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.

项目概要 该研究计划的重点是甲烷氧化细菌的生物无机化学，这些微生物 在其代谢途径的第一步中，将甲烷（一种强效温室气体）转化为甲醇。作为 作为自然界中主要的甲烷汇，甲烷氧化菌是减轻全球甲烷排放有害影响的有前途的工具。 气候变暖对人类健康的影响，可用于在甲烷中产生燃料和化学品 环保时尚。此外，一些甲烷氧化菌产生结合铜的天然产物， 正在作为治疗方法进行研究。拟议的项目综合了生物化学、生物物理、 结构和遗传学方法在分子水平上理解这些过程。 第一个项目研究颗粒甲烷单加氧酶 (pMMO) 的结构和功能， 一种完整的膜、铜依赖性酶，可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 铜的活性位点仍不清楚，底物、产物和还原剂结合的位点尚未确定 阐明了阐明化学机制的所有先决条件。实验方法包括 具有高度差异序列的新型 pMMO 的表征、脂质中 pMMO 的结构测定 维持酶活性的环境，以及天然甲烷氧化菌的基因操作。结果 将导致对这种极其重要的金属酶的全面了解，并将进一步 了解氨单加氧酶（AMO）等同系物，它是气候变化的另一个促成因素。 第二个项目重点关注核糖体产生的翻译后甲烷菌素 (Mbns) 由甲烷氧化菌分泌的改良天然产物，用于从环境中清除铜。机械 生物合成和运输 Mbns 是由 Mbn 操纵子编码的，这些操纵子也存在于多种非 甲烷氧化细菌，表明具有额外的功能和未开发的结构多样性。机械和 核心生物合成酶复合物、含铁 MbnBC 异二聚体的结构研究，以及 将进行其他生物合成蛋白质的表征。此外，还有其他操纵子的参与 将使用生化和体内策略研究从 Mbn 释放铜的蛋白质。采取 总之，这些结果将为天然产物生物合成提供新的见解，并将影响这些物质的使用 分子作为威尔逊病和其他铜代谢紊乱的治疗方法。