Structural and biochemical characterization of redox reactions within nitric oxid

一氧化氮内氧化还原反应的结构和生化表征

• 批准号: 8003819
• 负责人: Sarah C Hokanson
• 金额: $ 4.56万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-16 至 2013-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003819
• 关键词: Active Sites; Affinity; Alzheimer' s Disease; Arginine; Binding; Binding Sites; Biochemical; Biological Process; Blood Pressure; Blood Vessels; C-terminal; Calmodulin; Cardiovascular Diseases; Cells; Citrulline; Communication; Complex; Drug Design; Electron Transport; Enzymes; Flavins; Goals; Heme; Hemeproteins; Hypertension; Isoenzymes; Kinetics; Link; Malignant Neoplasms; Mixed Function Oxygenases; Modeling; N-terminal; NADP; Neurodegenerative Disorders; Neurons; Nitric Oxide; Nitric Oxide Synthase; Oxidases; Oxidation-Reduction; Oxidoreductase; Oxygen; Parkinson Disease; Process; Production; Proteins; Pterins; Reaction; Source; Specificity; Structure; System; angiogenesis; base; cofactor; hormone regulation; novel; pathogen; public health relevance; tetrahydrobiopterin; transmission process

DESCRIPTION (provided by applicant): Nitric oxide (NO) is a diffusible, reactive molecule that has many overlapping biological functions, including control of vascular tone and blood pressure, protection against pathogens and cancer, hormone regulation, nerve cell transmission, and angiogenesis. Nitric oxide synthase (NOS) proteins are heme-based monooxygenase enzymes that convert L-arginine to L-citrulline and nitric oxide (NO) by a two-step electron transfer process. Mammalian NOS enzymes are homodimers that contain an N-terminal oxidase domain (NOSox) and C-terminal reductase domain called NOSred. Crosstalk between the two domains is regulated by a calmodulin (CaM)-binding interface. NOSox binds the L-arginine substrate, heme, and the redox-active cofactor 6R-tetrahydrobiopterin (H4B), all of which are required for an active enzyme. NOSred has binding sites for flavin cofactors as well as NADPH, and acts as a source of reducing equivalents for oxygen binding and activation at the heme in NOSox. Controlling the communication between redox-active cofactors in the NOSox and NOSred domains regulates at least two mammalian NOS isozymes, though a structure of the two domains in complex has not yet been achieved. Bacterial NOS enzymes share many similarities to their mammalian counterparts, and because of their stripped-down domain structure and ease of purification, bacterial NOS proteins serve as useful models for investigating the mechanism of NO synthesis. The goal of this proposal is to provide a better understanding about the relationship between NOS structural arrangement, electron transfer and the mechanism of NO production by NOS enzymes. In aim 1, we will study a novel NOS enzyme from S. pcc7335 (spNOS), characterizing its steady state activity and yield of NO synthesis, the reaction kinetics of its NOSox domain, as well as the affinity and specificity of pterin substrates for its redox active site. In aim 2, we will obtain crystal structures of two bacterial NOS enzymes, spNOS and a NOS enzyme from S. cellulosum (scNOS), which contain a fused reductase domain never observed before in bacterial systems. Finally, Aim 3 will target specific redox intermediates in the NOS electron transfer mechanism for structural characterization. Specifically, we will determine detailed structures of two heme-oxy states occurring in G. stearothermophilus NOS (gsNOS). PUBLIC HEALTH RELEVANCE: Nitric oxide synthase (NOS) proteins convert L-arginine to L-citrulline and nitric oxide (NO). NO is a diffusible, reactive molecule that functions to control of vascular tone and blood pressure, protection against pathogens and cancer, hormone regulation, nerve cell transmission, and angiogenesis. NO production in cells is a target for drug design in many different capacities, as overproduction of NO has been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's diseases, and insufficient NO production has been linked to conditions such as hypertension and cardiovascular disease.

描述（由申请人提供）：一氧化氮（NO）是一种可扩散的，反应性的分子，具有许多重叠的生物学功能，包括控制血管张力和血压，防止病原体和癌症的保护，激素调节，神经细胞传播和血管生成。一氧化氮合酶（NOS）蛋白是基于血红素的单加氧酶，通过两步电子传递过程将L-精氨酸转化为L-精氨酸和一氧化氮（NO）。哺乳动物的NOS酶是含有N末端氧化酶结构域（Nosox）和C末端还原酶结构域的同型二聚体，称为Nosred。两个域之间的串扰由钙调蛋白（CAM）结合界面调节。 Nosox结合L-精氨酸底物，血红素和氧化还原活跃的辅因子6R-四氢异物蛋白（H4B），所有这些都是活性酶所需的。 Nosred具有黄酮辅因子和NADPH的结合位点，并且是减少Nosox中血红素的氧结合和激活等效物的来源。控制Nosox和Nosred结构域中的氧化还原活性辅助因子之间的通信至少调节两个哺乳动物的NOS同工酶，尽管尚未实现两个域中的两个结构。细菌NOS酶与哺乳动物对应物具有许多相似之处，并且由于其剥离的结构域结构并易于纯化，细菌NOS蛋白是研究无合成机制的有用模型。该提案的目的是更好地了解NOS结构排列，电子传输与NOS酶无生产机制之间的关系。在AIM 1中，我们将研究一种来自S. pcc7335（SPNOS）的新型NOS酶，表征其稳态活性和无合成的产量，其Nosox域的反应动力学以及其翼龙底物的亲和力和特异性，其REDOX活性位点的亲和力和特异性。在AIM 2中，我们将获得来自纤维素链球菌（SCNOS）的两种细菌NOS酶，SPNOS和NOS酶的晶体结构，这些酶包含在细菌系统中从未观察到的融合还原酶结构域。最后，AIM 3将针对NOS电子转移机理中特定的氧化还原中间体进行结构表征。具体而言，我们将确定在G. stearothermophilus nos（GSNOS）中出现的两个血红素氧状态的详细结构。 公共卫生相关性：一氧化氮合酶（NOS）蛋白将L-精氨酸转化为L-硫氨酸和一氧化氮（NO）。 NO是一种可扩散的，反应性的分子，可以控制血管张力和血压，防止病原体和癌症，激素调节，神经细胞传播和血管生成。细胞中没有生产是许多不同容量的药物设计的目标，因为NO的过量生产与诸如帕金​​森氏症和阿尔茨海默氏病这样的神经退行性疾病有关，并且不够与高血压和心血管疾病等疾病有关。