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

描述(申请人提供)：NADPH-细胞色素P450氧化还原酶 (CYPOR)和一氧化氮合酶异构体(NOSS)是哺乳动物的酶， 含有两种黄素，FMN和FAD，以及一个NADPH结合部位。CYPOR催化 还原当量从NADPH到细胞色素P450的转移是一种 微体细胞色素P450单加氧酶系统的基本成分。这个 系统催化药物、外源和内源性物质的氧化 底物，包括类固醇、类脂和前列腺素。尽管强度很大 近四十年来对细胞色素P450受体和细胞色素P450受体的机制和功能的研究 它与P450的相互作用，在我们的理解上仍然存在差距。在.期间 上个资助期，首席调查员的实验室确定了 有限胰酶处理增溶的大鼠CYPOR的晶体结构。基座 在这种结构上，建议进行以下研究：(1)CypoR突变体，以定义 特定残基在催化中的作用和(2)全息CYPOR确定其作用 细胞色素P450与细胞色素P450相互作用中膜结合区的研究 (3)利用电子顺磁共振波谱对可能的结构进行研究 细胞色素P450受体分子与P450结合时的重排及其生理意义 电子转移伙伴。神经元型一氧化氮合酶(NNOS)三种亚型，诱导型 一氧化氮合酶(INOS)和内皮型一氧化氮合酶(ENOS)催化NADPH依赖的血管生成 一氧化氮(NO)和L-瓜氨酸来自L-精氨酸和分子氧。不是的 神经元信号转导的介体(NNOS)、细胞毒剂(INOS)和 血管扩张剂(ENOS)，取决于酶的来源和产生的组织部位。 NNOS和eNOS都是结构性表达的，并被Ca++/CaM激活 而iNOS是由细胞因子转录激活的，并在 钙离子浓度正常。每种异构体都由一个血红素结构域(N-末端)组成 具有P450的共同特征，黄素区(C-末端)同源 在氨基酸序列和功能上与CYPOR结合，并与钙/钙结合 链接这两个域的区域。尽管有相似的基本化学机制， NO产生的总体反应速度和调节方式不同 在不同的亚型中有显著差异。要确定这些问题的结构基础 差异，建议测定(4)的晶体结构 Flavin结构域和(5)它们的三个NOS的变体，包含它们的 各自的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.