Friday, November 22, 2024
11/22/2024
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.
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