The goals of this work are to advance the understanding of the mechanisms of thioesterase loading-substrate and release-nucleophile selectivity and to develop an analytical methodology and modelling theory as tools for quantitative measures of thioesterase activity. The first aspect of this work is a bioinformatic examination of examples of thioesterases that catalyze different forms of release at different rates of reaction dependent on the type of thioester substrate presented to it. In addition, the type I thioesterase domain from the Type I modular polyketide synthase pathway responsible for the production of bacillaene was cloned, expressed, and purified for kinetic characterization as a model trans-AT type thioesterase. N-Acetylcysteamine and acyl carrier protein-bound acyl substrates were synthesized as substrates for hydrolysis catalysed by the bacillaene thioesterase. The rate of reaction was indirectly observed with a widely-adopted visual spectroscopy assay facilitated by the thiol indicator 5,5'-dithio-bis-[2-nitrobenzoic acid]. Variants of the bacillaene thioesterase coded from two different bacterial isolates with 57 % amino acid identity (73 % similarity) both exhibited rapid declining activity under the conditions used for Ellman’s, indicative of inhibition. This matches rare literature examples of other thioesterase domains under the same spectroscopic assay conditions. We demonstrate that initial rate of reaction data is insufficient to describe substrate selectivity in these systems and develop a time-dependent model capable of accounting for irreversible substrate inhibition during catalysis. This model was sufficient to reproduce the thioesterase activity observed between the bacillaene thioesterase and methyl valeric-SNAC but is insufficient to model the responses observed during the hydrolysis of trans-2-methyl-2-pentenoyl-SNAC or cis-3-methyl-2-pentenoyl-SNAC. Finally, we detailed synthetic and assay conditions for the production, cleavage, and detection of the ACP-tethered substrate equivalents. This contributes towards the development of complex thioesterase assays that better estimate type I bacterial thioesterase activity during polyketide synthesis.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/37711 |
| Date | 17 May 2018 |
| Creators | Horsman, Mark |
| Contributors | Boddy, Christopher |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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