Analysis of mechanisms of regulation of aspartate kinase and lysine biosyntheseis in Thermus thermophilus

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Aspartate kinase catalyzes the transfer of the γ-phosphate group of ATP to aspartic acid. That is the first step of the biosynthetic pathway for lysine, threonine, and methionine, and is known to be regulated by the end-metabolites through feedback inhibition. We identified amino acid residues involved in the catalytic reactions through site-directed mutagenesis of those conserved in aspartate kinases using Thermus aspartate kinase. For further understanding of the function and regulation in aspartate kinase, gene encoding aspartate kinase III, which is regulated by simultaneous presence of lysine and threonine, was cloned from Bacillus subtilis. All the aspartate kinases so far investigated are oligomaeric enzymes, while aspartate kinase III was found to be a monomer. This may suggest simple mechanism for the feedback inhibition in aspartate kinase III.Through the analyses on lysine-auxotrophic mutants and DNA fragment complementing the auxotrophy, lysine was shown to be synthesized through α-aminoadipate as an intermediate in T.thermophilus. Analysis of a gene cluster involved in the lysine biosynthesis suggested that the lysine biosynthesis through α-aminoadipate in Thermus thermophilus is not the same as that found in the lower eukaryotes, but is the mixture of the biosynthetic pathways for leucine and arginine. Cloned ArgD homolog, termed LysJ, catalyzed the reaction using an arginine pathway-related compound as the substrate for lysine synthesis. On the other hand, LysJ preferred N-acetylornithine an intermediate of arginine biosynthesis, to N-acetyllysine, the putative substrate in the lysine biosynthesis. All these results indicate that the lysine biosynthesis is evolutionarily related to the arginine biosynthesis.