MECHANISM OF METHYL TRANSFERS IN ACETYL-COA SYNTHESIS

热乙酸梭菌和其他产乙酸细菌产生 乙酸盐作为在H2/CO2、CO或有机基质上生长的唯一产物。 乙酰辅酶A途径，也称为伍德途径，是一个主要的机制， 在厌氧条件下的CO2固定。 它是一种非循环途径， C-C键的形成，其中主要中间体以酶结合的形式出现 （通常为有机金属）中间体。 森林有三个部分 途径：（1）CO2还原生成四氢叶酸甲酯（methyl-H4 folate）; (2)甲基-H4叶酸的甲基首先转移到C/Fe-SP 然后是CODH，这是一个涉及四种酶的反应序列- Corrinoid/铁硫蛋白（C-Fe-SP），甲基-H4叶酸：C/Fe-SP甲基- 转移酶（MeTr）、一氧化碳脱氢酶（CODH）和铁氧还蛋白 (Fd)和（3）合成的最后步骤，其中甲基化的CODH 结合CO和CoA，并从结合的甲基、CO和 CoA组。 这项建议的重点是机制酶学的转移 将甲基H4叶酸的甲基基团与C/Fe-SP结合，形成 甲基钴物质，然后转化为CODH，形成甲基-CODH。 形成 C/Fe-SP上的甲基钴物质由甲基转移酶催化， 它的独特之处在于它显然不包含结合的假体， 例如钴胺素或S-腺苷-L-甲硫氨酸。 该反应涉及 还原C/Fe-SP，然后用Co 1+亲核进攻C/Fe-SP， 叔胺的N-甲基键，5-甲基-H4叶酸。 形成 甲基-CODH似乎是乙酰辅酶A途径中的限速步骤 并且涉及在<-450 mV的电位下还原CODH上的金属中心 然后还原的金属中心亲核攻击 甲基钴酰胺中心，以在CODH上形成甲基金属中间体。 在 这里描述的工作，这些甲基转移步骤和蛋白质 催化它们的完全特征在于（1）稳态， 稳态前动力学分析;（2）初级结构分析 C/Fe-SP和MeTr的X射线晶体结构测定 MeTr和C/Fe-SP的配合比及钴的配位环境分析 （4）[4Fe-4S]的作用 C/Fe-SP中的簇，（5）钴酰胺结合位点的鉴定， [4Fe-4S]团簇在C/Fe-SP中的位置;（6）检测 钴酰胺的空间和配位状态要求， 用各种钴酰胺重构C/Fe-SP。

Clostridium thermoaceticum and other acetogenic bacteria produce acetate as the sole product of growth on H2/CO2, CO, or organic substrates. The acetyl-CoA pathway, also called the Wood pathway, is a major mechanism of C02 fixation under anaerobic conditions. It is a noncyclic pathway of C-C bond formation in which the major intermediates occur as enzyme-bound (often organometallic) intermediates. There are three parts to the Wood pathway: (1) reduction of CO 2 to methyltetrahydrofolate (methyl-H4folate); (2) transfer of the methyl group of methyl-H4folate first to the C/Fe-SP and then to CODH, a reaction sequence involving four enzymes- a corrinoid/iron-sulfur protein (C-Fe-SP), a methyl-H4folate: C/Fe-SP methyl- transferase (MeTr), carbon monoxide dehydrogenase (CODH), and ferredoxin (Fd); and (3) the final steps of the synthesis in which methylated CODH binds CO and CoA and synthesizes acetyl-CoA from the bound methyl, CO, and CoA groups. This proposal focuses on the mechanistic enzymology of the transfer of the methyl group of methylH4folate to the C/Fe-SP, forming a methylcobalt species, and then to CODH, forming methyl-CODH. Formation of the methylcobalt species on the C/Fe-SP is catalyzed by a methyltransferase which is unique in that it apparently does not contain bound prosthetic groups such as cobalamin or S-adenosyl-L-methionine. The reaction involves reduction of the C/Fe-SP followed by a nucleophilic attack of Co 1+ on the N-methyl bond of the tertiary amine, 5-methyl-H4folate. Formation of methyl-CODH appears to be the rate limiting step in the acetyl-CoA pathway and involves reduction of a metal center on CODH at potentials < - 450 mV followed by nucleophilic attack of the reduced metal center on the methylcobamide center to form a methyl-metal intermediate on CODH. In the work described here, these methyl transfer steps and the proteins catalyzing them will be fully characterized by (1) steady-state and presteady-state kinetic analyses, (2) analyses of the primary structures of the C/Fe-SP and MeTr, (3) determination of the X-ray crystal structures of MeTr and the C/Fe-SP and analyses of the cobalt coordination environment by X-ray absorption studies, (4) elucidation of the role of the [4Fe-4S] cluster in the C/Fe-SP, (5) identification of the cobamide binding site and the location of the [4Fe-4S] cluster in the C/Fe-SP, and (6) examination of the steric and coordination state requirements for the cobamide by reconstitution of the C/Fe-SP with various cobamides.