An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus kodakarensis

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and alpha-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic omega-aminotransferases that use L-ornithine (Orn), L-lysine (Lys), or gamma-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 omega-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 (Delta TK2101), 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.