Nobel stand-alone RAM-domain mediated catalytic control of anthranilate phosphoribosyltransferase in tryptophan biosynthesis in Thermus thermophilus
Nobel stand-alone RAM-domain mediated catalytic control of anthranilate phosphoribosyltransferase in tryptophan biosynthesis in Thermus thermophilus
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Nobel 独立 RAM 结构域介导的嗜热栖热菌色氨酸生物合成中邻氨基苯甲酸磷酸核糖基转移酶的催化控制
DOI：
10.1042/bcj20160699
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
M. Nishiyama
中科院分区：
文献类型：
--
作者：
T. Kubota;H. Matsushita;T. Tomita;S. Kosono;M. Yoshida;T. Kuzuyama;M. Nishiyama
参与中枢代谢的β-脱羧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。通过对84个物种的183个β-脱羧脱氢酶同源物的分子系统发育分析，选择了产自Kodakarensis的TK 0280作为祖先型β-脱羧脱氢酶的候选菌株。重组TK 0280的生化特性表明，该酶表现出脱氢酶活性高异柠檬酸，异柠檬酸和3-异丙基苹果酸，这对应于赖氨酸生物合成途径，三羧酸循环，亮氨酸生物合成途径，分别参与的关键反应。在T. kodakarensis中，KUW 1宿主菌株和TK 0280缺失菌株的生长特征表明，TK 0280参与了该古细菌中赖氨酸的生物合成。另一方面，以嗜热栖热菌（Thermus thermophilus）为宿主的基因互补分析表明，TK 0280在该生物体中同时具有异柠檬酸脱氢酶和高异柠檬酸脱氢酶的功能，但不具有3-异丙基苹果酸脱氢酶的功能，这很可能反映了其对3-异丙基苹果酸的低催化效率。对TK 0280结合各底物的晶体学研究表明，Thr 71和Ser 80在识别高异柠檬酸和异柠檬酸中起重要作用，而由Ile 82和Leu 83组成的疏水区负责识别3-异丙基苹果酸。这些分析还表明水介导的氢键网络对于稳定β3-α4环（包括Thr 71残基）的重要性，这与TK 0280底物特异性的混杂性有关。
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 fromThermococcus kodakarensiswas 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. InT. 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 usingThermus thermophilusas 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.