STRUCTURAL METALLOBIOCHEMISTRY OF NITRIC OXIDE SYNTHASES

描述：金属酶一氧化氮合酶（NOS）调节一氧化氮 氧化物（NO）的合成，从而其生物活性。 否具有双重 作为1）神经传递的可扩散生物信使， 长时程增强、血小板聚集和血压调节 和2）细胞毒性剂，用于防御肿瘤细胞和细胞内 寄生虫 一氧化氮合酶（NOS），发现于诱导型（iNOS），组成型 内皮型一氧化氮合酶（eNOS）和组成型神经元型一氧化氮合酶（nNOS）亚型，实现其 重要的生物功能，通过采用一个有趣的钙调节 催化机制，并结合了五个辅因子的独特组合： 血红素、四氢生物蝶呤（H4B）、FMN、FAD和NADPH。 NOS通过以下方式产生NO 在两步机制的每一步中消耗分子氧和NADPH： 首先，单加氧酶样反应将L-精氨酸转化为中间体 N4-羟基-L-精氨酸（NOH-arg），然后一个前所未有的反应转化为 NOH-arg与瓜氨酸和NO。 NOSs结构金属生物化学、综合晶体学和 建议对三种主要类型的NOS酶进行生物化学研究。 每个NOS亚基分为两个结构域， 铰链区：具有血红素、H4B和L-精氨酸结合的加氧酶结构域 形成NO生成催化中心的位点，以及还原酶结构域 其中NADPH、FAD和FMN结合位点向血红素提供电子。 从黄素到血红素的电子转移是由钙调素控制的 (CaM)，其实现了钙结合蛋白的新作用。 的 提出的研究首先旨在表征结构生物化学， 单个加氧酶和还原酶结构域，其已经过表达 然后用这些结果来确定 全长NOS和不同的类似同工酶结构域。 这种平行 结构-功能研究将建立共同特征和变异 在这三类NOS中。 对于每个NOS结构域或同工酶， 建议的工作耦合表达，纯化和生化 在施图尔实验室用晶体结构进行表征 在Getzoff和Tainer实验室进行测定和分析。 的 结果从这些耦合的分子生物学，光谱学，生物化学， 和晶体学实验将指导定点 突变体和选择合适的辅因子，底物，中间体 和抑制剂复合物用于进一步研究。 这种集成的、递归的 这种方法旨在增加对NOS催化作用的理解，并最终 有助于设计同工酶选择性NOS抑制剂，这将是非常宝贵的 用于发现体内同种型功能的工具， 用于控制血压、败血性休克和 炎性损伤

DESCRIPTION: The metalloenzyme nitric oxide synthase (NOS) regulates nitric oxide (NO) synthesis and thereby its biological activity. NO has a dual role as 1) a diffusible biological messenger for neurotransmission, long-term potentiation, platelet aggregation, and blood pressure regulation and 2) a cytotoxic agent for defense against tumor cells and intracellular parasites. NOS enzymes (NOSs), found in inducible (iNOS), constitutive endothelial (eNOS), and constitutive neuronal (nNOS) isoforms, achieve their important biological function by adopting an intriguing calcium-regulated catalytic mechanism and incorporating a unique assembly of five cofactors: heme, tetrahydrobiopterin (H4B), FMN, FAD and NADPH. NOSs generate NO by expending molecular oxygen and NADPH in each step of a two-step mechanism: first, a monooxygenase-like reaction converts L-arginine to the intermediate N4-hydroxy-L- arginine (NOH-arg), then an unprecedented reaction converts NOH-arg to citrulline and NO. To understand in atomic detail the unique structural metallobiochemistry of NOSs, integrated crystallographic and biochemical studies are proposed for the three major classes of NOS enzymes. Each NOS subunit is divided into two domains joined by a calmodulin-binding hinge region: an oxygenase domain with heme, H4B, and L-arginine binding sites forming the catalytic center for NO production, and a reductase domain with NADPH, FAD, and FMN binding sites supplying electrons to the heme. Electron transfer from the flavins to the heme is controlled by calmodulin (CaM), which fulfills a novel role for a calcium binding protein. The proposed studies first aim to characterize the structural biochemistry for individual oxygenase and reductase domains, which have been overexpressed and crystallized, and then to use these results to determine structures of full-length NOSs and of different analogous isozyme domains. Such parallel structure-function studies will establish common features and variations among these three classes of NOSs. For each NOS domain or isozyme, the proposed work couples expression, purification, and biochemical characterization in the Stuehr laboratory with crystallographic structure determination and analysis in the Getzoff and Tainer laboratories. The results from these coupled molecular biological, spectroscopic, biochemical, and crystallographic experiments will guide the design of site-directed mutants and the selection of appropriate cofactor, substrate, intermediate and inhibitor complexes for further research. This integrated, recursive approach aims to increase understanding of NOS catalysis and ultimately to aid the design of isozyme-selective NOS inhibitors, which will be invaluable tools for discovering isoform functions in vivo and are desirable as therapeutic agents for controlling blood pressure, septic shock, and inflammatory damage.