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; 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. !

项目摘要/摘要： 拟议的研究的目的是对蛋白质二甲基的黄素转移机械研究 硫化物单加氧酶（DMS单加氧酶）。 DMS单加氧酶催化二甲基的转化 硫化物至甲烷和甲醛。 DMS单加氧酶是两个组成的FMNH2依赖性 单加氧酶需要DMOA单加氧酶亚基和DMOB黄素还原酶亚基。两个都 亚基需要黄素单核苷酸（FMN）辅因子才能进行活性。黄素周围的机制 从DMOA转移到DMOB仍然难以捉摸。尽管有一些有关生物学功能的线索，但 围绕该酶机制的特定分子细节仍然未知。 最初的研究以鉴定从菌粒中的DMS单加氧酶的天然DMOB蛋白 磺诺维犬将通过三种方法来定义。有两个假定的黄素还原酶蛋白 DMO基因簇。定义活动所需的特定辅助因子的初始动力学实验将是 进行，然后进行氟化实验，以定量黄素结合和化学计量。终于结合了 将使用单独的DMOB候选者对DMOA亚基的活性测定测量。一次 已经确定了天然DMOB蛋白，将启动黄素转移机制研究。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。最初的 表征将通过固定在Ni-NTA列的His标记的DMOA来使用亲和力色谱以识别 强，静态蛋白结合伙伴。凝胶过滤研究将类似地用于识别强大的结合 合作伙伴。稳定的DMOA：DMOB蛋白复合物的形成将通过几种分析检测 包括Western印迹分析和本地页面在内的技术。最后，荧光各向异性测量 建议表征DMOA：DMOB相互作用，并将定量结合相互作用 两个亚基。 该提议通过制定替代策略来减轻变暖，与NIH的使命有关 温室气体排放引起的趋势。此外，该提案的结果将产生新模型 研究气候变化的系统，特别是挥发性有机硫的酶促降解 化合物（VOSC）。二甲基硫化物（DMS）是释放到我们的生物源的主要促成生物源 气氛，与气候冷却趋势有关。气候变暖对人类健康有直接影响 通过增加出生和昆虫传播疾病的病例。另外，硫酸盐气溶胶作为A VOSC释放的结果与肺部和心脏病有关。这项工作的结果将在体外发展 模仿DMS在环境中降解的模型，其在气候变化和最终人类健康中的作用。 呢