan). After screening of the recombinant plasmids by Southern hybridization, a positive plasmid containing the 4.2-kbp XbaI fragment, pNN10, was isolated. From a further Southern hybridization analysis, the AGT coding sequence existed within the XbaI-BamHI fragment in the insert DNA of pNN10. The 2.4-kbp XbaI-BamHI fragment was subcloned into the XbaI-BamHI site of pUC18, and p18XB was thus obtained. p18XB was used as a template for the DNA sequence. The nucleotides were sequenced by the dideoxy chain termination method (29) in an automated DNA sequencer (377A; Applied Biosystems). Sequence data were analyzed with Genetyx-SV/RC9.0 software (Software Development, Tokyo, Japan).

Sequence analysis revealed an open reading frame whose deduced amino acid sequence corresponded to the determined N-terminal protein sequence. The complete nucleotide sequence of the T. litoralis AGT gene comprises 1,221 bp coding for 407 amino acids with a calculated molecular weight of 45,169, which corresponds to the subunit molecular mass of about 42 kDa determined by SDS-PAGE. E. coli JM109 cells carrying p18XB exhibited AGT activity which was not lost by incubation at 80°C for 10 min. This confirmed that the gene encoded T. litoralis AGT (data not shown). Upon amino acid sequence alignment, T. litoralis AGT did not show similarity to any AGTs or SGTs that have been reported so far. On the other hand, homologues of the T. litoralis enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced. For example, T. litoralis AGT exhibited 44.9, 42.0, 41.8, 41.2, 41.1, 39.8, and 38.3% identities with the aminotransferase (ORF PAB2227) of Pyrococcus abyssi, aminotransferase class I (PAE2315) of Pyrobaculum aerophilum, a hypothetical protein (PH0207) of Pyrococcus horikoshii, a hypothetical kynulenine/alpha-aminoadipate aminotransferase (ST1411) of Sulfolobus tokodaii, the aspartate aminotransferase (SSO0104) of Sulfolobus solfataricus, a putative aspartate aminotransferase (PF0121) of Pyrococcus furiosus, and the aminotransferase (APE0169) of Aeropyrum pernix, respectively. In addition, homologues of the enzyme are also present in a few species of mesophilic archaea: the enzyme exhibited 38.1 and 38.9% identities with the aspartate aminotransferase-related protein (Ta1193) of Thermoplasma acidophilum and the aspartate aminotransferase (TVG0393535) of Thermoplasma volcanium, respectively. Genome information for these organisms is available at the Kyoto Encyclopedia of Genes and Genomes (http://www.genome.ad.jp/kegg/).

It has been reported that ORF PF0121 of Pyrococcus furiosus encodes an aromatic aminotransferase (AroAT-1) (1). This enzyme catalyzes the transamination of aromatic amino acids, using 2-oxoglutarate as the amino group acceptor. As described above, the enzyme exhibits relatively high sequence identity (39.8%) to T. litoralis AGT. Thus, we examined the aromatic aminotransferase activity of T. litoralis AGT by using a method based on the arsenate-catalyzed formation of aromatic 2-oxo acid-enol-borate complexes (7). However, activity was not detected for the transamination of l-phenylalanine, l-tyrosine, and l-tryptophan when 2-oxoglutarate was used as the amino group acceptor. This indicates that T. litoralis AGT is a totally different kind of enzyme from P. furiosus AroAT-1, despite their relatively high sequence identity.

With a phylogenetic aminotransferase family tree constructed by comparing the sequences of 51 aminotransferases, Mehta et al. (14) observed that aminotransferases can be classified into four subgroups as follows: subgroup I comprises aspartate, alanine, tyrosine, histidinol-phosphate, and phenylalanine aminotransferases; subgroup II comprises acetylornithine, ornithine, ω-amino acid, 4-aminobutyrate, and diaminopelargonate aminotransferases; subgroup III comprises d-alanine and branched-chain amino acid aminotransferases; and subgroup IV comprises serine and phosphoserine aminotransferases. We constructed a phylogenetic tree based on amino acid sequence alignment of the aminotransferases reported by Mehta et al. (14) and T. litoralis AGT. The sequence alignment and a neighbor-joining (25) tree of the aligned sequences were generated with Clustal X (11). The sequences used are summarized in Table Table2,2, and the phylogenetic tree is shown in Fig. Fig.2.2. The total arrangement of the phylogenetic tree was very similar to that reported by Mehta et al. (14), and the aminotransferases formed four clusters. Eucaryal AGT (SerATh) was classified into subgroup IV, as shown by Mehta et al. (14), and bacterial SGT (hmetSGT) also belonged to the same group (Fig. (Fig.2).2). On the other hand, surprisingly, T. litoralis AGT (TlitAGT) was clustered with Sulfolobus solfataricus aspartate aminotransferase (AspATss; ORF SSO0897), Bacillus sp. aspartate aminotransferase (AspATbs), rat alanine aminotransferase (AlaATr), and rat tyrosine aminotransferase (TyrATr). All of these enzymes are classified into subgroup I (Fig. (Fig.2).2). This indicates that the T. litoralis AGT is a novel type of AGT and that it might have evolved from an origin distinct from eucaryal AGTs and bacterial SGTs.

FIG. 2.
Phylogenetic tree of aminotransferases. The tree was constructed by the neighbor-joining method by ClustalX (11) based on an amino acid sequence alignment of the aminotransferases reported by Mehta et al. (14). T. litoralis AGT (TlitAGT) and H. methylovorum ...
Aminotransferases used for sequence alignment

We have demonstrated the presence of a novel glyoxylate reductase in T. litoralis (22). This enzyme catalyzes the reduction of glyoxylate in the presence of NADH. The enzyme activity for the oxidation of glycolate is very low compared to the forward reaction and could be detected only by activity staining (22). The presence of the glyoxylate reductase and AGT in T. litoralis suggests that glyoxylate metabolism may function in this organism, although the details of the metabolism are still unclear. Recently, the presence of a glyoxylate cycle in the aerobic thermoacidophilic archaeon Sulfolobus acidocaldarius was reported (34). Two key enzymes for the glyoxylate cycle, isocitrate lyase and malate synthase, were purified and characterized from the organism. Thus, we examined the presence of both of these enzyme activities in the crude extract of T. litoralis. We performed an assay as described previously (34); however, the activities were not detected. This suggests that a different metabolic pathway that includes glyoxylate as an intermediate might be present in T. litoralis. Our next aim is to shed light on the origin of glyoxylate and to clarify the physiological role of AGT and glyoxylate reductase.

Nucleotide sequence accession number.

The nucleotide sequence of T. litoralis AGT has been submitted to the DDBJ, GenBank, and EMBL data banks under accession number AB033996.


We thank Naoki Nunoura-Kominato for his technical assistance.

This study was supported in part by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science (no. 15560677) and the “National Project on Protein Structural and Functional Analysis” promoted by the Ministry of Education, Science, Sports, Culture, and Technology of Japan.


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