Analysis of novel lysine biosynthesis in Thermus and its regulation

我们发现在嗜热栖热菌中赖氨酸是通过非二氨基庚二酸（α-氨基己二酸）合成的。然而，该途径与真菌和酵母的途径不同，并且该途径的后半部分以类似于精氨酸生物合成的方式进行。我们以前的研究表明，赖氨酸是通过十个步骤的反应，使用2-酮戊二酸作为起始化合物。在本研究中，我们克隆了两个尚未被克隆的基因，编码高异柠檬酸脱氢酶和α-氨基己二酸氨基转移酶，高异柠檬酸脱氢酶将高异柠檬酸转化为α-酮己二酸，α-氨基己二酸氨基转移酶催化随后的反应产生α-氨基己二酸。为了克隆前一个基因，我们使用基于异柠檬酸脱氢酶和异丙基苹果酸脱氢酶的一级序列设计的简并引物进行聚合酶链反应，所述异柠檬酸脱氢酶和异丙基苹果酸脱氢酶被认为具有与高异柠檬酸脱氢酶相似的序列。后一个基因也通过PCR使用 ...更多信息 具有哺乳动物α-氨基己二酸氨基转移酶序列的引物组。通过破坏高异柠檬酸脱氢酶基因，细胞表现出赖氨酸营养缺陷型表型。另一方面，携带α-氨基己二酸氨基转移酶基因破坏的栖热菌细胞没有显示出完全的赖氨酸营养缺陷型表型，但细胞在基本培养基上具有缓慢的生长，通过添加赖氨酸或α-氨基己二酸部分恢复。接下来，我们建立了高异柠檬酸脱氢酶基因的高效表达系统，并分析了纯化酶的底物特异性。通过定点突变的基础上的三维结构的相关酶，氨基酸残基决定底物特异性进行了鉴定。我们还分析了高柠檬酸合成酶的催化特性，该酶催化该途径中的第一个反应，发现该酶可以催化柠檬酸合成酶反应，使用草酰乙酸作为底物，并被赖氨酸抑制。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