MOLECULAR GENETICS OF FLAVIN AND FE-S CONTAINING ENZYMES

含黄素和铁硫酶的分子遗传学

• 批准号: 5213170
• 负责人: ROBERT P GUNSALUS
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5213170
• 关键词: Escherichia coli; active sites; aminoacid; electron transport; enzyme mechanism; enzyme structure; flavins; gene expression; hydropathy; iron sulfur protein; ligands; mitochondrial membrane; model; molecular genetics; protein structure function; quinones; site directed mutagenesis; succinate dehydrogenase

The objective of this research project is to gain a clearer understanding of the structure and function of flavin and [Fe-S] containing enzymes that participate in electron transfer processes in both eucaryotic and procaryotic organisms. Major efforts center on the bacterial model systems including the fumarate reductase enzyme complex (Frd) and the succinate dehydrogenase enzyme complex (Sdh) of Escherichia coli. The two bacterial enzymes are powerful models to examine questions regarding how this class of enzymes inter-convert fumarate and succinate, and function in concert with the cellular electron transport machinery to accomplish ATP synthesis via oxidative phosphorylation processes. The bacterial enzymes are remarkably similar structurally although each is uniquely different with respect to their prosthetic group redox properties and the type of quinone used. Since the molecular and biochemical tools for the Escherichia coli Frd and Sdh systems are extremely well developed, their continued application should aid in elucidating the assembly and structure of this class of membrane bound oxidoreductase complexes that contain covalently linked flavin and multiple non-heme iron centers. We will evaluate the roles of specific amino acids in providing structaure and function at the [2Fe-2S] center, the [3Fe-4S] center, and the [4Fe-4S] centers of FrdB, the active site of FrdA, and the quinone interaction sites in the membrane association FrdCD subunits. Predictions from these studies will be evaluated with the Sdh complex of E. coli. Chimeric Frd and Sdh complexes will be generated by use of protein domain swapping methods to test whether determinants referring specific enzyme properties can be localized. Knowledge gained from these studies should provide a detailed molecular description of the structure and function of this class of membrane bound enzymes. We are using a non-animal system to perform basic research in a cost effective and timely manner to address human problems for which no equivalent experimental system has yet been development.

该研究项目的目的是获得更清晰的理解 黄素和[fe-s]的结构和功能 参与桉树和 倾向的生物。 细菌模型系统的主要努力中心 包括富马酸盐还原酶复合物（FRD）和琥珀酸酯 大肠杆菌的脱氢酶复合物（SDH）。 两个细菌 酶是强大的模型，可以检查有关该课程如何的问题 酶的酶间交换富马酸盐和琥珀酸酯，并在协调一致 使用细胞电子传输机械来完成ATP合成 通过氧化磷酸化过程。 细菌酶是 结构上非常相似，尽管每种都与 尊重其假体氧化还原特性和喹酮的类型 用过的。 由于大肠杆菌的分子和生化工具 FRD和SDH系统的发达非常好，它们继续 应用应有助于阐明其组装和结构 膜结合的氧化还原酶配合物，含有共价 连接的黄素和多个非血红素铁中心。 我们将评估 特定氨基酸在提供结构和功能的作用 [2FE-2S]中心，[3FE-4S]中心和[4FE-4] FRDB中心 FRDA的活性位点以及膜中的喹酮相互作用位点 协会FRDCD亚基。 这些研究的预测将是 用大肠杆菌的SDH复合物评估。 嵌合FRD和SDH复合物 将通过使用蛋白质域交换方法来测试是否生成 可以定位推荐特定酶特性的决定因素。 从这些研究中获得的知识应提供详细的分子 这类膜结合的结构和功能的描述 酶。 我们正在使用非动物系统来进行基础研究 具有成本效益和及时的方式来解决人类问题 等效实验系统尚未发展。