Enzymology of Bacteroides short and branched chain fatty acid metabolism

项目摘要 等位基因对使用元基因组技术解释新陈代谢提出了挑战 而微生物体内的调控网络作为重要的酶特性可能是 受核苷酸差异的影响，导致微小或细微的氨基酸替换。 初步工作表明，预测的丁酸激酶编码在基因组序列中 类杆菌thetaiotaomicron和Phocaeicola uggatus显示支链脂肪酸 (BCFA)酶活性和戊酸酸酶活性。这些未被认可的酶 与胞内辅酶A(CoA)利用率相关的活性和代谢潜能 对结肠微生物区系和人类健康都有重要影响。这项建议 重点描述了这些酶和相关的酶活性的生物化学和 遗传方法。具体目标1解决特定丁酸激酶变体的性质和 某些氨基酸取代对活性和功能的影响。与.一起 进一步鉴定了戊酸和BCFA激酶，这是一种从B. Mediterranesis与特征BCFA和戊酸酯具有很高的序列同源性 酶，但表现出典型的丁酸酶活性中心残基的序列保守性 将被描述为。这一具体目标的结果不仅将提供关于以下方面的见解 丁酸激酶的结构-功能关系，但也有等位基因特征 转化为其他结肠微生物区系。特异靶2测定细菌生化特性。 Taotaomicron和P.uggatus磷酸转丁酰酶，这是 它们相关的丁酸激酶变异体的细胞功能。这项工作提供了独立的 生物化学和预测的生理作用的确认和潜在的改进 丁酸激酶在这些细菌物种中特有的丁酸激酶变种。最后，具体目标3 重点研究了B.thetaiotaomicron和P.uggatus的发生和表型特征 缺乏这些酶的突变菌株。与支链氨基酸相关的生长表型 对B.thetaiotaomicron和P.thetaiotaomicron的酸发酵和戊酸酯利用进行评估。 Vallgatus，分别为。综上所述，这项提议将澄清关于脂肪酸的 人体关键细菌的代谢及其对辅酶A代谢通量的贡献 结肠微生物区系，同时也为本科生提供了出色的研究体验。

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.