Structure/Mechanism of an FMN- and FAD-containing Enzyme

含有 FMN 和 FAD 的酶的结构/机制

• 批准号: 6479566
• 负责人: JUNG JA P. KIM
• 金额: $ 22.5万
• 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 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6479566
• 关键词: NAD(H) phosphate; NAD(P)H oxidoreductase; X ray crystallography; active sites; binding sites; crystallization; cytochrome P450; electron spin resonance spectroscopy; electron transport; enzyme mechanism; enzyme structure; flavin adenine dinucleotide; flavin mononucleotide; isozymes; mutant; nitric oxide synthase; structural biology

DESCRIPTION (provided by applicant): NADPH-cytochrome P450 oxidoreductase (CYPOR) and nitric oxide synthase isoforms (NOSs) are mammalian enzymes that contain two flavins, FMN and FAD, and an NADPH-binding site. CYPOR 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 catalyzes the oxygenation of drugs, xenobiotics, and endogenous substrates, including steroids, lipids, and prostaglandins. Despite intensive studies over four decades to elucidate the mechanism and function of CYPOR and its interactions with P450s, gaps in our understanding still exist. During the last funding period, the principal investigator's laboratory determined the crystal structure of rat CYPOR, solubilized by limited trypsin treatment. Based on this structure, it is proposed to study: (1) mutants of CYPOR to define the role of specific residues in catalysis and (2) holo-CYPOR to determine the role of the membrane-binding domain in the interactions between CYPOR and P450 and (3) to initiate studies, by EPR spectroscopy, of possible structural rearrangement of the CYPOR molecule upon binding to P450s, its physiological electron-transfer partners. Three NOS isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) catalyze the NADPH-dependent formation of nitric oxide (NO) and L-citrulline from L-arginine and molecular oxygen. NO is a mediator of neuronal signaling (nNOS), a cytotoxic agent (iNOS), and a vasodilator (eNOS), depending on enzyme source and tissue site of production. Both nNOS and eNOS are constitutively expressed and are activated by Ca++/CaM whereas iNOS is transcriptionally activated by cytokines and is active at normal Ca++ concentrations. Each isoform consists of a heme domain (N-terminus) with characteristics common to P450s, a flavin domain (C-terminus) homologous to CYPOR in both amino acid sequence and function, and a Ca++/CaM-binding region linking the two domains. Despite similar basic chemical mechanisms, overall reaction rates and modes of regulation of NO production differ significantly among the isoforms. To determine the structural basis for these differences, it is proposed to determine the crystal structures of (4) the flavin domains and (5) their variants of the three NOSs, containing their respective Ca++/CaM-binding regions.

描述（由申请人提供）：NADPH-CYTOCHROME P450氧化还原酶 （CYPOR）和一氧化氮合酶同工型（NOSS）是哺乳动物酶 包含两个黄素，即FMN和FAD，以及一个NADPH结合位点。 Cypor催化 还原等效物从NADPH转移到细胞色素P450，是一个 微粒体细胞色素P450单加氧化酶系统的必要成分。这 系统催化药物，异种生物和内生的氧合 底物，包括类固醇，脂质和前列腺素。尽管很密集 在四十年的研究中，阐明了Cypor和 它与P450的相互作用，我们理解中的差距仍然存在。在 上次资助期，首席研究人员的实验室确定了 大鼠Cypor的晶体结构，通过有限的胰蛋白酶处理溶解。基于 在这种结构上，建议研究：（1）CYPOR的突变体定义 特定残基在催化中的作用和（2）Holo-cypor确定作用 Cypor和P450与P450与P450之间的相互作用的膜结合域 （3）通过EPR光谱启动可能的结构 Cypor分子与P450结合后的重新排列，其生理 电子转移合作伙伴。三个NOS同工型，神经元NOS（NNO），可诱导 NOS（iNOS）和内皮NOS（ENOS）催化NADPH依赖性形成 来自L-精氨酸和分子氧的一氧化氮（NO）和L-硫氨酸。不 神经元信号传导（NNOS），细胞毒性剂（iNOS）和A的介体 血管扩张剂（ENOS），具体取决于生产的酶来源和组织部位。 NNOS和ENOS均以组成型表达，并被Ca ++/CAM激活 而iNOS是通过细胞因子转录激活的，并且在 正常的Ca ++浓度。每个同工型都由血红素结构域（N末端）组成 具有P450的特征，黄素域（C-terminus）同源 在氨基酸序列和功能和CA ++/CAM结合中均以CYPOR 连接两个域的区域。尽管采用了类似的基本化学机制 总体反应率和无生产调节模式不同 在同工型中显着。确定这些结构基础 差异，建议确定（4）的晶体结构 黄素域和（5）它们的三个noss的变体 各自的CA ++/CAM结合区域。