Purification of three aminotransferases from Hydrogenobacter thermophilus TK‐6 – novel types of alanine or glycine aminotransferase

Abstract

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 aminotransferase, 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 glutamate:glyoxylate aminotransferases are members of family Iγ. 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 lineages within family I, because neither high sequence similarity nor putative substrate‐binding residues are shared by these two enzymes. To our knowledge, this study is the first report of the primary structure of bacterial glutamate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase, and demonstrates the presence of novel types of aminotransferase phylogenetically distinct from known eukaryotic and archaeal isozymes.

Discovery and Analysis of Cofactor-dependent Phosphoglycerate Mutase Homologs as Novel Phosphoserine Phosphatases in Hydrogenobacter thermophilus

“…Evidence for the existence of a phosphoserine anabolic pathway in this bacterium has also been provided from the annotation of its complete genome sequence (13), which indicates that 3-phosphoglycerate can be synthesized by gluconeogenesis (Embden-Meyerhof-Parnas pathway) and that orthologs of PGDH and PSAT exist. Furthermore, PSAT activity has also been detected biochemically (14), and we confirmed the existence of phosphoserine by metabolome analysis. 3 However, because H. thermophilus and other members of Aquificales lack an ortholog of dPSP (Table 1), we hypothesized that H. thermophilus may possess a novel type of PSP.…”

Mechanism for folate‐independent aldolase reaction catalyzed by serine hydroxymethyltransferase

“…Gel chromatography enabled us to estimate the molecular mass of native htSHMT as 74 kDa. Because the molecular masses of thermophilic enzymes are often underestimated by gel filtration [18,19], htSHMT is probably a homodimer, as are bsSHMT and the other prokaryotic SHMTs.…”

Analysis of the Enzymatic Properties of a Broad Family of Alanine Aminotransferases

“…AlaAT is widespread, with homologues in all three biological domains ( Eukarya , Archaea and Eubacteria ) and functions as a part of several major metabolic pathways. Existing in both the cytosol and the mitochondria, AlaAT plays a critical role in linking carbon and nitrogen metabolism (assimilation and catabolism) within both eukaryotes and prokaryotes [3]. AlaAT is involved in a number of cellular processes including glycolysis, gluconeogenesis, amino acid metabolism [2], hepatocellular damage in mammals [4], photorespiration in plants [5] and nitrogen use efficiency (NUE) in plants, including cereal crops [6], [7], [8].…”