The essential role of copper in bacterial methane oxidation: characterising a novel copper protein involved in storage and the soluble copper proteome

铜在细菌甲烷氧化中的重要作用：表征参与储存的新型铜蛋白和可溶性铜蛋白质组

• 批准号: BB/K008439/1
• 负责人: Christopher Dennison
• 金额: $ 41.8万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK008439%2F1
• 关键词: essential; role; copper; bacterial; methane

Methane-oxidising bacteria use methane, a potent greenhouse gas, as a source of carbon and energy. To enable these organisms to perform this key function most effectively they require large quantities of copper. Methane-oxidising bacteria have evolved novel mechanisms that ensure they are able to accumulate the copper they need to metabolise methane. This includes the secretion of small peptides called methanobactins (mbtins) that are able to sequester the minute amounts of copper present in the organism's surrounding environment. Methane-oxidising bacteria use mbtin to uptake copper, with one of the main uses being for the enzyme that oxidises methane (a methane monooxygenase). How these organisms handle copper, which is potentially toxic as well as being essential, has not been studied in any detail. Whilst analysing extracts from Methylosinus trichosporium OB3b, a methane-oxidising bacterium, for mbtin we have found a number of high abundance soluble copper pools containing proteins that bind this metal. The presence of large amounts of soluble copper in M. trichosporium OB3b was unanticipated as only two low-abundance soluble copper proteins are predicted (the copper-containing methane monooxygenase is membrane bound). One of the high abundance copper pools has been found to contain a novel copper protein, potentially involved in storing copper ions, herein called Csp1. The other soluble copper pools have not yet been studied in any detail.In the proposed work we will study copper binding to Csp1 both in M. trichosporium OB3b and also using protein that we have produced in large amounts via an over-expression host. We will analyse the structure of Csp1 and how this is influenced by copper binding. We will investigate the role of Csp1 in copper storage by genetically engineering M. trichosporium OB3b, as well as by looking at how copper levels during growth influence the production of Csp1. Proteins similar to Csp1 are present in methane-oxidising bacteria and also in other bacteria. The Csp1 homologues from M. trichosporium OB3b and Bacillus subtilis, an organism in which copper handling has been studied and that is more tractable for genetic engineering approaches, will be studied. The other soluble copper pools that have been identified in M. trichosporium OB3b will also be analysed with the aim of finding additional novel copper-binding proteins. The proposed studies will provide insight into copper management in a relatively simple system of environmental importance and therefore has applications in helping to understand the oxidation of methane in Nature and the mitigation of the contribution this greenhouse gas makes to global warming. Furthermore, vast gaseous methane reserves are untapped as a feedstock for liquid fuels and chemical production due to the expense and difficulties associated with the available processes that can facilitate the conversion to methanol, a reaction readily performed by methane-oxidising bacteria. Understanding how these organisms handle an essential cofactor, copper, for the oxidation of methane therefore has potential biotechnological applications.

甲烷氧化细菌利用甲烷作为碳和能量的来源，甲烷是一种强效温室气体。为了使这些生物体能够最有效地执行这一关键功能，它们需要大量的铜。甲烷氧化细菌已经进化出新的机制，确保它们能够积累代谢甲烷所需的铜。这包括分泌一种叫做甲烷氧化菌素（mbtins）的小肽，这种肽能够隔离存在于生物体周围环境中的微量铜。甲烷氧化细菌利用mbtin吸收铜，主要用途之一是氧化甲烷的酶（甲烷单加氧酶）。这些生物如何处理铜，这是潜在的毒性以及是必不可少的，还没有进行任何详细的研究。在分析甲烷氧化细菌Methylosinus trichosporium OB3b的提取物时，我们发现了许多含有结合这种金属的蛋白质的高丰度可溶性铜池。M.丝孢霉OB3b的发现是出乎意料的，因为仅预测了两种低丰度的可溶性铜蛋白（含铜的甲烷单加氧酶是膜结合的）。已发现高丰度铜池之一含有一种新的铜蛋白，其可能参与储存铜离子，本文称为Csp 1。其他可溶性铜池尚未进行任何详细的研究。毛孢菌OB3b，并且还使用我们通过过表达宿主大量产生的蛋白质。我们将分析Csp1的结构，以及它如何受到铜结合的影响。我们将通过基因工程方法研究Csp1在铜储存中的作用。毛孢菌OB3b，以及通过观察生长过程中铜水平如何影响Csp1的产生。类似于Csp1的蛋白质存在于甲烷氧化细菌和其他细菌中。Csp1同源物来自M.将研究毛孢霉OB3b和枯草芽孢杆菌，枯草芽孢杆菌是一种已经研究过铜处理的生物，并且对于基因工程方法更容易处理。在M.还将分析毛孢菌OB3b，目的是发现其他新的铜结合蛋白。拟议的研究将在一个相对简单的环境重要性系统中深入了解铜的管理，因此有助于了解自然界中甲烷的氧化和减轻这种温室气体对全球变暖的贡献。此外，由于与可促进转化为甲醇的可用方法相关的费用和困难，大量的气态甲烷储量未被开发作为液体燃料和化学生产的原料，甲醇是一种容易由甲烷氧化细菌进行的反应。因此，了解这些生物如何处理甲烷氧化的重要辅因子铜具有潜在的生物技术应用。

项目成果

Visualizing Biological Copper Storage: The Importance of Thiolate-Coordinated Tetranuclear Clusters.

• DOI: 10.1002/anie.201703107
• 发表时间: 2017-07-17
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Baslé A;Platsaki S;Dennison C
• 通讯作者: Dennison C

A four-helix bundle stores copper for methane oxidation.

• DOI: 10.1038/nature14854
• 发表时间: 2015-09-03
• 期刊: Nature
• 影响因子: 64.8
• 作者: Vita N;Platsaki S;Baslé A;Allen SJ;Paterson NG;Crombie AT;Murrell JC;Waldron KJ;Dennison C
• 通讯作者: Dennison C

Intracellular copper storage and delivery in a bacterium

细菌细胞内铜的储存和输送

• DOI: 10.1101/2022.01.05.475036
• 发表时间: 2022
• 作者: Lee J

Important Structural Features of Thiolate-Rich Four-Helix Bundles for Cu(I) Uptake and Removal.

• DOI: 10.1021/acs.inorgchem.2c04490
• 发表时间: 2023-05-01
• 期刊: INORGANIC CHEMISTRY
• 影响因子: 4.6
• 作者: Lee, Jaeick;Dalton, Rosemary A.;Basle, Arnaud;Vita, Nicolas;Dennison, Christopher;Subtilis, Bacillus
• 通讯作者: Subtilis, Bacillus

Copper delivery to an endospore coat protein of Bacillus subtilis.

• DOI: 10.3389/fcell.2022.916114
• 发表时间: 2022
• 期刊: Frontiers in cell and developmental biology
• 影响因子: 5.5