Elucidating the mechanism of particulate methane monooxygenase

阐明颗粒甲烷单加氧酶的机制

• 批准号: 8262694
• 负责人: Megen A Culpepper
• 金额: $ 5.22万
• 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 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8262694
• 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. PUBLIC HEALTH RELEVANCE: The studies proposed in this work are significant because they address the growing concern in our society on the effect environmental contamination and the global climate change has on human health. The development of greener, safer catalysis is essential in diminishing these environmental health concerns. The strategies proposed here are the first steps in developing these catalysts and minimizing the effects environmental stressors pose on human health and wellbeing.

描述（由申请人提供）：拟议研究的目的是阐明整合膜金属酶颗粒甲烷单加氧酶（pMMO）的机制。 pMMO 在环境条件下有效催化甲烷选择性氧化为甲醇。我们的中心假设是 pMMO 使用一种新的机制途径催化甲烷的选择性氧化。催化途径的核心是氧桥二铜物质，与之前表征的二铜酶和模型化合物有些相似。然而，pMMO 二铜中心的配位环境与所有其他已知二铜酶的配位环境显着不同，并且可能定义了一类全新的酶。可能会出现新的活性位点和新的 O2 活化化学，从而影响生物无机化学和催化。 pMMO 机制将通过三种方法来定义。初步表征将使用各种光谱技术研究 pMMO 的二铜位点和可溶性 pmoB 结构域 (spmoB) 的重组构建体上的 O2 结合。 spmoB 将在研究中用作 pMMO 的功能模型，并且将制作位点特异性变体以进一步探测活性位点的特性。一旦确定了 O2 结合的特征，就可以使用气相色谱法和停流光谱法来确定酶动力学。这些数据将定义膜在 pMMO 中的作用并捕获反应途径中的快速时间尺度中间体。在进行生化研究的同时，还将采用氧化和还原 pMMO 和 spmoB 的高分辨率晶体结构。所有拟议的研究都将在存在和不存在合适底物的情况下进行，以研究甲烷进入和氧化的位置。 该提案与美国国立卫生研究院的使命相关，即制定新战略来减少导致癌症的环境污染和气候变化引起的疾病。 pMMO 在环境条件下分解最惰性的碳氢化合物，即甲烷，因此在开发绿色催化剂以实现生物修复和最大限度地减少温室气体排放方面是一个有吸引力的目标。 pMMO可有效降解对人类健康构成威胁的卤代烃污染物，如三氯乙烯（TCE）和氯乙烯（VC）。据疾病控制中心称，氯化烃与内分泌失调和多种癌症有关。此外，pMMO 代表了最大限度减少温室气体排放的目标，温室气体排放通过加剧地球气候而对人类健康构成威胁。温室气体排放导致的气候变化增加了水源性疾病和通过昆虫传播的疾病，如腹泻、营养不良、疟疾和登革热。 公共健康相关性：这项工作中提出的研究意义重大，因为它们解决了我们社会日益关注的环境污染和全球气候变化对人类健康影响的问题。开发更绿色、更安全的催化对于减少这些环境健康问题至关重要。这里提出的策略是开发这些催化剂并最大限度地减少环境压力对人类健康和福祉造成的影响的第一步。