Structure and function of benzoyl-coenzyme A reductases

苯环代表地球上第二丰富的生物产生的结构（在陆地站点为25%），并且存在于木质素、化石油和许多环境相关的芳族化合物（例如，溶剂、杀虫剂、杀虫剂）。在厌氧条件下，例如在海洋/淡水沉积物或受污染的含水层中，只有厌氧原核生物能够完全降解芳香族化合物。在这些生物体中，大量的单环芳香族化合物转化为中心中间体苯甲酰辅酶A，其通过还原酶脱芳构化为二烯酰辅酶A。这种还原在生物学中的负氧化还原电位极限下进行，并且需要与放能过程耦合。存在两种根本不同的用于酶促苯甲酰辅酶A还原的生物解决方案，其由I类和II类苯甲酰辅酶A还原酶（BCR）催化。I类BCR存在于兼性厌氧菌中;它们由四个BcrABCD亚基组成，并含有三个[4Fe-4S]簇。I类BCR将从还原铁氧还蛋白到芳环的吸能电子转移与化学计量的ATP水解偶联。高度复杂的1 MDa II类BCR由8个BamBCDEFGHI亚基组成，并含有W、Zn、FeS簇、Se和FAD。他们建议通过黄素为基础的电子转移过程驱动苯甲酰辅酶A还原。该项目旨在了解催化有机化学合成中需要极其苛刻条件的反应（使用碱金属作为还原剂的桦木还原）的酶的结构和功能。将对来自不同生物体的I类和II类BCR以及使用新开发的表达/操作平台产生的分子变体进行大量光谱、动力学和结构研究。该项目将申请人20年的经验与参与厌氧芳香族代谢的酶与众多国家/国际合作伙伴提供的最先进的结构和光谱方法协同连接起来。

The benzene ring represents the second most abundant biologically produced structure on earth (25% at terrestrial sites), and is present in lignin, fossil oil and many environmentally relevant aromatic compounds (e.g., solvents, pesticides, insecticides). Under anaerobic conditions, e.g. at marine/freshwater sediments or contaminated aquifers only anaerobic prokaryotes are capable of fully degrading aromatic compounds. In these organisms the multitude of monocyclic aromatic compounds are converted to the central intermediate benzoyl-coenzyme A that is dearomatized to a dienoyl-CoA by reductases. This reduction proceeds at the negative redox potential limit in biology and requires a coupling to an exergonic process. There are two fundamentally different biological solutions for enzymatic benzoyl-CoA reduction, catalyzed by the class I and II benzoyl-CoA reductases (BCRs). Class I BCRs are present in facultative anaerobes; they are composed of four BcrABCD subunits and contain three [4Fe-4S] clusters. Class I BCRs couple the endergonic electron transfer from reduced ferredoxin to the aromatic ring to a stoichiometric ATP-hydrolysis. The highly complex 1 MDa class II BCRs are composed of the eight BamBCDEFGHI subunits and contain W, Zn, FeS clusters, Se and FAD. They are proposed to drive benzoyl-CoA reduction by a flavin-based electron transfer process. The project aims understand the structure and function of enzymes catalyzing a reaction that in organic chemical synthesis requires extremely harsh conditions (Birch reduction using alkali metals as reductants). Numerous spectroscopic, kinetic, and structural studies will be carried out with class I and II BCRs from different organisms, and with molecular variants generated by the use of newly developed expression/manipulation platforms. The project synergistically connects the 20 year experience of the applicant with enzymes involved in anaerobic aromatic metabolism with state-of-the-art structural and spectroscopic methodologies provided by numerous national/international collaboration partners.