Vitamin B12 trafficking and selectivity in gut bacteria

维生素 B12 在肠道细菌中的运输和选择性

• 批准号: 10660958
• 负责人: Romila Nina Mascarenhas
• 金额: $ 11.99万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-06 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10660958
• 关键词: Address; Affinity; Anabolism; Bacteria; Bacteroides thetaiotaomicron; Bacteroidetes; Binding; Biochemical; Biochemical Reaction; Biological Assay; Carbon; Carrier Proteins; Cells; Cobalamin; Communities; Complex; Corrinoids; Disease; Ecosystem; Environment; Enzymes; Equilibrium; Escherichia coli; Fluorescence; Gene Expression Regulation; Genes; Gram-Negative Bacteria; Health; Homologous Gene; Human; Human Genome; Individual; Individual Differences; Kinetics; Lipoproteins; Measures; Membrane; Membrane Transport Proteins; Metabolic; Metabolic Pathway; Metabolism; Metagenomics; Methylmalonyl-CoA Mutase; Molecular; Molecular Chaperones; Nitrogen; Operon; Oxidation-Reduction; Periplasmic Binding Proteins; Phase; Play; Predisposition; Process; Proteins; Reaction; Research; Resources; Ribonucleotide Reductase; Role; Shapes; Signal Transduction; Site; Specificity; Structure; Surface; System; Testing; Therapeutic; Thermodynamics; Vitamin B 12; Vitamins; beta barrel; biophysical techniques; cobamamide; cofactor; cost; dysbiosis; fitness; gut bacteria; gut microbiome; insight; interest; microbial; microbial community; microbial composition; microbiome; microbiota; model organism; paralogous gene; preference; protein protein interaction; protein transport; symbiont; trafficking; treatment response; uptake

Project Summary The human gut microbiome is inhabited by trillions of bacteria that encode over 150-fold more genes than the human genome itself. The inter-individual differences in microbial composition can be significant, and the factors contributing to this diversity are not well understood. Metagenomic studies suggest that the microbiome might play an important role in determining an individual’s predisposition to disease and responses to treatments. The paucity of understanding how cofactors and other factors influence microbial composition limit strategies to rationally alter it for therapeutic purposes. Much of our current understanding of the factors that shape the gut microbial flora composition derives form studies on how bacteria generate energy, maintain redox balance and acquire carbon and nitrogen. The enzymatic reactions that support these metabolic processes often rely on cofactors that are in short supply. Vitamin B12 is an example of one such cofactor that is essential for many bacteria that are unable to biosynthesize it and lack parallel B12-independent metabolic pathways to circumvent its absence. So, one approach to the targeted manipulation of the gut microbiome is via altering the levels of available corrinoids. In this proposal, I seek to elucidate the corrinoid selectivity of transport systems to provide needed insights into how gut bacteria compete with each other and their hosts for a critical resource in a complex ecosystem. My studies will focus on Bacteroidetes thetaiotaomicron, a common gut bacterium, which lacks the genes required for de novo synthesis of vitamin B12 but encodes multiple B12-dependent enzymes. 5’-Deoxyadenosylcobalamin is the active cofactor form that is utilized by some B12 dependent enzymes and is synthesized by BtuR in B. thetaiotaomicron. The chaperone and catalytic activities are uncharacterized and will be addressed in Aim 1. It also encodes three copies of the outer membrane B12-transporter BtuB with each system displaying a different preference for corrinoid derivatives. The bacterium also possesses additional transport machinery that is not observed in E. coli, a model organism in which studies on B12 transport in gram-negative bacteria have been focused. Using a combination of biochemical and biophysical approaches, I propose to elucidate the mechanism of B12 transport by the B12-uptake (Btu) system in Aim 2. The kinetic and thermodynamic studies in Aims 1 and 2 will define the selectivity of the Btu proteins for cobamides and provide insights into protein-protein interactions. Combined with the structures determined in Aims 1 and 2, my studies will furnish mechanistic insights into how a precious and rare cofactor is relayed from the environment across two layers of bacterial membranes to support the metabolic needs of a common gut bacterium.

项目摘要 人类肠道微生物组被数万亿个细菌感染，该细菌的编码超过150倍以超过150倍 人类基因组本身。微生物组成的个体间差异可能很重要，并且 导致这种多样性的因素尚不清楚。宏基因组研究表明 微生物组可能在确定个人对疾病的易感性和 对治疗的反应。了解辅助因子和其他因素如何影响微生物的匮乏 组成限制策略以理性的目的为治疗目的而改变。我们目前的大部分理解 塑造肠道微生物菌群组成的因素，得出了有关细菌如何产生的研究 能量，维持氧化还原平衡并获取碳和氮。支持的酶促反应 这些代谢过程通常依赖于供应短缺的辅助因子。维生素B12是 对于许多无法生物合成且缺乏平行的细菌至关重要的辅助因子 非依赖于B12的代谢途径，以规避其缺失。因此，一种方法 肠道微生物组的操纵是通过改变可用的类似石的水平。在这个建议中，我寻求 阐明运输系统的类con阳性选择性，以提供所需的见解，以了解肠道细菌 相互竞争，他们的主人在复杂的生态系统中获得关键资源。我的学习会 专注于一种常见的肠道细菌Thetaiotaomicron，缺乏DE基因 维生素B12的NOVO合成，但编码多种B12依赖性酶。 5'-脱氧核生酸盐 是由某些B12依赖性酶使用的活性辅因子形式，并由BTUR合成 B. Thetaiotaomicron。伴侣和催化活性未经表征，将在 AIM 1。它还编码每个系统的三个外膜B12转运蛋白BTUB的副本 表现出对类似衍生物的不同偏好。细菌还具有额外的 在大肠杆菌中未观察到的传输机制，这是一种模型生物体，其中B12转运中的研究 革兰氏阴性细菌已聚焦。结合生化和生物物理 方法，我建议阐明AIM中B12摄取（BTU）系统的B12运输机制 2。目标1和2中的动力学研究将定义BTU蛋白的选择性 共氨酰胺并提供对蛋白质 - 蛋白质相互作用的见解。结合确定的结构 在目标1和2中，我的研究将提供机械的见解 从两层细菌机制的环境中，以支持A的代谢需求 常见的肠道细菌。

项目成果

Architecture of the human G-protein-methylmalonyl-CoA mutase nanoassembly for B 12 delivery and repair.

用于 B 12 递送和修复的人类 G 蛋白-甲基丙二酸单酰辅酶 A 变位酶纳米组件的结构。

• DOI: 10.1101/2023.03.23.533963
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Mascarenhas,Romila;Ruetz,Markus;Gouda,Harsha;Heitman,Natalie;Yaw,Madeline;Banerjee,Ruma
• 通讯作者: Banerjee,Ruma