英国《生物化学杂志》（Biochemical Journal）分别于2008年10月和2009年6月发表了中国科学院上海生命科学研究院生物化学与细胞研究所丁建平研究员课题组和植物生理生态研究所赵国屏研究员课题组的二项合作研究成果，该研究揭示了病原微生物问号钩端螺旋体（Leptospira interrogans）中一条特殊的亮氨酸合成途径关键酶——α-甲基苹果酸合酶（citramalate synthase，CMS）的催化反应与反馈抑制的分子机制。
问号钩端螺旋体能引起一种在世界各地都广泛流行的人畜共患病——钩端螺旋体病（Leptospirosis）。在我国，“赖型问号钩端螺旋体”（L. interrogans Serotype lai）曾引起钩端螺旋体病的多次大流行，病人有高热、淋巴结肿大、咯血等症状，严重时可导致患者因肺部大面积溶血而死亡。前人研究表明，钩端螺旋体体内有一种不同于绝大多数微生物的异亮氨酸生物合成途径——丙酮酸途径，关键酶CMS催化了该合成途径的第一步反应——丙酮酸与乙酰辅酶A的缩合。由于异亮氨酸属于人体不能合成的必需氨基酸，所以该途径可以成为研发针对问号钩端螺旋体的抗生素药物的作用靶标。另外，对它的结构解析可以为研究历史悠久、却仍未被完全理解的反馈抑制的变构调节机制提供更多的结构信息。
丁建平和赵国屏课题组合作，运用结构生物学和生物化学的方法分别研究了CMS的催化结构域与其底物丙酮酸、乙酰辅酶A、金属辅基的四元复合物的晶体结构，和CMS的调节结构域与反馈抑制剂异亮氨酸的复合物的晶体结构，以及全酶与这些配体相互结合的动力学性质。研究结果表明CMS的催化反应为羟醛缩合反应，其结合底物侧链的疏水口袋大小决定了酶对底物的高度专一性。抑制剂分子的侧链与周围氨基酸残基形成的疏水空间相契合，保证了抑制剂结合的特异性。抑制剂的结合可能导致调节结构域的二聚体接触面发生构象变化，并通过连接区诱导催化结构域的二聚体接触面和活性中心发生构象变化，从而影响底物和辅酶的结合，最终导致抑制的发生。这些研究结果不仅揭示了CMS的催化机理、底物识别和反馈抑制机制的分子基础，同时为以CMS为靶标的抗生素药物的设计提供了结构基础。
该项研究工作得到了国家科技部、基金委 、中国科学院和上海市科委的经费支持。(Bioon.com)
生物谷推荐原始出处：
Biochem. J. (2009) 421 (133–143)
Molecular basis of the inhibitor selectivity and insights into the feedback inhibition mechanism of citramalate synthase from Leptospira interrogans
Peng Zhang*?12, Jun Ma??1, Zilong Zhang??, Manwu Zha*, Hai Xu?3, Guoping Zhao?§5 and Jianping Ding*5
*State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China, ?Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China, ?Laboratory of Microbial Molecular Physiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng-Lin Road, Shanghai 200032, China, and §Shanghai-MOST Key Laboratory for Health and Disease Genomics, Chinese National Human Genome Center, Shanghai 201203, China
LiCMS (Leptospira interrogans citramalate synthase) catalyses the first reaction of the isoleucine biosynthesis pathway in L. interrogans, the pathogen of leptospirosis. The catalytic reaction is regulated through feedback inhibition by its end product isoleucine. To understand the molecular basis of the high selectivity of the inhibitor and the mechanism of feedback inhibition, we determined the crystal structure of LiCMSC (C-terminal regulatory domain of LiCMS) in complex with isoleucine, and performed a biochemical study of the inhibition of LiCMS using mutagenesis and kinetic methods. LiCMSC forms a dimer of dimers in both the crystal structure and solution and the dimeric LiCMSC is the basic functional unit. LiCMSC consists of six β-strands forming two anti-parallel β-sheets and two α-helices and assumes a βαβ three-layer sandwich structure. The inhibitor isoleucine is bound in a pocket at the dimer interface and has both hydrophobic and hydrogen-bonding interactions with several conserved residues of both subunits. The high selectivity of LiCMS for isoleucine over leucine is primarily dictated by the residues, Tyr430, Leu451, Tyr454, Ile458 and Val468, that form a hydrophobic pocket to accommodate the side chain of the inhibitor. The binding of isoleucine has inhibitory effects on the binding of both the substrate, pyruvate, and coenzyme, acetyl-CoA, in a typical pattern of K-type inhibition. The structural and biochemical data from the present study together suggest that the binding of isoleucine affects the binding of the substrate and coenzyme at the active site, possibly via conformational change of the dimer interface of the regulatory domain, leading to inhibition of the catalytic reaction.
