Structure and function of benzoyl-coenzyme A reductases

苯环是地球上第二丰富的生物产生结构（在陆地地点占25%），并且存在于木质素，化石油和许多与环境相关的芳香族化合物（例如溶剂，杀虫剂，杀虫剂）中。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被转化为中心中间体苯甲酰辅酶A，再被还原酶脱芳化为二烯酰辅酶A。在生物学中，这种还原在负氧化还原电位极限处进行，并且需要耦合到一个exergonic过程。有两种根本不同的酶促苯甲酰辅酶a还原的生物解决方案，由I类和II类苯甲酰辅酶a还原酶（bcr）催化。一类bcr存在于兼性厌氧菌中；它们由4个BcrABCD亚基组成，包含3个[4Fe-4S]簇。一类bcr将还原的铁氧还蛋白到芳香环的自能电子转移与atp水解化学计量相结合。高度复杂的1 MDa II类bcr由8个BamBCDEFGHI亚基组成，含有W、Zn、FeS簇、Se和FAD。它们被提议通过基于黄素的电子转移过程来驱动苯甲酰辅酶a的还原。该项目旨在了解酶的结构和功能，催化有机化学合成中需要极端恶劣条件的反应（使用碱金属作为还原剂的桦树还原）。将对来自不同生物的I类和II类bcr进行大量的光谱、动力学和结构研究，并使用新开发的表达/操作平台产生分子变异。该项目将申请人在厌氧芳香代谢酶方面的20年经验与众多国家/国际合作伙伴提供的最先进的结构和光谱方法相结合。

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.