Metalloenzymes and metal homeostasis

金属酶和金属稳态

• 批准号: 9894812
• 负责人: AMY C. ROSENZWEIG
• 金额: $ 57.91万
• 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/9894812
• 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; Consumption; Crystallography; Global Warming; Goals; Halogenated Hydrocarbons; Health; Homeostasis; Human; Hydrocarbons; Integral Membrane Protein; Ion Transport; Ions; Link; Membrane; Metal Binding Site; Metalloproteins; Metals; Methane; Methane Metabolism Pathway; Methane hydroxylase; Methanol; Molecular; Organism; Oxides; Oxygen; Particulate; Play; Process; Property; Protein Subunits; Proteins; Recombinants; Research; Role; Scaffolding Protein; Site; Source; Specificity; Spectrum Analysis; Structure; Substrate Specificity; Variant; Virulence; human disease; human pathogen; in vitro activity; in vivo; metal metabolism; metalloenzyme; oxidation; 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）：该研究计划侧重于生物无机化学的两个领域：P1 B-ATP酶的金属转运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理解膜金属蛋白的结构和功能，阐明这些蛋白质中金属位点的原子细节，以及建立金属离子转运或金属离子催化的分子机制。这两个过程是根本不同的：运输需要一个高度特异性的金属结合位点，具有动态变化的亲和力，而催化需要一个专门为化学转化量身定制的位点。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。 P1 B-ATP酶是利用ATP水解的能量跨膜转运金属离子的膜蛋白，在所有生物体的金属稳态中起关键作用。特别是，P1 B-ATP酶与人类金属代谢疾病和人类病原体的毒力有关。尽管它们具有普遍的重要性，但与P1 B-ATP酶结构和功能相关的基本问题仍未得到解决，包括金属离子特异性的分子基础和转运机制。这些问题将通过表征一系列运输不同金属底物的P1 B-ATP酶来解决。实验方法包括生物化学表征、金属结合研究、光谱学、体外活性测定、体内分析、光谱学和晶体学。 颗粒甲烷单加氧酶（pMMO）是一种寡聚的、完整的膜金属酶，其在甲烷营养细菌中将甲烷转化为甲醇，甲烷营养细菌是利用甲烷作为其唯一碳源和能量的生物体。甲烷氧化菌是缓解全球变暖对人类健康有害影响的潜在手段。此外，甲烷氧化菌可以氧化其他底物，包括卤代烃，因此已成为生物修复应用的目标。pMMO结构和功能的主要问题将得到解决，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，以及底物特异性的分子基础。实验方法涉及天然pMMO、重组变体pMMO和重组可溶性pMMO结构域的生物化学、光谱学、机械学和晶体学表征。