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: 2883026
• 负责人: JUNG JA P. KIM
• 金额: $ 17万
• 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 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2883026
• 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）催化还原性 从NADPH到细胞色素P450的当量，是一种必需的成分 微粒体细胞色素P450单加氧酶系统。系统携带 各种药物、外源性物质和一些 内源性底物，包括类固醇、脂质和洋地黄素。CPR 和一氧化氮合酶是已知的仅有的两种 含有黄素辅基，FMN和FAD，以及NADPH结合 绝佳的价钱尽管在过去的三十年里进行了深入的研究， 阐明CPR的机制、结构和遗传调控， 我们的理解仍然存在差距。详细结构信息 高分辨率X射线分析将使我们能够将生物化学 蛋白质的结构，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， 在E.大肠杆菌和溶解的有限 胰蛋白酶分解我们还得到了两个重原子衍生物。我们 建议确定CPR的三维结构， 在其天然和NADP+/NADPH结合的农场中的分离度。结构的 设计位点特异性突变体以探测其催化机制， 监管也将确定。晶体学的开始 还建议对完整CPR进行研究，以研究其相互作用 CPR与其生理电子受体细胞色素P450之间的关系。