Methyltransfer reactions in the reductive acetyl-Coenzym A pathway

还原性乙酰辅酶 A 途径中的甲基转移反应

• 批准号: 186145375
• 负责人: Professor Dr. Holger Dobbek
• 金额: --
• 依托单位: Arbeitsgruppe Strukturbiologie und Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/186145375?language=en
• 关键词: Methyltransfer; reactions; reductive; acetyl; Coenzym

Anaerobic bacteria and archaea use homologous enzymes to convert CO2 and H2 to acetyl-CoA and methane, which determine since the dawn of life the global carbon cycle on earth. In the last funding period, we focused on three proteins involved in methyl transfer in the reductive acetyl-CoA pathway. We could show that orf7 of the gene cluster of the acetyl-CoA pathway encodes the reductive activator of the central B12-dependent methyl group acceptor and donor CoFeSP. To achieve activation, the activator catalyzes an ATP-dependent intermolecular electron transfer against a redox potential gradient between a [2Fe2S] cluster and a Co(II) ion. The structure of the complex between activator and CoFeSP together with spectroscopic and kinetic studies have demonstrated a novel way of how an activator alters the redox potential of the cofactor in the target protein to render electron transfer favorable. For CoFeSP, we have developed a FRET assay and combined it with PELDOR measurements now allowing to determine the magnitude of the conformational changes (PELDOR spectroscopy) together with the kinetics of the change (stopped-flow kinetics FRET). The final reaction in acetyl-CoA formation is catalyzed by acetyl-CoA synthase, whose unstable Ni-Ni-[4Fe4S] center we could analyze with a resolution (dmin = 1.4 Å) using a truncated construct.In the next funding period, we want to clarify (I) the mechanism of the reductive activator such as the function and mechanism of ATP hydrolysis and the role of dissociation of the CoFeSP-activator complex for electron transfer. The rate-determining step of the reaction needs to be determined, as well as the position of the electron-donating [2Fe2S] cluster domain in the complex. (II) Where the substrates and intermediates CO, CH3CO+, CH3+ and coenzyme A bind on the Ni-Ni-[4Fe4S] cluster of acetyl-CoA synthase is still unknown and we want to use the good quality of our crystals to gain a first look into the substrate binding place and mechanism. (III) We want to investigate the homologous enzymes from methanogenic archaea that, in contrast to the bacterial proteins, form an approximately 2.5 MDa complex, containing acetyl-CoA synthase, carbon monoxide dehydrogenase and CoFeSP. The reactions of the three enzymes are coordinated in the complex in an as yet unknown way. While bacterial CoFeSP is methylated by methyltetrahydrofolate-dependent methyltransferase, the CoFeSP homologs from methanogens can methylate itself using CH3-methanopterin as substrate. Comparing the enzymes of bacteria and archaea will give further insight into the early stages in the evolution of B12-dependent methyltransferases and the origin of methanogenesis and acetogenesis.

厌氧细菌和古细菌使用同源酶将CO2和H2转化为乙酰辅酶A和甲烷，这决定了自生命诞生以来地球上的全球碳循环。在上一个资助期间，我们专注于还原乙酰辅酶A途径中参与甲基转移的三种蛋白质。我们可以证明乙酰辅酶A途径基因簇的orf7编码中心B12依赖性甲基受体和供体CoFeSP的还原激活剂。为了实现活化，活化剂催化ATP依赖的分子间电子转移对[2Fe2S]簇和Co（II）离子之间的氧化还原电位梯度。激活剂和CoFeSP之间的复合物的结构以及光谱和动力学研究已经证明了激活剂如何改变靶蛋白中辅因子的氧化还原电位以使电子转移有利的新方法。对于CoFeSP，我们已经开发了一种FRET检测，并将其与PELDOR测量相结合，现在可以确定构象变化的幅度（PELDOR光谱）以及变化的动力学（停流动力学FRET）。乙酰辅酶A形成的最终反应是由乙酰辅酶A合酶催化的，其不稳定的Ni-Ni-[4Fe4S]中心我们可以用分辨率分析。（dmin = 1.4）。在下一个供资期，我们希望阐明（I）还原激活剂的机制，如ATP水解的功能和机制以及CoFeSP-用于电子转移的激活剂复合物。需要确定反应的速率决定步骤，以及络合物中供电子[2Fe2S]簇域的位置。(II)底物和中间体CO，CH3CO+，CH3+和辅酶A在乙酰辅酶A合酶的Ni-Ni-[4Fe4S]簇上的结合位置仍然未知，我们希望利用我们的晶体的良好质量来初步了解底物结合位置和机制。(III)我们想研究产甲烷古菌的同源酶，与细菌蛋白质相反，形成约2.5 MDa的复合物，含有乙酰辅酶A合酶，一氧化碳脱氢酶和CoFeSP。这三种酶的反应以一种未知的方式在复合物中协调。虽然细菌CoFeSP被甲基四氢叶酸依赖性甲基转移酶甲基化，但来自产甲烷菌的CoFeSP同系物可以使用CH 3-甲烷蝶呤作为底物使自身甲基化。比较细菌和古细菌的酶将进一步了解B12依赖的甲基转移酶的进化的早期阶段和甲烷生成和乙酸生成的起源。

项目成果

Complex formation with the activator RACo affects the corrinoid structure of CoFeSP.

与活化剂 RACo 形成复合物会影响 CoFeSP 的类咕啉结构

• DOI: 10.1021/bi300795n
• 发表时间: 2012
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Meister;Hennig;Jeoung;Lendzian;Dobbek;Hildebrandt
• 通讯作者: Hildebrandt

ATP-induced electron transfer by redox-selective partner recognition

• DOI: 10.1038/ncomms5626
• 发表时间: 2014-08-01
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Hennig, Sandra E.;Goetzl, Sebastian;Dobbek, Holger
• 通讯作者: Dobbek, Holger

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

• DOI: 10.1073/pnas.1117126109
• 发表时间: 2012-04-03
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Hennig, Sandra E.;Jeoung, Jae-Hun;Dobbek, Holger
• 通讯作者: Dobbek, Holger

ATP Binding and a Second Reduction Enables a Conformationally Gated Uphill Electron Transfer

• DOI: 10.1021/acscatal.1c01038
• 发表时间: 2021-06-30
• 期刊: ACS CATALYSIS
• 影响因子: 12.9
• 作者: Neumann，Felix;Dobbek，Holger
• 通讯作者: Dobbek，Holger