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-细胞色素 P450 还原酶 (CPR) 催化还原酶的转移 NADPH 与细胞色素 P450 的等价物，是重要组成部分 微粒体细胞色素 P450 单加氧酶系统。系统搭载 各种药物、异生素和许多物质的氧化代谢 内源性底物，包括类固醇、脂质和前列腺素。心肺复苏 和一氧化氮合酶是已知的仅有的两种哺乳动物酶 含有黄素辅基 FMN 和 FAD，以及 NADPH 结合 地点。尽管过去三十年进行了深入研究 阐明 CPR 的机制、结构和遗传调控，大 我们的理解仍然存在差距。详细的结构信息 高分辨率 X 射线分析将使我们能够将生物化学联系起来 功能对蛋白质的结构和定义催化 机制。我们获得了大鼠肝脏 CPR 的 X 射线质量晶体， 已在大肠杆菌中克隆和表达，并通过有限的溶解 胰蛋白酶溶解。我们还获得了两种重原子衍生物。我们 提议确定高位心肺复苏的三维结构 其天然和 NADP+/NADPH 结合农场的分辨率。的结构 旨在探测其催化机制的位点特异性突变体 监管也将确定。晶体学的起始 还建议对完整的 CPR 进行研究以研究相互作用 CPR 与其生理电子受体细胞色素 P450 之间。