STRUCTURE AND MECHANISM OF AN FMN AND FAD CONTAINING EN

含EN的FMN和FAD的结构和机制

• 批准号: 2378295
• 负责人: JUNG JA P. KIM
• 金额: $ 15.74万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2378295
• 关键词: NAD(H) phosphate; NAD(P)H oxidoreductase; X ray crystallography; active sites; cofactor; computer simulation; crystallization; cytochrome P450; electron transport; enzyme mechanism; enzyme structure; enzyme substrate complex; flavin adenine dinucleotide; flavin mononucleotide; microsomes; mutant; nitric oxide synthase; physical model; structural biology

NADPH-cytochrome P450 reductase (CPR) catalyzes the transfer of reducing equivalents from NADPH to cytochromes P450 and is an essential component of the microsomal cytochrome P450 monooxygenase system. The system carries out oxidative metabolism of various drugs, xenobiotics, and a number of endogenous substrates, including steroids, lipids, and prostaglandins. CPR and nitric oxide synthase are the only two mammalian enzymes known to contain both flavin prosthetic groups, FMN and FAD, and an NADPH binding site. Despite the intensive studies over the last three decades to elucidate the mechanism, structure, and genetic regulation of CPR, large gaps in our understanding still exist. Detailed structural information from high resolution X-ray analysis will enable us to relate biochemical functions to the structure of the protein and to define the catalytic mechanism. We have obtained X-ray quality crystals of rat liver CPR that has been cloned and expressed in E. coli and solubilized by limited trypsinolysis. We have also obtained two heavy atom derivatives. We propose to determine the three-dimensional structure of CPR at high resolution in its native and NADP+/NADPH bound farms. Structures of the site-specific mutants designed to probe its catalytic mechanism and regulation will also be determined. Initiation of the crystallographic studies on the intact CPR is also proposed to study the interactions between CPR and its physiological electron acceptor, cytochromes P450.

NADPH-CYTOCHROME P450还原酶（CPR）催化还原的转移 从NADPH到细胞色素P450的等效物，是必不可少的组成部分 微粒细胞色素P450单加氧酶系统的。系统携带 氧化各种药物，异种生物和许多代谢的氧化代谢 内源性底物，包括类固醇，脂质和前列腺素。 CPR 一氧化氮合酶是仅有的两种已知的哺乳动物酶 同时包含黄素假体，FMN和FAD，以及NADPH结合 地点。尽管在过去三十年中进行了深入研究 阐明CPR的机制，结构和遗传调节 我们理解中的差距仍然存在。详细的结构信息 通过高分辨率X射线分析将使我们能够联系生化 蛋白质结构的功能并定义催化 机制。我们已经获得了大鼠肝脏CPR的X射线质量晶体 已被克隆并在大肠杆菌中表达，并被有限的 胰蛋白酶溶解。我们还获得了两个重原子衍生物。我们 提议确定CPR在高的三维结构 本地和NADP+/NADPH绑定的农场的分辨率。结构 旨在探究其催化机制的位点特异性突变体和 也将确定法规。晶体学的启动 还提出了关于完整CPR的研究以研究相互作用 在CPR及其生理电子受体之间，细胞色素P450。