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. !

项目摘要/摘要： 拟议研究的目的是启动蛋白质二甲基黄素转移机制的研究 硫化物单加氧酶（DMS 单加氧酶）。 DMS单加氧酶催化二甲基转化 硫化物生成甲硫醇和甲醛。 DMS 单加氧酶是一种依赖 FMNH2 的双组分 需要 DmoA 单加氧酶亚基和 DmoB 黄素还原酶亚基的单加氧酶。两个都 亚基需要黄素单核苷酸 (FMN) 辅助因子才能发挥活性。黄素的作用机制 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， 围绕这种酶机制的具体分子细节仍然未知。！ 鉴定丝菌属 DMS 单加氧酶天然 DmoB 蛋白的初步研究 食磺菌将通过三种方法来定义。有两种假定的黄素还原酶蛋白位于 dmo 基因簇。定义活性所需的特定辅助因子的初始动力学实验将是 进行，然后进行荧光实验以定量黄素结合和化学计量。终于耦合了 将使用单独的 DmoB 候选物对 DmoA 亚基进行活性测定。一次 天然DmoB蛋白已确定，黄素转移机制研究将启动。蛋白质- DMS 单加氧酶的蛋白质相互作用研究将通过三种方法来定义。最初的 表征将利用固定在 Ni-NTA 柱上的 His 标记 DmoA 的亲和层析来鉴定 强、静态的蛋白质结合伙伴。凝胶过滤研究将类似地用于识别强结合 合作伙伴。稳定的 DmoA:DmoB 蛋白复合物的形成将通过多种分析检测 技术包括蛋白质印迹分析和天然 PAGE。最后，荧光各向异性测量 建议描述 DmoA:DmoB 相互作用的特征，并将量化 DmoA:DmoB 相互作用之间的结合相互作用 两个亚基。 该提案与 NIH 的使命相关，即制定缓解变暖的替代策略 温室气体排放引起的趋势。此外，该提案的结果将产生一个新的模型 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）。二甲硫醚 (DMS) 是释放到我们体内的生物 VOSC 的主要贡献者 大气，并与气候变冷趋势有关。气候变暖直接影响人类健康 水传播疾病和昆虫传播疾病的病例增加。此外，硫酸盐气溶胶吸入作为 VOSC 释放的结果与肺病和心脏病有关。这项工作的结果将在体外开发 模拟 DMS 在环境中的降解、其在气候变化以及最终人类健康中的作用的模型。 ！