Metalloenzymes and metal homeostasis

金属酶和金属稳态

• 批准号: 9069232
• 负责人: AMY C. ROSENZWEIG
• 金额: $ 52.95万
• 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 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9069232
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Active Sites; Address; Affinity; Area; Bacteria; Binding; Biochemical; Bioinorganic Chemistry; Biological; Biological Assay; Bioremediations; Carbon; Carcinogens; Catalysis; Chemicals; Crystallography; Global Warming; Goals; Halogenated Hydrocarbons; Health; Homeostasis; Human; Hydrocarbons; Integral Membrane Protein; Ion Transport; Ions; Link; Membrane; Metal Binding Site; Metalloproteins; Metals; Methane Metabolism Pathway; Methane hydroxylase; Methanol; Molecular; Organism; Oxygen; Particulate; Play; Process; Property; Protein Subunits; Proteins; Recombinants; Research; Role; Scaffolding Protein; Site; Source; Specificity; Spectrum Analysis; Structure; Substrate Specificity; Variant; Virulence; base; human disease; in vitro activity; in vivo; metal metabolism; metalloenzyme; oxidation; pathogen; programs; public health relevance

 DESCRIPTION (provided by applicant): This research program focuses on two areas of bioinorganic chemistry: metal transport by P1B-ATPases and biological methane oxidation by particulate methane monooxygenase (pMMO). Common themes of these two projects include understanding the structure and function of integral membrane metalloproteins, elucidating the atomic details of metal sites within these proteins, and establishing molecular mechanisms of metal ion transport or of catalysis by metal ions. These two processes are fundamentally different: transport requires a highly specific metal binding site with dynamically changing affinities whereas catalysis demands a site specifically tailored for chemical transformations. In both cases, the long term goal is to understand how the larger context of the protein scaffold confers these functional properties. The P1B-ATPases, integral membrane proteins that use the energy of ATP hydrolysis to transport metal ions across membranes, play a key role in metal homeostasis in all organisms. In particular, P1B-ATPases are linked to human diseases of metal metabolism and to the virulence of human pathogens. Despite their universal importance, fundamental issues related to P1B-ATPase structure and function remain unresolved, including the molecular basis of metal ion specificity and the mechanism of transport. These questions will be addressed by characterizing a range of P1B-ATPases that transport different metal substrates. Experimental approaches include biochemical characterization, metal binding studies, spectroscopy, in vitro activity assays, in vivo analysis, spectroscopy, and crystallography. Particulate methane monooxygenase (pMMO) is an oligomeric, integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria, organisms that utilize methane as their sole source of carbon and energy. Methanotrophs are a potential means to mitigate the deleterious effects of global warming on human health. In addition, methanotrophs can oxidize other substrates, including halogenated hydrocarbons, and have therefore been targeted for bioremediation applications. Major questions central to pMMO structure and function will be addressed, including the atomic details of the active site, the chemical mechanisms of oxygen and methane activation, the roles of the different protein subunits, and the molecular basis for substrate specificity. The experimental approach involves biochemical, spectroscopic, mechanistic, and crystallographic characterization of native pMMOs, recombinant variant pMMOs, and recombinant soluble pMMO domains.

 描述（由申请人提供）：该研究项目重点关注生物无机化学的两个领域：P1B-ATP酶的金属运输和颗粒甲烷单加氧酶（pMMO）的生物甲烷氧化这两个项目的共同主题包括了解整体的结构和功能。膜金属蛋白，阐明这些蛋白质内金属位点的原子细节，并建立金属离子运输或金属离子催化的分子机制。这两个过程本质上是不同的：运输需要具有动态变化的亲和力的高度特异性的金属结合位点，而催化需要专门针对化学转化的位点。在这两种情况下，长期目标是了解蛋白质支架的更大背景如何赋予。这些功能特性是 P1B-ATP 酶，利用 ATP 水解的能量跨膜运输金属离子，在所有生物体的金属稳态中发挥着关键作用。尽管它们具有普遍重要性，但与金属离子特异性的分子基础和转运机制有关的基本问题仍未得到解决。将通过表征一系列转运不同金属底物的 P1B-ATP 酶来解决，实验方法包括生化表征、金属结合研究、光谱学、体外活性测定、体内分析、光谱学、颗粒甲烷单加氧酶 (pMMO) 是一种低聚整体膜金属酶，可在甲烷氧化菌中将甲烷转化为甲醇，甲烷氧化菌是利用甲烷作为其唯一碳和能源来源的生物体，是减轻全球有害影响的潜在手段。此外，甲烷氧化菌还可以氧化其他底物，包括卤代烃，因此已成为防治目标。将解决 pMMO 结构和功能的核心问题，包括活性位点的原子细节、氧和甲烷活化的化学机制、不同蛋白质亚基的作用以及底物实验特异性的分子基础。这些方法涉及天然 pMMO、重组变体 pMMO 和重组可溶性 pMMO 结构域的生化、光谱、机械和晶体学表征。