Browsing by Author "Kellow-Webb, Sarah Maggie"

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    The expression, purification and characterization of escherichia coli NudL, a putative coenzyme A hydrolase
    (Stellenbosch : Stellenbosch University, 2021-04) Kellow-Webb, Sarah Maggie; Strauss, Erick; De Villiers, Marianne; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Coenzyme A (CoA) is an essential cofactor that is synthesized by a conserved five-step pathway from pantothenic acid (vitamin B5). The regulation of CoA levels is vital in supporting normal cellular function. In higher order organisms, CoA degradation and the recycling of CoA have proven efficient for the dynamic control of CoA levels, but in prokaryotes the contribution of CoA degradation to the regulation of CoA levels remains largely unstudied. Escherichia coli is known to make significant quantities of 4'-phosphopantetheine (PPanSH), a CoA biosynthesis intermediate, the excess of which is irreversibly exported to the extracellular environment. Early studies revealed that the source of the high PPanSH levels is both from the biosynthesis and the degradation of CoA. In E. coli, CoA degradation can be mediated via an indirect mechanism through acyl carrier protein (ACP) prosthetic group turnover, where AcpH releases the PPanSH moiety from the holo-ACP, or via a direct mechanism by an unidentified enzyme. One possible mechanism for direct degradation of CoA is by the action of a CoA hydrolase, a Nudix hydrolase subfamily member. Nudix hydrolases are a superfamily of housekeeping enzymes that regulate metabolic intermediates or remove toxic nucleotide metabolites. Nudix hydrolases specific for CoA are able to degrade CoA at the pyrophosphate moiety into 3',5'-ADP and PPanSH. E. coli has 13 predicted Nudix enzymes of which NudL is the only one yet to be characterized. NudL is encoded by the yeaB gene and contains both the Nudix box and NuCoA motif that points to its selectivity for CoA and strongly suggests that it is a CoA hydrolase. The first part of this study focused on optimizing the production of pure, soluble NudL protein for testing its putative CoA hydrolase activity. The protein was found to be unstable and significant challenges were experienced with soluble recombinant protein expression. NudL could therefore only be partially purified for the purpose of activity testing. The second part of this study focussed on characterizing NudL’s activity. Both the partially purified enzyme and E. coli lysate containing overexpressed NudL was found to be able to degrade CoA and form PPanSH. These results provide strong evidence that CoA hydrolysis into PPansH is mediated by NudL and confirms the presence of CoA hydrolase activity in E. coli lysate. CoA hydrolase activity was dependent on the presence of a metal cofactor and NudL appeared to prefer Mn²⁺ over Mg²⁺. Following this, the physiological relevance of NudL was investigated. The results of metabolomic studies revealed that NudL contributes significantly to the intracellular PPanSH levels as a knockout mutant lacking the enzyme had considerably lower intracellular PPanSH levels. An ∆acpH mutant also showed a similar trend, confirming that CoA degradation contributes to the intracellular PPanSH pool. Conditions which promote CoA degradation were also investigated. Transcription of the NudL- encoding gene yeaB did not appear to be influenced by oxidative stress; however, a significant growth defect was observed for the mutant strains when acetate was the carbon source, suggesting that CoA degradation by NudL is important during growth on acetate. The results of this study support a direct mechanism of CoA degradation in E. coli, specifically by the NudL enzyme, being the last E. coli Nudix hydrolase to be experimentally characterized. Further investigations are needed to understand how this enzyme is regulated. This may provide insight into how CoA levels are regulated in E. coli in general.