Analysis of novel lysine biosynthesis in Thermus and its regulation

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Previously we found that lysine is synthesized via non-diaminopimelate (α-aminoadipate) in Thermus thermophilus. However, the pathway is different from that of fungi and yeast, and the latter half of the pathway proceeds in a way similar to that of arginine biosynthesis. Our previous studies suggest that lysine is synthesized through ten steps of the reactions using 2-oxoglutarate as a starting compound. In the present study, we cloned two genes, which have not yet been cloned, encoding homoisocitrate dehydrogenase which converts homoisocitrate to α-ketoadipate and α-aminoadipate aminotransferase catalyzing the following reaction to produce α-aminoadipate. For cloning of the former gene, we carried out polymerase chain reaction using degenerate primersn designed based on the primary sequences of isocitrate dehydrogenase and isopropylmalate dehydrogenase that are thought to have the sequences similar to that of homoisocitrate dehydrogenase. The latter gene was also cloned by PCR using a … More set of primers with the sequence for mammalian α-aminoadipate aminotransferase. By disruption of the homoisocitratre dehydrogenase gene, the cells showed lysine-auxotrophic phenotype. On the other hand, Thermus cells carrying the disruption of α-aminoadipate aminotransferase gene did not show complete lysine-auxotrophic phenotype but the cells possessed slow growth on minimal medium that was restored in part by addition of lysine or α-aminoadipate. We next established efficient expression systems for homoisocitrate dehydrogenase gene and analyzed substrate specificity for purified enzyme. By site-directed mutagenesis based on the three dimensional structures of related enzymes, amino acid residues determining substrate specificity were identified . We also analyzed the catalytic properties of homocitrate synthase, a enzyme catalyzing the first reaction in this pathway, and found that the enzyme could catalyze the citrate synthase reaction using oxaloacetate as a substrate and was inhibited by lysine. Less