Enzymology of Bacteroides short and branched chain fatty acid metabolism

拟杆菌短链和支链脂肪酸代谢的酶学

• 批准号: 10651505
• 负责人: Robert Barber
• 金额: $ 37.25万
• 依托单位: UNIVERSITY OF WISCONSIN PARKSIDE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651505
• 关键词: Active Sites; Acyl Coenzyme A; Acyltransferase; Address; Alleles; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Bacterial Genome; Bacteroides; Bacteroides thetaiotaomicron; Binding; Biochemical; Branched-Chain Amino Acids; Butyrates; Catabolism; Cell physiology; Cloning; Coenzyme A; Conserved Sequence; Data Analyses; Diet; Disease; Enzymatic Biochemistry; Enzymes; Exhibits; Family; Fatty Acids; Fermentation; Generations; Genes; Genome; Goals; Growth; Health; Human; In Vitro; Isoleucine; Leucine; Link; Liquid substance; Measurement; Measures; Metabolic; Metabolic Pathway; Metabolism; Metagenomics; Minor; Nature; Nucleotides; Pathway interactions; Phenotype; Phosphate Acetyltransferase; Phosphotransferases; Physiological; Physiology; Population; Positioning Attribute; Property; Role; Scheme; Structure; Structure-Activity Relationship; Substrate Specificity; Techniques; Testing; Translating; Valerates; Valine; Variant; Volatile Fatty Acids; Work; branched chain fatty acid; colon microbiota; design; enzyme activity; experimental study; fatty acid metabolism; genetic approach; genetic variant; gut microbiota; improved; insight; microbial; microbiome; microbiome research; mutant; n-pentanoic acid; novel; preference; screening; undergraduate research experience

Project Summary Alleles present a challenge regarding use of metagenomic techniques for interpreting metabolic and regulatory networks within microbiomes as important enzymatic properties may be influenced by nucleotide differences leading to minor or nuanced amino acid substitutions. Preliminary work indicates predicted butyrate kinases encoded within the genome sequences of Bacteroides thetaiotaomicron and Phocaeicola vulgatus exhibit branched chain fatty acid (BCFA) kinase and valerate kinase activity, respectively. These unrecognized enzymatic activities and metabolic potentials associated with intracellular coenzyme A (CoA) availability have important implications for both colonic microbiota and human health. This proposal focuses on the characterization of these and related enzyme activities using biochemical and genetic approaches. Specific Aim 1 addresses the nature of specific butyrate kinase variants and the influence certain amino acid substitutions exert regarding activity and function. Along with further characterization of the valerate and BCFA kinase, a predicted butyrate kinase from B. mediterraneensis sharing high sequence identity with the characterized BCFA and valerate kinase, but exhibiting sequence conservation of the typical butyrate kinase active site residues will be characterized. Results from this specific aim will provide not only insight regarding structure-function relationships for butyrate kinases, but also allele characterizations that translate to other colonic microbiota. Specific Aim 2 measures the biochemical properties of B. thetaiotaomicron and P. vulgatus phosphotransbutyrylases, which are essential enzymes for the cellular function of their associated butyrate kinase variants. This work provides independent confirmation and potential refinement of the biochemical and predicted physiological roles for the characterized butyrate kinase variants in these bacterial species. Lastly, Specific Aim 3 focuses on the generation and phenotypic characterization of B. thetaiotaomicron and P. vulgatus mutant strains lacking these enzymes. Growth phenotypes associated with branched chain amino acid fermentation and valerate utilization will be assessed in B. thetaiotaomicron and P. vulgatus, respectively. Taken together, this proposal will deliver clarity concerning fatty acid metabolism and its contribution to CoA metabolic flux in key bacterial species among the human colonic microbiota, while also providing an outstanding research experience for undergraduates.

项目摘要 等位基因提出了使用宏基因组技术来解释代谢的挑战 微生物组中的调节网络可能是重要的酶特性 受核苷酸差异的影响，导致氨基酸取代次数或细微差别。 初步工作表明在基因组序列中编码的预测丁酸酯激酶 细菌thetaiotaomicron和phocaeicola vulgatus表现出分支链脂肪酸 （BCFA）激酶和瓣膜激酶活性。这些无法识别的酶促 与细胞内辅酶A（COA）可用性相关的活动和代谢电位 对结肠微生物群和人类健康都有重要意义。这个建议 专注于使用生化和相关酶活性的表征 遗传方法。特定目的1解决了特定丁酸酯激酶变体的性质和 影响某些氨基酸取代方面对活性和功能发挥作用。以及 瓣膜和BCFA激酶的进一步表征。 与特征BCFA和valerate共享高序列身份的地中海。 激酶，但表现出典型丁酸激酶活性位点残基的序列保守 将被描述。这个特定目标的结果不仅会提供有关有关的见解 丁酸酯激酶的结构功能关系，但也是等位基因特征 转化为其他结肠微生物群。特定目标2测量B的生化特性。 Thetaiotaomicron和P. vulgatus phosphotransbutylylases，它们是必不可少的酶 其相关丁酸酯激酶变体的细胞功能。这项工作提供了独立 确认和潜在的生化和预测生理作用的细化 这些细菌物种中的特征性丁酸酯激酶变体。最后，特定目标3 侧重于B. thetaiotaomicron和P. vulgatus的产生和表型表征 缺乏这些酶的突变菌株。与分支链氨基相关的生长表型 将在B. thetaiotaomicron和P.中评估酸发酵和脱气利用。 Vulgatus分别。综上所述，该提案将提供有关脂肪酸的清晰度 代谢及其对人类关键细菌物种中COA代谢通量的贡献 结肠微生物群，同时还为大学生​​提供出色的研究经验。