Particulate Methane Monooxygenase

颗粒甲烷单加氧酶

• 批准号: 7942225
• 负责人: AMY C. ROSENZWEIG
• 金额: $ 5.1万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7942225
• 关键词: Active Sites; Affinity; Ammonia; Architecture; Arts; Bacteria; Bathing; Binding; Binding Sites; Bioinorganic Chemistry; Biological Assay; Bioremediations; Carbon; Carcinogens; Chemistry; Copper; Crystallization; Data; Energy-Generating Resources; Enzymes; Escherichia coli; Essential Amino Acids; Fab Immunoglobulins; Freezing; Gases; Global Warming; Goals; Health; Human; Hydrocarbons; In Vitro; Knowledge; Ligands; Lipids; Location; Membrane; Membrane Proteins; Metabolic Pathway; Metals; Methane; Methane hydroxylase; Methanol; Methods; Methylococcus capsulatus; Methylocystis; Methylosinus trichosporium; Microfluidics; Nature; Nitrosomonas; Organism; Particulate; Phage Display; Play; Preparation; Procedures; Proteins; Raman Spectrum Analysis; Research; Research Personnel; Resolution; Rhodobacter sphaeroides; Role; Sampling; Screening procedure; Site-Directed Mutagenesis; Soil; Structure; Substrate Specificity; System; Techniques; Testing; Transmembrane Domain; Variant; Water; Work; ammonia monooxygenase; analog; carboxylate; catalyst; enzyme activity; improved; oxidation; programs; protein complex; public health relevance; reconstitution; research study

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.

描述(申请人提供)：甲烷营养细菌利用甲烷作为其唯一的碳源和能源。其代谢途径的第一步是甲烷单加氧酶(MMO)系统将甲烷氧化为甲醇。除了一种以外，所有的甲烷氧化菌都能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及其同源酶氨单加氧酶(AMO)的结构和作用机制一直是生物无机化学中尚未解决的主要问题之一。了解pMMO如何激活O2以氧化甲烷和其他碳氢化合物是本研究计划的长期目标。尽管已经有了晶体结构和广泛的光谱数据，但关于金属含量和活性中心身份的关键问题仍然没有得到回答。这些问题对生物无机铜化学具有基本的重要性，并对甲烷氧化菌在生物修复中的使用产生了影响。此外，甲烷氧化菌在全球碳循环中发挥着关键作用，可以帮助缓解全球变暖对人类健康的有害影响。拟议的研究包括从多个生物体中纯化和鉴定pMMO和AMO。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，以进行定点突变实验。最后，将优化体外酶活性并启动机理研究。与公共健康相关：消耗甲烷气体的细菌在缓解全球变暖方面发挥着重要作用，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质污染的土壤和水的生物修复也很有用。这个项目将调查这些细菌如何将甲烷转化为甲醇的细节。