Abstract
Amino acid metabolism in archaea in many cases differs from those reported in bacteria and eukaryotes. The hyperthermophilic archaeon Thermococcus kodakarensis possesses an incomplete tricarboxylic cycle, and the biosynthesis pathway of aspartate is unknown. Here, four Class I aminotransferases in T. kodakarensis encoded by TK0186, TK0548, TK1094, and TK2268 were examined to identify the enzyme(s) responsible for the conversion of oxaloacetate to aspartate. Among the four proteins, the TK2268 protein (TK2268p) was the only protein to recognize oxaloacetate as the amino acceptor. With oxaloacetate, TK2268p only recognized glutamate as the amino donor. The protein also catalyzed the reverse reaction, the transamination between aspartate and 2-oxoglutarate. Substrate inhibition was observed in the presence of high concentrations of oxaloacetate or 2-oxoglutarate. Aminotransferase activity between oxaloacetate and glutamate was observed in cell extracts of the T. kodakarensis host strain KU216. Among the individual gene disruption strains of the four aminotransferases, a significant decrease in activity was only observed in the ΔTK2268 strain. T. kodakarensis KU216 does not display growth in synthetic amino acid medium when aspartate/asparagine are absent. Growth was restored upon the addition of both oxaloacetate and glutamate. Although this restoration in growth was maintained in ΔTK0186, ΔTK0548, and ΔTK1094, growth was not observed in the ΔTK2268 strain. Our results suggest that TK2268p is the predominant aminotransferase responsible for the conversion of oxaloacetate to aspartate. The growth experiments and tracer-based metabolomics using 13C3-pyruvate indicated that pyruvate is a precursor of aspartate and that this conversion is dependent on TK2268p. Importance: Based on genome sequence, the hyperthermophilic archaeon Thermococcus kodakarensis possesses an incomplete tricarboxylic cycle, raising questions on how this organism carries out the biosynthesis of aspartate and glutamate. The results of this study clarify two main points related to aspartate biosynthesis. We show that aspartate can be produced from oxaloacetate and identify TK2268p as the aminotransferase responsible for this reaction. The other point demonstrated in this study is that pyruvate can act as the precursor for oxaloacetate synthesis. Together with previous results, we can propose some of the roles of the individual aminotransferases in T. kodakarensis. TK0548p and TK0186p are involved in amino acid catabolism, with the latter along with TK1094p involved in the conversion of glyoxylate to glycine. TK2268p is responsible for the biosynthesis of aspartate from oxaloacetate.