Shimizu Tetsu, Yin Lulu, Yoshida Ayako, Yokooji Yuusuke, Hachisuka Shin-Ichi, Sato Takaaki, Tomita Takeo, Nishida Hiromi, Atomi Haruyuki, Kuzuyama Tomohisa, Nishiyama Makoto
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Biochem J. 2017 Jan 1;474(1):105-122. doi: 10.1042/BCJ20160699. Epub 2016 Nov 9.
β-Decarboxylating dehydrogenases, which are involved in central metabolism, are considered to have diverged from a common ancestor with broad substrate specificity. In a molecular phylogenetic analysis of 183 β-decarboxylating dehydrogenase homologs from 84 species, TK0280 from Thermococcus kodakarensis was selected as a candidate for an ancestral-type β-decarboxylating dehydrogenase. The biochemical characterization of recombinant TK0280 revealed that the enzyme exhibited dehydrogenase activities toward homoisocitrate, isocitrate, and 3-isopropylmalate, which correspond to key reactions involved in the lysine biosynthetic pathway, tricarboxylic acid cycle, and leucine biosynthetic pathway, respectively. In T. kodakarensis, the growth characteristics of the KUW1 host strain and a TK0280 deletion strain suggested that TK0280 is involved in lysine biosynthesis in this archaeon. On the other hand, gene complementation analyses using Thermus thermophilus as a host revealed that TK0280 functions as both an isocitrate dehydrogenase and homoisocitrate dehydrogenase in this organism, but not as a 3-isopropylmalate dehydrogenase, most probably reflecting its low catalytic efficiency toward 3-isopropylmalate. A crystallographic study on TK0280 binding each substrate indicated that Thr71 and Ser80 played important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 was responsible for the recognition of 3-isopropylmalate. These analyses also suggested the importance of a water-mediated hydrogen bond network for the stabilization of the β3-α4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280.
参与中心代谢的β-脱羧脱氢酶被认为是从具有广泛底物特异性的共同祖先分化而来的。在对来自84个物种的183个β-脱羧脱氢酶同源物进行的分子系统发育分析中,来自嗜热栖热菌的TK0280被选为祖先型β-脱羧脱氢酶的候选物。重组TK0280的生化特性表明,该酶对同型异柠檬酸、异柠檬酸和3-异丙基苹果酸表现出脱氢酶活性,它们分别对应于赖氨酸生物合成途径、三羧酸循环和亮氨酸生物合成途径中的关键反应。在嗜热栖热菌中,KUW1宿主菌株和TK0280缺失菌株的生长特性表明,TK0280参与了这种古菌的赖氨酸生物合成。另一方面,以嗜热栖热菌为宿主的基因互补分析表明,TK0280在该生物体中既作为异柠檬酸脱氢酶又作为同型异柠檬酸脱氢酶发挥作用,但不作为3-异丙基苹果酸脱氢酶发挥作用,这很可能反映了其对3-异丙基苹果酸的催化效率较低。对结合每种底物的TK0280进行的晶体学研究表明,Thr71和Ser80在同型异柠檬酸和异柠檬酸的识别中起重要作用,而由Ile82和Leu83组成的疏水区域负责3-异丙基苹果酸的识别。这些分析还表明,水介导的氢键网络对于稳定包括Thr71残基在内的β3-α4环对于TK0280底物特异性的混杂性很重要。