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

研究自由基 SAM 酶 PqqE 在 PQQ 生物发生中的作用

• 批准号: 7272447
• 负责人: Stephen Robert Wecksler
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-10 至 2009-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7272447
• 关键词: Active Sites; Address; Aerobic; Antibiotics; Attention; Binding; Biochemical; Biochemical Pathway; Biogenesis; Carbon; Class; Color; Cyclization; Deuterium; Dicarboxylic Acids; Enzymes; Escherichia coli; Family; Figs - dietary; Freezing; Genes; Goals; Gram-Negative Bacteria; Growth and Development function; Health; High Pressure Liquid Chromatography; Human; In Vitro; Iron; Klebsiella pneumonia bacterium; Label; Mammals; Mus; Object Attachment; Operon; Organism; Oxidation-Reduction; Oxidoreductase; Oxygenases; PQQ Cofactor; Pathway interactions; Peptide Synthesis; Peptides; Phase; Plasmids; Production; Prosthesis; Proteins; Rate; Reaction; Recombinants; Research Proposals; Role; Solid; Sugar Alcohols; Sulfur; System; Vitamin B Complex; analog; insight; interest; knockout gene; oxidation; periplasm; research study; tool

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.

描述（由申请人提供）：吡咯并喹啉醌（PQQ）是一类细菌脱氢酶的非共价结合辅基，其催化革兰氏阴性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可能是哺乳动物必需的B族维生素。PQQ作为原核生物氧化还原辅因子的作用受到了极大的关注，但PQQ的生物起源却知之甚少。本研究计划将通过PqqE的体外研究来研究PQQ生物合成机制中的主要步骤，以分离和表征该反应的中间体和产物。蛋白质同源性研究表明PqqE是一类被称为自由基SAM（S-腺苷甲硫氨酸）酶的蛋白质的一部分，其在活性位点中含有[4Fe-4S]簇。这些酶通过铁硫簇还原活化SAM形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。固相肽合成将用于合成PqqE的肽底物（PqqA），沿着产生用于捕获反应机制中提出的中间体的结构类似物。HPLC和LC-MS将与各种自由基清除剂结合使用，以分离和表征厌氧和好氧反应的产物。稳态和冷冻淬灭EPR研究将提供一个额外的工具，了解自由基中间体在PqqE反应过程中形成的。这些将与专门标记有氘和碳13的底物结合进行，以确定反应机制期间形成的自由基中间体的归属。本项目的长期目标是阐明PQQ生产的生化步骤和机制。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菌特异性生化途径的深入了解，这可能为一类新的抗生素靶标提供额外的工具。了解PqqE反应中的生化机制也可能产生关于自由基SAM超家族的其他酶的重要信息，这些酶与人类的各种不同反应有关。具体来说，我们想解决的一个问题是，PqqE是否可以作为一个假定的自由基SAM酶和加氧酶。