Particulate Methane Monooxygenase

描述（由申请人提供）：甲烷营养细菌利用甲烷作为碳和能量的唯一来源。在其代谢途径的第一步，甲烷单加氧酶（MMO）酶系统将甲烷氧化为甲醇。所有的甲烷氧化菌都会产生一种膜结合的含铜酶，称为颗粒甲烷单加氧酶（pMMO）。虽然pMMO是自然界中主要的甲烷氧化催化剂，但它已被证明难以分离，大多数研究人员选择研究可溶性甲烷单加氧酶（sMMO），这是一种二铁羧酸桥接酶，比pMMO更容易处理，但不那么普遍。pMMO的结构和作用机理一直是生物无机化学领域未解决的主要问题之一。除甲烷外，pMMO还氧化多种烷烃和烯烃，包括三氯乙烯（TCE），一种有毒和致癌的地下水污染物。因此，甲烷氧化菌是污染土壤和饮用水生物修复的理想候选者。由于pMMO是这些过程中的主要生物催化系统，因此了解其结构和机制有助于设计具有不同底物选择性的微生物，以用于未来的生物修复应用。甲烷氧化菌在全球甲烷循环中也起着关键作用，它阻止大量甲烷进入大气层，而甲烷是一种强有力的温室气体。温室气体排放增加导致的全球变暖可能对人类健康产生有害影响。对pMMO进行了一些生物化学和生物物理方面的研究，但关于其分子结构和活性部位的大部分问题仍未得到解答。迄今为止的证据表明铜离子的主要催化作用以及可能存在的功能性铁中心。pMMO配合物中金属离子的确切化学计量、位置和配体环境尚未阐明。本研究的具体目的是对多株甲烷营养细菌中的活性pMMO进行溶解和纯化，表征pMMO中的金属中心，获得衍射良好的pMMO晶体，并确定其x射线结构。

DESCRIPTION (provided by applicant): Methanotrophic bacteria utilize methane as their sole source of carbon and energy. In the first step of their metabolic pathway, methane monooxygenase (MMO) enzyme systems oxidize methane to methanol. All methanotrophs produce a membrane-bound, copper-containing enzyme called particulate methane monooxygenase (pMMO). Although pMMO is the predominant methane oxidation catalyst in nature, it has proved difficult to isolate, and most investigators have instead opted to study soluble methane monooxygenase (sMMO), a diiron carboxylate-bridged enzyme that is more tractable, but less universal, than pMMO. The structure and mechanism of pMMO remains one of the major unsolved problems in bioinorganic chemistry. In addition to methane, pMMO oxidizes a variety of alkanes and alkenes, including trichloroethylene (TCE), a toxic and carcinogenic groundwater pollutant. Therefore, methanotrophs are excellent candidates for bioremediation of contaminated soil and drinking water. Since pMMO is the primary biocatalytic system in these processes, an understanding of its structure and mechanism could help to engineer microorganisms with different substrate selectivities for future bioremediation applications. Methanotrophs also play a key role in the global methane cycle, preventing large quantities of methane from reaching the atmosphere where it is a potent greenhouse gas. Global warming due to increasing greenhouse gas emissions can have detrimental effects on human health. Some biochemical and biophysical studies of pMMO have been conducted, but most questions regarding the molecular structure and active site remain unanswered. The evidence to date points to a primary catalytic role for copper ions as well as the possible existence of a functional iron center. The exact stoichiometry, location, and ligand environment of the metal ions in the pMMO complex have not been elucidated. The specific aims of the proposed research are to solubilize and purify active pMMO from multiple strains of methanotrophic bacteria, to characterize the metal centers in pMMO, to obtain well diffracting crystals of pMMO, and to determine its X-ray structure.