Particulate Methane Monooxygenase

描述（由申请人提供）：甲烷营养细菌利用甲烷作为其唯一的碳和能量来源。其代谢途径的第一步是通过甲烷单加氧酶（MMO）酶系统将甲烷氧化为甲醇。除了一种甲烷氧化菌外，所有的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及其同源酶氨单加氧酶（AMO）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。了解pMMO如何激活O2氧化甲烷和其他碳氢化合物是本研究项目的长期目标。尽管有晶体结构和广泛的光谱数据，但关于金属含量和活性位点身份的关键问题仍未得到解答。这些问题对生物无机铜化学具有重要意义，对甲烷氧化菌在生物修复中的应用具有重要意义。此外，甲烷氧化菌在全球碳循环中发挥关键作用，有助于减轻全球变暖对人类健康的有害影响。拟建的研究涉及从多种生物中分离pMMO和AMO的纯化和表征。最先进的膜蛋白结晶技术将应用于这些酶。此外，还将开发表达系统以实现定点诱变实验。最后，优化体外酶活性并开展机制研究。公共卫生相关性：消耗甲烷气体的细菌在减缓全球变暖方面发挥着重要作用，而全球变暖对人类健康具有有害影响。这些细菌对被碳氢化合物致癌物污染的土壤和水的生物修复也很有用。该项目将研究这些细菌如何将甲烷转化为甲醇的细节。

DESCRIPTION (provided by applicant): Methanotrophic bacteria utilize methane as their sole carbon and energy source. The first step in their metabolic pathway is the oxidation of methane to methanol by methane monooxygenase (MMO) enzyme systems. All but one genus of 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 and the homologous enzyme ammonia monooxygenase (AMO) remain one of the major unsolved problems in bioinorganic chemistry. Understanding how pMMO activates O2 for oxidation of methane and other hydrocarbons is the long term goal of this research program. Despite the availability of a crystal structure and extensive spectroscopic data, key questions regarding the metal content and active site identity remain unanswered. These issues are of fundamental importance to bioinorganic copper chemistry and have implications for the use of methanotrophs in bioremediation. In addition, methanotrophs play a key role in the global carbon cycle and could help mitigate the deleterious effects of global warming on human health. The proposed research involves purification and characterization of pMMO and AMO from multiple organisms. State-of-the-art crystallization techniques for membrane proteins will be applied to these enzymes. In addition, expression systems will be developed to enable site-directed mutagenesis experiments. Finally, in vitro enzyme activity will be optimized and mechanistic studies initiated. PUBLIC HEALTH RELEVANCE: Bacteria that consume methane gas play an important role in mitigating global warming, which has deleterious effects on human health. These bacteria also are useful for bioremediation of soil and water polluted with hydrocarbon carcinogens. This project will investigate the details of how these bacteria transform methane into methanol.