Analysis of novel lysine biosynthesis in Thermus and its regulation

以前我们发现，在嗜热嗜热菌中，赖氨酸是通过非二氨基己二酸酯(α-氨基己二酸酯)合成的。然而，该途径与真菌和酵母的途径不同，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，赖氨酸是以2-氧代戊二酸为起始化合物，经过十步反应合成的。在本研究中，我们克隆了两个尚未被克隆的基因，它们编码高异柠檬酸脱氢酶和α-氨基己二酸氨基转移酶，高异柠檬酸脱氢酶可将高异柠檬酸转化为α-酮基己二酸酯，而氨基己二酸氨基转移酶可催化后续反应生成α-氨基己二酸酯。为了克隆前者的基因，我们根据被认为具有与高异柠檬酸脱氢酶相似的序列的异柠檬酸脱氢酶和异丙基苹果酸脱氢酶的初级序列设计了简并引物，进行了聚合酶链式反应。后一种基因也被用…进行聚合酶链式反应克隆。更多的带有哺乳动物α-氨基己酸氨基转移酶序列的引物。由于同种异柠檬酸脱氢酶基因的中断，细胞呈现出赖氨酸营养缺陷型。另一方面，携带α-氨基己二酸氨基转移酶基因突变的Thermus细胞没有表现出完全的赖氨酸营养缺陷型，但细胞在最低限度的培养基上生长缓慢，这种生长可以通过添加赖氨酸或α-氨基己二酸酯部分恢复。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，并对纯化的酶进行了底物专一性分析。通过基于相关酶的三维结构的定点突变，确定了决定底物专一性的氨基酸残基。我们还分析了催化该途径中第一反应的酶-高柠檬酸合成酶的催化性质，发现该酶能催化以草酰乙酸酯为底物的柠檬酸合成酶反应，并被赖氨酸抑制。较少

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