Elucidating the mechanism of particulate methane monooxygenase

阐明颗粒甲烷单加氧酶的机制

• 批准号: 8634182
• 负责人: Megen A Culpepper
• 金额: $ 5.39万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8634182
• 关键词: Active Sites; Address; Bacteria; Binding; Binding Sites; Biochemical; Bioinorganic Chemistry; Biological Assay; Biological Models; Bioremediations; Cancer Etiology; Carbon; Catalysis; Centers for Disease Control and Prevention (U.S.); Chemistry; Chlorinated Hydrocarbons; Climate; Crystallization; Data; Dengue; Development; Diarrhea; Disease; Endocrine System Diseases; Environment; Environmental Health; Environmental Pollution; Enzyme Kinetics; Enzymes; Gas Chromatography; Halogenated Hydrocarbons; Health; Heating; High temperature of physical object; Human; Hydrocarbons; Hydrogen Bonding; Insecta; Kinetics; Length; Malaria; Malignant Neoplasms; Malnutrition; Membrane; Methane; Methane hydroxylase; Methanol; Mission; Modeling; Particulate; Pathway interactions; Property; Proteins; Reaction; Recombinants; Research; Resolution; Role; Running; Site; Site-Directed Mutagenesis; Societies; Spectrum Analysis; Structure; Substrate Specificity; Techniques; Tertiary Protein Structure; Time; Transition Elements; Trichloroethylene; United States National Institutes of Health; Variant; Water; Work; catalyst; climate change; environmental stressor; enzyme model; greenhouse gases; member; metalloenzyme; novel; oxidation; planetary Atmosphere; pollutant; public health relevance; research study; tool

DESCRIPTION (provided by applicant): The objective of the proposed research is to elucidate the mechanism of the integral membrane metalloenzyme particulate methane monooxygenase (pMMO). pMMO efficiently catalyzes the selective oxidation of methane to methanol under ambient conditions. Our central hypothesis is that pMMO catalyzes the selective oxidation of methane using a novel mechanistic pathway. Central to the catalytic pathway is an oxo-bridged dicopper species somewhat similar to that in previously characterized dicopper enzymes and model compounds. However, the coordination environment of the pMMO dicopper center is significantly different from that in all other known dicopper enzymes, and likely defines a completely new class of enzymes. A novel active site and new O2 activation chemistry will likely emerge, impacting both bioinorganic chemistry and catalysis. The pMMO mechanism will be defined by three approaches. Initial characterization will investigate the O2 binding at the dicopper site of pMMO and a recombinant construct of the soluble pmoB domain (spmoB) using various spectroscopic techniques. spmoB will be used in the studies as a functional model for pMMO and site-specific variants will be made to further probe the properties of the active site. Once the O2 binding has been characterized, enzyme kinetics using gas chromatography and stopped-flow spectroscopy will be determined. These data will define the role of the membrane in pMMO and trap fast timescale intermediates on the reaction pathway. In parallel to the biochemical studies, the high-resolution crystal structures of oxidized and reduced pMMO and spmoB will be employed. All the proposed studies will be run in the presence and absence of a suitable substrate to investigate the site of methane entry and oxidation. This proposal is relevant to the mission of the NIH by developing new strategies to diminish both cancer causing environmental contaminates and diseases induced by climate change. pMMO breaks down the most inert hydrocarbon, methane, under ambient conditions and therefore represents an attractive target in the development of green catalysts to target bioremediation and minimize greenhouse gas emissions. Halogenated hydrocarbon pollutants, such as trichloroethylene (TCE) and vinylchloride (VC) that pose a threat to human health are effectively degraded by pMMO. According to the Centers of Disease Control, chlorinated hydrocarbons are implicated in endocrine disorders and many forms of cancer. Additionally, pMMO represents a target for minimizing greenhouse gas emissions that pose a threat to human health by increasing the earth<s climate. Climate changes due to greenhouse gas emissions increase water borne diseases and diseases transmitted through insects such as diarrhea, malnutrition, malaria, and dengue.

描述（由申请人提供）：拟议的研究的目的是阐明整体膜金属酶颗粒甲烷单加仑酶（PMMO）的机制。 PMMO在环境条件下有效催化甲烷对甲醇的选择性氧化。我们的中心假设是，PMMO使用新型的机械途径催化甲烷的选择性氧化。催化途径的核心是一种与先前特征的二十二杯酶和模型化合物相似的氧桥式双齿物种。然而，PMMO DiCopper中心的协调环境与所有其他已知的二氧化​​碳酶的协调环境显着不同，并且可能定义了一类全新的酶。一种新型的活跃部位和新的O2激活化学可能会影响生物无机化学和催化。 PMMO机制将通过三种方法定义。最初的表征将使用各种光谱技术研究PMMO的Dicopper位点的O2结合以及可溶性PMOB结构域（SPMOB）的重组构建体。 SPMOB将在研究中用作PMMO的功能模型，将进行特定于位点的变体，以进一步探测活性位点的性质。一旦表征了O2结合，将确定使用气相色谱和停止流光谱法的酶动力学。这些数据将定义膜在PMMO中的作用，并在反应途径上捕获快速时间尺度中间体。与生化研究并行，将采用氧化和减少PMMO和SPMOB的高分辨率晶体结构。所有提出的研究将在存在和不存在合适的底物的情况下进行研究，以研究甲烷进入和氧化位点。 该提案与NIH的使命有关，通过制定新的策略来减少引起环境污染物和由气候变化引起的疾病的癌症。 PMMO在环境条件下分解了最惰性的碳氢化合物，甲烷，因此代表了绿色催化剂靶向生物修复并最大程度减少温室气体排放的有吸引力的靶标。 PMMO有效地降低了对人类健康构成威胁的卤代污染物，例如三氯乙烯（TCE）和乙烯基氯化物（VC）。根据疾病控制的中心，氯化烃与内分泌疾病和多种形式的癌症有关。此外，PMMO代表了最大程度地减少温室气体排放的目标，该温室气体排放量通过增加地球气候对人类健康构成威胁。由于温室气体排放引起的气候变化增加了通过腹泻，营养不良，疟疾和登革热等昆虫传播的水传播疾病和疾病。