Analysis of novel lysine biosynthesis in Thermus and its regulation

以前我们发现赖氨酸是通过非二氨基己二酸（α-氨基己二酸）在嗜热菌中合成的。然而，该途径与真菌和酵母的途径不同，后半部分的途径与精氨酸生物合成的方式相似。我们以前的研究表明，赖氨酸是通过10步反应合成的，以2-氧葡萄糖酸酯为起始化合物。在本研究中，我们克隆了两个尚未被克隆的基因，分别编码高异柠檬酸脱氢酶，将高异柠檬酸转化为α-酮己二酸；编码α-氨基己二酸转氨酶，催化以下反应生成α-氨基己二酸。对于前者基因的克隆，我们利用异柠檬酸脱氢酶和异丙酸脱氢酶原序列设计的简并引物进行聚合酶链反应，认为其序列与同型异柠檬酸脱氢酶相似。用α-氨基己二酸转氨酶（α-氨基己二酸转氨酶）序列的引物进行PCR克隆。通过破坏同异柠檬酸脱氢酶基因，细胞显示赖氨酸营养不良表型。另一方面，携带α-氨基己二酸转氨酶基因破坏的热细胞没有表现出完全的赖氨酸营养不良表型，但细胞在添加赖氨酸或α-氨基己二酸后部分恢复的微量培养基上生长缓慢。接下来，我们建立了高效的同型异柠檬酸脱氢酶基因表达系统，并分析了纯化酶的底物特异性。通过基于相关酶的三维结构的定点诱变，确定了决定底物特异性的氨基酸残基。我们还分析了该途径中催化第一反应的同柠檬酸合成酶的催化性能，发现该酶可以催化以草酰乙酸为底物的柠檬酸合成酶反应，并且被赖氨酸抑制。少

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