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

项目摘要/摘要： 这项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的转化 硫化物变成甲硫醇和甲醛。DMS单加氧酶是一种双组分的FMNH2依赖酶 需要DmoA单加氧酶亚基和DmoB黄素还原酶亚基的单加氧酶。两者都有 亚基的活性需要黄素单核苷酸(FMN)辅因子。围绕着黄化的机理 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不清楚。 产生菌DMS单加氧酶天然DmoB蛋白的初步鉴定 将通过三种方法定义磺胺类动物。有两种可能的黄素还原酶蛋白位于 DMO基因簇。确定活动所需的特定辅因子的初始动力学实验将为 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 将进行DmoA亚基与单独的DmoB候选者的活性测定。一次 天然的DmoB蛋白已经确定，黄素转移机制的研究将启动。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。首字母 表征将利用通过将His标记的DmoA固定在Ni-NTA柱上的亲和层析来鉴定 强大的、静态的蛋白质结合伙伴。凝胶过滤研究将类似地用于确定强结合 合伙人。稳定的DmoA：DmoB蛋白复合体的形成将通过几种分析方法来检测 包括蛋白质印迹分析和本地PAGE等技术。最后，荧光各向异性测量 提出了用来表征DmoA：DmoB相互作用的方法，并将量化 两个亚单位。 这项提议与美国国立卫生研究院的任务有关，因为它制定了减缓全球变暖的替代战略 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 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)是释放到大气中的VOSC的主要来源 大气层，并与气候变冷的趋势有关。气候变暖对人类健康有直接影响 由于水生和昆虫传播疾病的病例增加。此外，硫酸盐气雾剂吸入作为一种 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在环境中的退化及其在气候变化和最终人类健康中的作用的模型。 好了！

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. !