Particulate Methane Monooxygenase

描述(申请人提供)：甲烷营养细菌利用甲烷作为其唯一的碳和能源来源。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)。尽管pMMO是自然界中主要的甲烷氧化催化剂，但事实证明很难分离出它，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联的酶，比pMMO更容易处理，但通用性较差。PMMO的结构和作用机理一直是生物无机化学中尚未解决的主要问题之一。除了甲烷，pMMO还氧化各种烷烃和烯烃，包括三氯乙烯(TCE)，这是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极佳候选者。由于pMMO是这些过程中的主要生物催化系统，了解其结构和机理将有助于在未来的生物修复应用中设计具有不同底物选择性的微生物。甲烷氧化菌还在全球甲烷循环中发挥关键作用，阻止大量甲烷进入大气，在大气中它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。人们已经对pMMO进行了一些生化和生物物理研究，但关于其分子结构和活性部位的大多数问题仍未得到解答。到目前为止的证据表明，铜离子起到了主要的催化作用，而且可能存在一个功能铁中心。PMMO络合物中金属离子的确切化学计量比、位置和配体环境尚未阐明。本研究的具体目的是从多株甲烷营养细菌中分离纯化活性pMMO，表征pMMO中的金属中心，获得衍射性良好的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.