Structural Biology of Prokaryotic NO Synthases

说明书(申请人提供)：一氧化氮(NO)是一种重要的新型气态信使分子，可调节人体的生理和病理反应。NO调节健康人的血压，也由健康跳动的心脏不断合成，以发挥其正确的功能。尽管NO的受控生产对个人福祉是必不可少的，但过量生产可能会导致严重的问题。在哺乳动物中，一氧化氮是由一个统称为一氧化氮合酶(NOS)的三种酶家族产生的。一氧化氮合酶是一种双核蛋白，由一个含血红素的催化结构域通过连接钙调蛋白的连接子共价融合到一个还原酶结构域上。NO的生理作用是由其血红素蛋白受体--可溶性鸟苷酸环化酶介导的，该酶被NO激活并催化第二信使cGMP的生物合成。NO可通过cGMP依赖和非依赖两种机制介导其作用。后者包括一氧化氮杀死细菌和病毒病原体的能力。NO对细菌的毒性导致假设没有信号通路是真核生物所特有的。我们发现，一些革兰氏阳性细菌编码一种一氧化氮合酶样基因，其产物与哺乳动物中发现的一氧化氮合酶催化血红素结构域具有很强的序列和结构相似性。这项建议的主要长期目标是了解细菌一氧化氮合酶催化和分子识别的结构基础。我们将特别关注炭疽杆菌和金黄色葡萄球菌的一氧化氮合酶，炭疽杆菌是炭疽病的病原体，金黄色葡萄球菌是一种对抗生素表现出显著抗药性的微生物。第二个长期目标是利用细菌一氧化氮合酶作为模型系统，以获得对哺乳动物一氧化氮合酶工作原理的分子洞察。第三个长期目标是了解一氧化氮合酶在细菌病原体中的功能，并发现它可能参与的新途径。为了实现这些目标，我们计划利用一系列X射线结晶学、生化、光谱、分子生物学和遗传学方法。从拟议的研究中获得的知识不仅将产生关于细菌如何产生和处理氮氧化物的关键信息，而且还将为我们开发打击生物战的新战略提供关键线索。

DESCRIPTION (provided by applicant): Nitric oxide (NO) has emerged as an important new gaseous messenger molecule that can mediate physiological and pathological responses in humans. NO regulates blood pressure in healthy human subjects and is also constantly synthesized by a healthy beating heart for its proper function. While controlled production of NO is essential for an individual's well being, an overproduction can lead to severe problems. In mammals NO is produced by a family of three enzymes collectively known as nitric oxide synthases (NOS). NOS is a bidomain protein made up of a heme-containing catalytic domain covalently fused to a reductase domain via a linker that binds caldmodulin. Physiological actions of NO are mediated by its heme protein receptor, soluble guanylyl cyclase, which gets activated by NO and catalyzes the biosynthesis of second messenger, cGMP. NO can mediate its actions via both cGMP-dependent and -independent mechanisms. The latter includes the ability of NO to kill bacterial and viral pathogens. The toxicity of NO to bacteria has given rise to the assumption that NO signaling pathway is indigenous to eukaryotes. We have discovered that some gram-positive bacterial pathogens encode a NOS-like gene whose product bears strong sequence and structural resemblance to the catalytic heme domain of NOS found in mammals. The major long-term goal of this proposal is to understand the structural bases of catalysis and molecular recognition by bacterial NOS. We will particularly focus our efforts on NOS from Bacillus anthracis, the etiological agent of anthrax, and Staphylococcus aureus, an organism that exhibits remarkable resistance to antibiotics. A secondary long-term goal is to utilize bacterial NOS as a model system to gain molecular insights into the workings of mammalian NOS. A tertiary long-range goal is to understand the function of NOS in bacterial pathogens and to discover novel pathways in which it might participate. To achieve these goals, we plan to utilize a battery of X-ray crystallographic, biochemical, spectroscopic, molecular biological, and genetic methods. Knowledge gained from the proposed research will not only generate key information regarding how bacteria generate and deal with nitrogen oxides, but also provide us with critical leads for the development of novel strategies to combat biological warfare.