Abstract

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene-disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.

Highlights

  • Aminotransferases are pyridoxal 5؅-phosphate– dependent enzymes that catalyze reversible transamination reactions between amino acids and ␣-keto acids, and are important for the cellular metabolism of nitrogen

  • The structures of ␻-amino acids differ from those of ␣amino acids, and the amino group involved in the transamination reaction catalyzed by ␻-aminotransferase is distal to the carboxyl group [9]. ␻-Aminotransferases constitute the majority of the class III aminotransferases, which belong to the fold type I of pyridoxal 5Ј-phosphate (PLP)-dependent enzymes [10]. ␻-Aminotransferases can further be divided into groups according to their natural substrates, such as putrescine, ␥-aminobutyrate (GABA), N-acetylornithine, ornithine (Orn), and lysine (Lys), and they play a pivotal physiological role in their metabolism

  • In our efforts to elucidate the mechanism of coenzyme A (CoA) biosynthesis in archaea [23,24,25,26,27], we identified a glutamate decarboxylase homolog encoded by TK1814 from T. kodakarensis, which catalyzed the decarboxylation of aspartate and Glu to ␤-alanine and GABA, respectively, in vitro [26]

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Summary

Edited by Chris Whitfield

Aminotransferases are pyridoxal 5؅-phosphate– dependent enzymes that catalyze reversible transamination reactions between amino acids and ␣-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ␻-aminotransferases that use L-ornithine (Orn), L-lysine (Lys), or ␥-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. We examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis. Aminotransferases are pyridoxal 5Ј-phosphate (PLP)3– dependent enzymes that catalyze reversible transamination reactions between amino acids and ␣-keto acids and play an important role in the cellular metabolism of nitrogen. ␻-Aminotransferases can further be divided into groups according to their natural substrates, such as putrescine, ␥-aminobutyrate (GABA), N-acetylornithine, ornithine (Orn), and lysine (Lys), and they play a pivotal physiological role in their metabolism. Genetic analysis of TK2101 confirmed its physiological role as an Orn-AT involved in the biosynthesis of L-Pro

Results
Relative activity
Kinetic studies
Discussion
Experimental procedures
Enzyme assay
Effects of pH and temperature on enzyme activity
Phylogenetic analysis
Full Text
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