Journal of Bacteriology, August 2004, p . 5513-5518, Vol . 186, No . 16

Novel Archaeal Alanine:Glyoxylate Aminotransferase from Thermococcus litoralis

Haruhiko Sakuraba, Ryushi Kawakami, Hajime Takahashi, and Toshihisa Ohshima^*

Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan

Received 14 February 2004/ Accepted 5 May 2004

 
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 2.6.1.44) catalyzes the transfer of the -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 2.6.1.45) 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 1.1.1.27) . 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 x 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 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 x 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 x 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 . 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 . 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 .

 
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, ß-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, ß-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 K_m 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 [-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, -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 2, and the phylogenetic tree is shown in Fig . 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. 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 . 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 .

 
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 .

 
* Corresponding author . Mailing address: Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan . Phone: 81 88 656 7518 . Fax: 81 88 656 9071 . E-mail: ohshima@bio.tokushima-u.ac.jp .

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