Particulate Methane Monooxygenase

描述（由申请人提供）：甲烷营养菌利用甲烷作为其唯一的碳源和能源。在其代谢途径的第一步中，甲烷单加氧酶（MMO）酶系统将甲烷氧化成甲醇。所有甲烷氧化菌都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，大多数研究人员转而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更易处理，但不那么通用。pMMO的结构和作用机制是生物无机化学中尚未解决的主要问题之一。除甲烷外，pMMO还氧化各种烷烃和烯烃，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优秀候选者。由于pMMO是在这些过程中的主要生物催化系统，其结构和机制的理解可以帮助工程微生物与不同的底物选择性，为未来的生物修复应用。甲烷氧化菌在全球甲烷循环中也发挥着关键作用，阻止大量甲烷进入大气层，而甲烷是一种潜在的温室气体。由于温室气体排放量增加而导致的全球变暖可能对人类健康产生不利影响。已经对pMMO进行了一些生物化学和生物物理学研究，但关于分子结构和活性位点的大多数问题仍然没有答案。迄今为止的证据表明，铜离子的主要催化作用以及功能性铁中心的可能存在。pMMO络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。拟议研究的具体目标是从多种甲烷氧化细菌菌株中溶解和纯化活性pMMO，表征pMMO中的金属中心，获得良好衍射的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.