Initial flavin transfer studies on the sulfur-degrading enzyme Dimethyl Sulfide Monooxygenase

硫降解酶二甲硫醚单加氧酶的初步黄素转移研究

• 批准号: 9244915
• 负责人: Megen A Culpepper
• 金额: $ 6.27万
• 依托单位: APPALACHIAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9244915
• 关键词: Address; Aerosols; Affect; Affinity Chromatography; Back; Binding; Binding Proteins; Biological Assay; Biological Models; Biological Process; Breathing; Catalysis; Cell Nucleus; Climate; Complex; Coupled; Data; Development; Diarrhea; Disease; Electron Transport; Environment; Environmental Pollution; Enzyme Kinetics; Enzymes; Escherichia coli; Flavin Mononucleotide; Flavins; Fluorescence; Fluorescence Anisotropy; Formaldehyde; Gases; Gel Chromatography; Gene Cluster; Global Warming; Health; Human; Hyphomicrobium; Insecta; Kinetics; Label; Link; Malaria; Malnutrition; Measurement; Measures; Mission; Mixed Function Oxygenases; Modeling; Molecular; NADH; National Institute of Environmental Health Sciences; Operon; Oxidoreductase; Pathway interactions; Pattern; Physical condensation; Planet Earth; Proteins; Public Health; Pulmonary Heart Disease; Radiation; Recombinants; Regulation; Research; Role; Running; Solar Energy; Substrate Specificity; Sulfur; Sulfur Compounds; System; Techniques; Testing; Titrations; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Water; Western Blotting; Work; Zika Virus; analog; anthropogenesis; biophysical techniques; chemical reaction; climate change; climate impact; cofactor; dimethyl sulfide; enzyme mechanism; enzyme substrate; experimental study; greenhouse gases; in vitro Model; model development; permissiveness; planetary Atmosphere; protein complex; protein protein interaction; stoichiometry; trend

Project Summary/Abstract: The objective of the proposed research is to initiate flavin transfer mechanistic studies on the protein dimethyl sulfide monooxygenase (DMS monooxygenase). DMS monooxygenase catalyzes the conversion of dimethyl sulfide to methanethiol and formaldehyde. DMS monooxygenase is a two-component FMNH2-dependent monooxygenase that requires a DmoA monooxygenase subunit and a DmoB flavin reductase subunit. Both subunits require a flavin mononucleotide (FMN) cofactor for activity. The mechanism surrounding the flavin transfer from DmoA to DmoB remains elusive. Though there are some clues regarding biological function, the specific molecular details surrounding this enzyme mechanism remain unknown.! Initial studies to identify the native DmoB protein of DMS monooxygenase from Hyphomicrobium sulfonivorans will be defined by three approaches. There are two putative flavin reductase proteins located on the dmo gene cluster. Initial kinetic experiments to define the specific cofactors required for activity will be performed, followed by fluorimetric experiments to quantitate flavin binding and stoichiometry. Finally coupled activity assay measurements of the DmoA subunit with the separate DmoB candidates will be performed. Once the native DmoB protein has been determined, flavin transfer mechanism studies will be initiated. The protein- protein interactions studies of DMS monooxygenase will be defined by three approaches. Initial characterization will utilize affinity chromatography by His-tagged DmoA affixed to a Ni-NTA column to identify strong, static protein binding partners. Gel filtration studies will be used similarly to identify strong binding partners. The formation of a stable DmoA:DmoB protein complex will be detected by several analytical techniques including western blot analysis and native PAGE. Finally, fluorescence anisotropy measurements are proposed to characterize the DmoA:DmoB interaction, and will quantitate the binding interaction among the two subunits. This proposal is relevant to the mission of the NIH by developing alternate strategies to mitigate warming trends caused by greenhouse gas emissions. In addition, the results of this proposal will produce a new model system for studying climate change, in particular the enzymatic degradation of volatile organic sulfur compounds (VOSC). Dimethyl sulfide (DMS) is the major contributing biogenic VOSC released into our atmosphere, and is implicated in climate cooling trends. Climate warming has a direct effect on human health by increasing cases of water-born and insect transmitted diseases. Additionally, sulfate aerosol inhalation as a result of VOSC release is linked to pulmonary and heart disease. The results of this work will develop in vitro models to mimic DMS degradation in the environment, its role in climate change and ultimately human health. !

项目概要/摘要： 该研究的目的是启动黄素转移机制的研究蛋白质二甲基 硫化物单加氧酶（DMS单加氧酶）。DMS单加氧酶催化二甲基 硫化物转化为甲硫醇和甲醛。DMS单加氧酶是一种双组分FMNH 2依赖性 在一些实施方案中，DmoB是需要DmoA单加氧酶亚基和DmoB黄素还原酶亚基的单加氧酶。两 亚基的活性需要黄素单胞苷肽（FMN）辅因子。围绕黄素的机制 从DmoA到DmoB的转移仍然难以捉摸。尽管有一些关于生物功能的线索，但 围绕这种酶机制的具体分子细节仍然未知。 鉴定丝微菌DMS单加氧酶天然DmoB蛋白的初步研究 将通过三种方法来定义磺胺类药物。有两个假定的黄素还原酶蛋白位于 DMO基因簇。确定活性所需的特定辅因子的初始动力学实验将在 进行，然后进行荧光实验以定量黄素结合和化学计量。最终耦合 将进行DmoA亚基与单独的DmoB候选物的活性测定测量。一旦 天然DmoB蛋白已被确定，黄素转移机制的研究将开始。蛋白质- DMS单加氧酶的蛋白质相互作用研究将由三种方法定义。初始 表征将利用通过附着到Ni-NTA柱上的His标记的DmoA的亲和色谱来鉴定 强的、静态的蛋白质结合伴侣。凝胶过滤研究将以类似方式用于鉴定强结合 伙伴稳定的DmoA：DmoB蛋白复合物的形成将通过几种分析方法检测。 包括蛋白质印迹分析和非变性PAGE的技术。最后，荧光各向异性测量 提出表征DmoA：DmoB相互作用，并将定量DmoA：DmoB之间的结合相互作用。 两个小组。 这项建议与国家卫生研究院的使命有关，即制定减缓气候变暖的替代战略 温室气体排放造成的趋势。此外，这项提案的结果将产生一种新的模式 用于研究气候变化，特别是挥发性有机硫的酶降解的系统 化合物（VOSC）。二甲基硫（DMS）是主要的贡献生物VOSC释放到我们的 大气层，并与气候变冷趋势有关。气候变暖直接影响人类健康 通过增加水传播和昆虫传播的疾病。此外，硫酸盐雾化吸入作为一种 VOSC释放的结果与肺部和心脏疾病有关。这项工作的结果将在体外发展 模拟二甲硫醚在环境中的降解及其在气候变化和最终人类健康中的作用的模型。 !