Browsing by Author "Goosen, Rene"

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    A comparative analysis of CoA biosynthesis in selected organisms: a metabolite study
    (Stellenbosch : Stellenbosch University, 2016-03) Goosen, Rene; Strauss, Erick; Snoep, Jacky L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH SUMMARY: This study investigated the biochemical regulation of CoA production because it is an essential pathway that presents an important target for antimicrobial drug discovery studies. Currently, the specific life-sustaining functions of CoA are not clearly defined and a better understanding of the regulation of the CoA biosynthesis pathway would aid in the understanding of the relevance of maintaining specific CoA levels for survival. Regulation of CoA production was investigated on two levels. First, it was determined if production is up-regulated under conditions predicted to be associated with increased demand in S. aureus. Second, regulation of production of CoA by the salvage pathway in E. coli was investigated. In S. aureus it was found that CoA production is up-regulated under conditions of oxidative stress by an as yet unidentified mechanism. This led to an investigation of the regulation in the CoA biosynthesis pathway to understand how production is controlled. At present, the regulation of CoA production is thought to occur at a single, rate-limiting step identified as the first enzyme in the pathway, pantothenate kinase (PanK). Failure of inhibition of PanK to result in growth inhibition suggested that a re-evaluation of this premise is required. To this end, a systems analysis approach was taken in this study to elucidate the control of CoA production by the salvage pathway. Previously, a lack of analytical tools to measure the intermediates of CoA biosynthesis hampered investigations into regulation of the pathway and a holistic study has not been performed to elucidate the control profile. Consequently a method was also developed for the quantification of all the intermediates of the CoA salvage pathway based on derivatization with a fluorescent thiol probe and HPLC analysis. This method allowed for time course analysis of the reconstituted pathway to be performed to provide a holistic interpretation of CoA production. A kinetic model of the pathway was constructed from rate equations parameterized with a combination of experimentally determined values and values reported in the literature. Time course profiles were used to validate the model for subsequent control analyses. Both time course profiles and predictions made by the model indicated that PanK is unlikely to control the rate of CoA production under most conditions, and that it is in fact dephospho-CoA kinase (DPCK), the last enzyme in the pathway, that controls the rate under physiological conditions. This implies that DPCK is the best target for inhibition of the CoA biosynthetic pathway because it is far more likely to be in control of the rate of CoA production under physiological conditions. This finding is significant to antimicrobial drug development efforts because it suggests that the target focus should be shifted from PanK to DPCK. Therefore the findings of this study represent a major shift in our current understanding of the regulation of the rate of CoA production. It also highlights the importance of conducting a detailed systems analysis when studying metabolic pathways from both regulatory and drug development perspectives.