Some of the biggest contributors to cellular respiration (and cellular metabolism in general) are acyl-CoA derivatives, a subclass of biological thioesters. Known to function in a variety of pathways, the regulation of their formation and breakdown are critical, carried about by acyl-CoA synthetases and thioesterases, respectively. The work reported within this dissertation will focus on functional divergence within two enzyme superfamilies -- the hot dog-fold and acyl-adenylate-forming superfamilies — and can be broken down into two main parts. Part one will look at tracking the functional divergence within the hot dog-fold superfamily thioesterases. A highly evolved thioesterase, flK, has been found to function in the critical, and highly specific role of fluoroacetate detoxification within the fluorometabolite-producing bacteria, S. cattleya. Using an extensive bioinformatics analysis, flK orthologs were identified and tracked throughout all three domains of life, primarily in bacteria that make up the gut microbiome. Additionally, sequence and structural analyses revealed distinct flK scaffolds, a further indication of divergent functionality. Various flK orthologs were then isolated, cloned and subjected to substrate screening by measuring their individual steady-state kinetic parameters kcat, Km and kcat/Km. Combined with gene context analyses, divergent in vivo functionality was assigned to members of the flK subfamily, as they were proposed to be involved in supplying formate for the one-carbon pool. Part two will focus on the functional characterization of the acyl-CoA synthetases (ligases) in Pseudomonas aeruginosa, the dominant pathogen present in all patients with cystic fibrosis and the leading cause of morbidity and mortality within this afflicted population. Nine freestanding ligases were cloned, isolated and subjected to an extensive substrate screening for acyl-CoA synthetase activity using a novel high-throughput assay. Individual activities were verified by measuring the steady-state kinetic parameters. Combing these results with extensive gene context analyses, in vivo functions were proposed for the tested ligases, implicating them in a variety of nutrient scavenging pathways as well as in virulence factor production.
Enzymology, Thioesterase, Acyl-CoA Ligase, Pseudomonas aeruginosa, Functional Divergence, Protein Evolution
Level of Degree
Department of Chemistry and Chemical Biology
First Committee Member (Chair)
Second Committee Member
Third Committee Member
Zimney, Lucas. "Case Studies of the Hot Dog-Fold and Acyl-Adenylate-Forming Superfamilies: Characterizing the Importance of Functional Divergence in Cellular Metabolism." (2015). https://digitalrepository.unm.edu/chem_etds/12