Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by Author "Bouwer, Wilmarie"

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    Investigation and characterisation of dephospho-coenzyme A kinase: a potential drug target
    (Stellenbosch : Stellenbosch University, 2018-12) Bouwer, Wilmarie; De Villiers, Marianne; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Due to the rise in antimicrobial resistance, the coenzyme A (CoA) biosynthesis pathway has been identified as a metabolic process of interest as a potential new antimicrobial drug target that could aid in relieving the threat to global healthcare. The CoA biosynthesis pathway consists of five enzymes, each a potential novel drug target. The focus of this study was on dephospho-Coenzyme A kinase (DPCK), the last enzyme in the pathway, as it is thought to hold the most control over the flux through the CoA pathway and is largely unexplored. Particular focus was given to DPCK from Staphylococcus aureus and Plasmodium falciparum. Both these organisms have shown resistance to current drug treatments available in a clinical setting, thus causing resistant strains to become more prevalent and pose a greater threat to global healthcare. The aims of this study were to expand current knowledge on these two organisms’ DPCK enzymes, as neither of these enzymes have been characterised previously. The first aim of this study focussed on characterising S. aureus DPCK, whilst comparing the results to previously characterised DPCK enzymes from Corynebacterium ammoniagenes, Corynebacterium glutamicum, and Escherichia coli. From this we were able to determine the quaternary structure of DPCKs from S. aureus, C. ammoniagenes and C. glutamicum to be trimers in solution, whereas DPCK from E. coli was confirmed to be a monomer as previously reported in literature. Following this, we have determined that the specific activity of S. aureus DPCK is very low, in contrary to what is found for C. ammoniagenes DPCK. Techniques to aid in measuring the low activity of the enzyme were further explored. This led to the use of high performance liquid chromatography (HPLC) assays that incorporated all the CoA biosynthesis pathway enzymes to determine specific activity. We concluded that the presence of all of the other pathway enzymes, at physiological ratios, aided the specific activity of S. aureus DPCK. In the second aim of this study we wanted to determine the activity of P. falciparum DPCK, as this enzyme is the only CoA biosynthesis pathway enzyme that does not localise to the cytosol of the parasite. Further, little information is available on the activity of the parasite’s CoA biosynthesis enzymes as they are difficult to express recombinantly using bacterial expression vectors. We successfully determined conditions for that yielded soluble expression and purification conditions of P. falciparum DPCK, however the enzyme is not very stable in solution. This unfortunately prevented us from determining the activity of the enzyme. The results from this study have shed more light on methods to determine the activity of DPCK enzymes and have established soluble expression conditions for P. falciparum DPCK from a bacterial expression vector. Therefore, it can form the basis in future investigations on DPCK as a potential drug target.