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 该研究计划集中于甲状腺营养细菌的生物学化学，微生物， 将甲烷（一种有效的温室气）转换为代谢途径的第一步。作为 本质中的主要甲烷下水道，甲烷营养是有希望减轻全球有害影响的有前途的工具 对人类健康的变暖，可以部署以从甲烷中产生燃料和化学物质 环保的时尚。此外，某些甲烷营养产生的铜结合天然产品 正在接受治疗。拟议的项目采用综合的生化，生物物理， 在分子水平上理解这些过程的结构和遗传方法。 第一个项目解决了颗粒甲烷单加氧酶（PMMO）的结构和功能， 一种整体膜的铜依赖性酶，可催化甲烷氧化为甲醇。尽管 PMMO晶体结构以及一系列光谱数据的可用性，位置和原子细节 铜的活性位点尚不清楚，底物，产物和还原剂结合的位置尚未 阐明了所有先决条件，以阐明化学机制。实验方法涉及 具有高度不同序列的新PMMO的表征，脂质中PMMO的结构测定 维持酶活性的环境以及对天然甲嗜营养物的遗传操纵。结果 将导致对这一至关重要的金属酶的全面理解，并将进一步 了解诸如氨基氧酶（AMO）之类的同源物，这是气候变化的另一个原因。 第二个项目侧重于甲烷素（MBN），核糖体产生的核糖体。 通过甲烷营养分泌的改性天然产品从环境中清除铜。机械 生物合成和运输MBN在MBN操纵子中编码，这些操纵子也存在于广泛的非 - 甲状腺营养细菌，提示了其他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的结构研究，含铁MBNBC异二聚体 将进行其他生物合成蛋白的表征。此外，其他操纵子的参与 将使用生化和体内策略研究释放MBN铜的蛋白质。拍摄 结果将共同提供对天然产品生物合成的新见解，并会影响这些的使用 分子作为威尔逊疾病和其他铜代谢疾病的疗法。