Metalloenzymes and metal homeostasis

金属酶和金属稳态

• 批准号: 10376838
• 负责人: AMY C. ROSENZWEIG
• 金额: $ 62.84万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10376838
• 关键词: Active Sites; Address; Ammonia; Anabolism; Bacteria; Binding; Biochemical; Bioinorganic Chemistry; Biology; Biophysics; Biosynthetic Proteins; Chemicals; Chemistry; Consumption; Copper; Crystallization; Disease; Environment; Enzymes; Gases; Global Warming; Health; Hepatolenticular Degeneration; Homeostasis; Human; Investigation; Iron; Lipids; Location; Membrane; Metabolic Pathway; Metabolism; Metals; Methane; Methane hydroxylase; Methanol; Microbe; Mixed Function Oxygenases; Molecular; Multienzyme Complexes; Natural Products; Nature; Operon; Particulate; Process; Proteins; Reducing Agents; Research; Research Project Summaries; Ribosomes; Site; Structure; Therapeutic; climate change; genetic approach; genetic manipulation; greenhouse gases; in vivo; insight; metal chelator; metalloenzyme; oxidation; programs; spectroscopic data; tool

Project Summary This research program centers on the bioinorganic chemistry of methanotrophic bacteria, microbes that convert methane, a potent greenhouse gas, to methanol in the first step of their metabolic pathway. As the primary methane sink in nature, methanotrophs are promising tools to mitigate the deleterious effects of global warming on human health, and may be deployed to generate fuels and chemicals from methane in an environmentally-friendly fashion. Moreover, some methanotrophs produce copper-binding natural products that are under investigation as therapeutics. The proposed projects take an integrated biochemical, biophysical, structural, and genetic approach to understanding these processes on the molecular level. The first project addresses the structure and function of particulate methane monooxygenase (pMMO), an integral membrane, copper-dependent enzyme that catalyzes the oxidation of methane to methanol. Despite the availability of pMMO crystal structures and a range of spectroscopic data, the location and atomic details of the copper active site remain unclear, and the sites of substrate, product, and reductant binding have not been elucidated, all prerequisites for elucidating the chemical mechanism. The experimental approach involves characterization of new pMMOs with highly divergent sequences, structural determination of pMMOs in a lipid environment that maintains enzymatic activity, and genetic manipulation of native methanotrophs. The results will lead to a comprehensive understanding of this critically important metalloenzyme and will further understanding of homologs such as ammonia monooxygenase (AMO), another contributor to climate change. The second project focuses on methanobactins (Mbns), ribosomally produced, post-translationally modified natural products secreted by methanotrophs to scavenge copper from the environment. The machinery to biosynthesize and transport Mbns is encoded in Mbn operons, which are also present in a wide range of non- methanotrophic bacteria, suggesting additional functions and unexplored diversity in structure. Mechanistic and structural studies of the core biosynthetic enzyme complex, the iron-containing MbnBC heterodimer, along with characterization of other biosynthetic proteins will be conducted. In addition, the involvement of other operon proteins in release of copper from Mbn will be investigated using both biochemical and in vivo strategies. Taken together, the results will provide new insights into natural products biosynthesis and will impact the use of these molecules as therapeutics for Wilson disease and other disorders of copper metabolism.

项目摘要 这项研究计划集中在甲烷营养细菌的生物无机化学上，这些微生物 甲烷是一种强有力的温室气体，在他们代谢途径的第一步，将甲烷转化为甲醇。作为 甲烷氧化菌在自然界中吸收初级甲烷，是缓解全球气候变化有害影响的有希望的工具 变暖对人类健康的影响，并可能被部署在一个由甲烷产生燃料和化学品的 环保时尚。此外，一些甲烷氧化菌产生与铜结合的天然产物 作为治疗学正在接受研究。拟议的项目采用了综合生化、生物物理、 在分子水平上理解这些过程的结构和遗传方法。 第一个项目涉及颗粒甲烷单加氧酶(PMMO)的结构和功能， 一种完整的膜，依赖于铜的酶，催化甲烷氧化成甲醇。尽管 PMMO晶体结构和一系列光谱数据的可用性、位置和原子细节 铜的活性部位尚不清楚，底物、产物和还原剂的结合部位也不清楚。 阐明了，阐明化学机理的所有先决条件。实验方法包括 具有高度差异序列的新pMMOS的表征，脂类中pMMOS的结构测定 维持酶活性的环境，以及对本地甲烷氧化菌的遗传操作。结果是 将导致对这种至关重要的金属酶的全面了解，并将进一步 了解同系物，如氨单加氧酶(AMO)，气候变化的另一个贡献者。 第二个项目的重点是甲烷结合蛋白(MBN)，核糖体产生，翻译后 由甲烷氧化菌分泌的天然修饰产物，用于从环境中清除铜。这台机器 生物合成和运输MBN是在MBN操纵子中编码的，这些操纵子也存在于广泛的非 甲烷益生菌，暗示了额外的功能和结构上的未知多样性。机械和 核心生物合成酶复合体-含铁的MbnBC杂二聚体的结构研究 将对其他生物合成蛋白质进行表征。此外，其他操作员的参与 从MBN释放铜的蛋白质将使用生物化学和体内策略进行研究。已被占用 总而言之，这些结果将为天然产品的生物合成提供新的见解，并将影响这些产品的使用 分子作为治疗威尔逊病和其他铜代谢障碍的药物。