Investigating the role of the Radical SAM enzyme PqqE in PQQ biogenesis

描述(申请人提供)：吡咯喹啉醌(PQQ)是一类细菌脱氢酶的非共价结合的修复基，催化革兰氏阴性细菌周质中醇和糖的氧化。PQQ也被证明是小鼠正常生长和发育所必需的，最近的证据表明，PQQ可能是哺乳动物必不可少的B类维生素。PQQ作为原核生物氧化还原辅助因子的作用已受到广泛关注，但其生物发生机制尚不清楚。这项研究计划将通过对PqqE的体外研究来分离和表征这一反应的中间产物和产物，从而探索PQQ生物发生机制的主要步骤。蛋白质同源性研究表明，PqqE是一类被称为自由基SAM(S-腺苷蛋氨酸)酶的蛋白质的一部分，该酶在活性部位含有一个[4Fe-4S]簇。这些酶通过铁硫簇还原活化SAM生成有机自由基来催化独特的反应。重组表达系统将用于表达PqqE，并将启动体外研究，以了解特定的生化中间体、速率常数和产物。固相多肽合成将被用来合成PqqE的多肽底物(PqqA)，并创建结构类似物，这些结构类似物将用于捕获反应机理中提出的中间体。高效液相色谱和LC-MS将与各种自由基清除剂一起使用，以分离和表征厌氧和好氧反应的产物。稳态和冷冻猝灭EPR研究将为理解PqqE反应中形成的自由基中间体提供额外的工具。这些工作将与特别标记了氢和碳-13的底物一起完成，以确定在反应机理中形成的自由基中间体的归属。该项目的长期目标是阐明PQQ产生的生化步骤和机制。这些研究将对人类健康产生重要影响，因为它们将提供对革兰氏阴性细菌特有的生化途径的洞察，这可能会为一类新的抗生素靶点提供额外的工具。了解PqqE反应中的生化机制也可能获得有关自由基SAM超家族的其他酶的重要信息，这些酶参与了人类的各种不同反应。具体地说，我们想要解决的一个问题是，PqqE是否可以作为一种假定的自由基SAM酶和加氧酶发挥作用。

DESCRIPTION (provided by applicant): Pyrrolo-quinoline quinone (PQQ) is a non-covalently bound prosthetic group of a class of bacterial dehydrogenases that catalyze the oxidation of alcohols and sugars in the periplasm of Gram-negative bacteria. PQQ has also been shown to be necessary for proper growth and development in mice, and recent evidence has been presented that suggests PQQ may be an essential B vitamin in mammals. The role of PQQ as a prokaryotic redox co-factor has received significant attention, but the biogenesis of PQQ is much less understood. This research proposal will investigate the primary steps in the mechanism for PQQ biogenesis by using in vitro studies of PqqE to isolate and characterize the intermediates and products of this reaction. Protein homology studies have indicated that PqqE is part of a class of proteins known as Radical SAM (S- adenosylmethionine) enzymes, that contain a [4Fe-4S] cluster in the active site. These enzymes catalyze unique reactions via the formation of an organic radical from reductive activation of SAM by the iron sulfur cluster. Recombinant expression systems will be used to express PqqE, and in vitro studies will be initiated to understand the specific biochemical intermediates, rate constants, and products. Solid phase peptide synthesis will be invoked to synthesize the peptide substrate (PqqA) for PqqE, along with creating structural analogs that will be used to trap the intermediates proposed in the reaction mechanism. HPLC and LC-MS will be used in conjunction with various radical scavenging agents to isolate and characterize the products of the anaerobic and aerobic reactions. Steady state and freeze-quench EPR studies will provide an added tool for understanding the radical intermediates formed during the PqqE reaction. These will be done in conjunction with substrates specifically labeled with deuterium and carbon-13 to make definitive assignments of the radical intermediates formed during the reaction mechanism. The long term goal of this project is to elucidate the biochemical steps and mechanisms for PQQ production. These studies will have important implications for human health because they will provide insight into a biochemical pathway specific for gram-negative bacteria, which may yield added tools for a new class of antibiotic targets. Understanding the biochemical mechanisms in the PqqE reaction may also yield important information concerning other enzymes of the Radical SAM super family, that have been implicated in a variety of different reactions in humans. Specifically, one question we would like to address is, whether PqqE can function as a putative Radical SAM enzyme and an oxygenase.