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: 6272526
• 负责人: ROBERT P GUNSALUS
• 金额: $ 21.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6272526
• 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了解 黄素和[Fe-S]含酶的结构和功能 参与真核生物和生物体内的电子转移过程 原核生物。 主要工作集中在细菌模型系统上 包括富马酸还原酶复合物 (Frd) 和琥珀酸 大肠杆菌脱氢酶复合物 (Sdh)。 两种细菌 酶是检验此类问题的强大模型 多种酶相互转化富马酸和琥珀酸，并协同发挥作用 与细胞电子传递机制一起完成 ATP 合成 通过氧化磷酸化过程。 细菌酶是 尽管每个都有独特的不同，但结构上非常相似 关于它们的辅基氧化还原特性和醌的类型 用过的。 由于大肠杆菌的分子和生化工具 Frd 和 SDh 系统非常发达，它们的持续发展 应用程序应有助于阐明该组件的组装和结构 一类膜结合氧化还原酶复合物，包含共价键 连接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中的作用 FrdB的[2Fe-2S]中心、[3Fe-4S]中心和[4Fe-4S]中心， FrdA 的活性位点和膜中的醌相互作用位点 关联FrdCD亚基。 这些研究的预测将是 用大肠杆菌的 Sdh 复合物进行评估。 嵌合 Frd 和 Sdh 复合物 将通过使用蛋白质结构域交换方法来生成以测试是否 可以定位涉及特定酶特性的决定因素。 从这些研究中获得的知识应该提供详细的分子 此类膜结合物的结构和功能描述 酶。 我们正在使用非动物系统来进行基础研究 以具有成本效益和及时的方式解决人类问题 等效的实验系统尚未开发。