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

性状（由申请方提供）：NADPH-细胞色素P450氧化还原酶 CYPOR和一氧化氮合酶同工型（NOS）是哺乳动物的酶， 含有两种黄素，FMN和FAD，以及一个NADPH结合位点。CYPOR催化 还原当量从NADPH转移到细胞色素P450，是一种 微粒体细胞色素P450单加氧酶系统的重要组成部分。的 系统催化药物、外源性物质和内源性物质的氧化， 底物，包括类固醇、脂质和甘草素。尽管密集 四十多年来的研究，以阐明CYPOR的机制和功能， 它与P450的相互作用，我们的理解仍然存在差距。期间 上一个资助期，主要研究者的实验室确定了 大鼠CYPOR的晶体结构，通过有限的胰蛋白酶处理溶解。基于 在此基础上，建议研究：（1）CYPOR的突变体，以确定CYPOR的结构。 特定残基在催化中的作用和（2）确定作用的全CYPOR CYPOR和P450之间相互作用的膜结合结构域， (3)开始研究，通过EPR光谱， CYPOR分子在与P450结合后的重排，其生理学 电子转移合作伙伴三种NOS亚型，神经型NOS（nNOS），诱导型NOS 一氧化氮合酶（iNOS）和内皮型一氧化氮合酶（eNOS）催化NADPH依赖的 一氧化氮（NO）和L-瓜氨酸来自L-精氨酸和分子氧。no是 神经元信号传导介质（nNOS）、细胞毒性剂（iNOS）和 血管扩张剂（eNOS），取决于酶来源和组织产生部位。 nNOS和eNOS都是组成性表达的，并被Ca ++/CaM激活 而iNOS被细胞因子转录激活， 正常Ca ++浓度。每个异构体由一个血红素结构域（N-末端）组成。 具有P450的共同特征，一个黄素结构域（C-末端）同源 在氨基酸序列和功能上与CYPOR结合，并且与Ca ++/CaM结合。 连接两个域的区域。尽管基本的化学机制相似， 总的反应速率和NO产生的调节方式不同 在同种型中显著。为了确定这些的结构基础， 差异，建议确定（4）的晶体结构 黄素结构域和（5）它们的变体的三个NOS，含有它们的 相应的Ca ++/CaM结合区。

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.