Enzymology of Bacteroides short and branched chain fatty acid metabolism - 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.
