Structural Biology of Prokaryotic NO Synthases

描述（由申请人提供）：一氧化氮（NO）已经成为一种重要的新的气体信使分子，可以介导人类的生理和病理反应。NO调节健康人受试者的血压，并且也由健康跳动的心脏不断合成以发挥其适当的功能。虽然控制NO的产生对于个体的健康是必不可少的，但过量产生可能导致严重的问题。在哺乳动物中，NO由统称为一氧化氮合酶（NOS）的三种酶的家族产生。NOS是由含血红素的催化结构域通过结合钙调蛋白的接头共价融合到还原酶结构域的双酶蛋白。NO的生理作用是通过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可以通过cGMP依赖性和非依赖性机制介导其作用。后者包括NO杀死细菌和病毒病原体的能力。NO对细菌的毒性已经引起了这样的假设，即NO信号通路是真核生物固有的。我们已经发现，一些革兰氏阳性细菌病原体编码一氧化氮合酶样基因，其产物具有很强的序列和结构相似的催化血红素结构域的NOS在哺乳动物中发现。该提案的主要长期目标是了解细菌NOS催化和分子识别的结构基础。我们将特别集中精力研究炭疽杆菌（炭疽的病原体）和金黄色葡萄球菌（一种对抗生素具有显著耐药性的生物体）的NOS。第二个长期目标是利用细菌NOS作为模型系统，以获得对哺乳动物NOS工作的分子见解。第三个长期目标是了解NOS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射线晶体学、生物化学、光谱学、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，还将为我们开发对抗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.