英國(guó)《生物化學(xué)雜志》（Biochemical Journal）分別于2008年10月和2009年6月發(fā)表了中國(guó)科學(xué)院上海生命科學(xué)研究院生物化學(xué)與細(xì)胞研究所丁建平研究員課題組和植物生理生態(tài)研究所趙國(guó)屏研究員課題組的二項(xiàng)合作研究成果,，該研究揭示了病原微生物問(wèn)號(hào)鉤端螺旋體（Leptospira interrogans）中一條特殊的亮氨酸合成途徑關(guān)鍵酶——α-甲基蘋果酸合酶（citramalate synthase,，CMS）的催化反應(yīng)與反饋抑制的分子機(jī)制,。
問(wèn)號(hào)鉤端螺旋體能引起一種在世界各地都廣泛流行的人畜共患病——鉤端螺旋體病（Leptospirosis）,。在我國(guó),，“賴型問(wèn)號(hào)鉤端螺旋體”（L. interrogans Serotype lai）曾引起鉤端螺旋體病的多次大流行，病人有高熱,、淋巴結(jié)腫大,、咯血等癥狀，嚴(yán)重時(shí)可導(dǎo)致患者因肺部大面積溶血而死亡,。前人研究表明,，鉤端螺旋體體內(nèi)有一種不同于絕大多數(shù)微生物的異亮氨酸生物合成途徑——丙酮酸途徑，關(guān)鍵酶CMS催化了該合成途徑的第一步反應(yīng)——丙酮酸與乙酰輔酶A的縮合,。由于異亮氨酸屬于人體不能合成的必需氨基酸,，所以該途徑可以成為研發(fā)針對(duì)問(wèn)號(hào)鉤端螺旋體的抗生素藥物的作用靶標(biāo)。另外,，對(duì)它的結(jié)構(gòu)解析可以為研究歷史悠久,、卻仍未被完全理解的反饋抑制的變構(gòu)調(diào)節(jié)機(jī)制提供更多的結(jié)構(gòu)信息。
丁建平和趙國(guó)屏課題組合作,，運(yùn)用結(jié)構(gòu)生物學(xué)和生物化學(xué)的方法分別研究了CMS的催化結(jié)構(gòu)域與其底物丙酮酸,、乙酰輔酶A、金屬輔基的四元復(fù)合物的晶體結(jié)構(gòu),，和CMS的調(diào)節(jié)結(jié)構(gòu)域與反饋抑制劑異亮氨酸的復(fù)合物的晶體結(jié)構(gòu),，以及全酶與這些配體相互結(jié)合的動(dòng)力學(xué)性質(zhì)。研究結(jié)果表明CMS的催化反應(yīng)為羥醛縮合反應(yīng),，其結(jié)合底物側(cè)鏈的疏水口袋大小決定了酶對(duì)底物的高度專一性,。抑制劑分子的側(cè)鏈與周圍氨基酸殘基形成的疏水空間相契合，保證了抑制劑結(jié)合的特異性,。抑制劑的結(jié)合可能導(dǎo)致調(diào)節(jié)結(jié)構(gòu)域的二聚體接觸面發(fā)生構(gòu)象變化,，并通過(guò)連接區(qū)誘導(dǎo)催化結(jié)構(gòu)域的二聚體接觸面和活性中心發(fā)生構(gòu)象變化，從而影響底物和輔酶的結(jié)合,，最終導(dǎo)致抑制的發(fā)生,。這些研究結(jié)果不僅揭示了CMS的催化機(jī)理、底物識(shí)別和反饋抑制機(jī)制的分子基礎(chǔ),，同時(shí)為以CMS為靶標(biāo)的抗生素藥物的設(shè)計(jì)提供了結(jié)構(gòu)基礎(chǔ),。
該項(xiàng)研究工作得到了國(guó)家科技部、基金委 ,、中國(guó)科學(xué)院和上海市科委的經(jīng)費(fèi)支持,。(Bioon.com)
生物谷推薦原始出處：
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.
