Doctoral Degrees (Biochemistry)

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Browsing Doctoral Degrees (Biochemistry) by browse.metadata.advisor "De Villiers, Marianne"

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    Membrane permeability and transport studies of coenzyme A, its precursors and antimetabolites.
    (Stellenbosch : Stellenbosch University, 2020-04) Mogwera, Koketso Seipelo Precious; Strauss, Erick; De Villiers, Marianne; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Coenzyme A (CoA) is a cofactor essential to several metabolic processes, including central energy and fatty acid metabolism. In addition, there is increasing evidence that CoA also plays an important role in the rate of neurodegeneration and aging. CoA is usually biosynthesized from vitamin B5, but recently it was shown that dietary CoA can also replenish intracellular CoA levels through degradation of exogenous CoA by ecto-nucleoside pyrophosphatases into 3',5’-ADP and 4'-phosphopantetheine (P-PantSH), which is subsequently taken up by cells. Importantly, permeability studies suggested that this uptake occurs by passive diffusion, an observation that was further confirmed by nutrient complementation studies in Drosophila S2 cells containing a mutation that results in CoA depletion. Moreover, investigation of CoA homeostasis in Escherichia coli suggested that this bacterium irreversibly exports P-PantSH to regulate intracellular CoA levels. This suggests an interplay between eukaryotes and their microbiome regarding the maintenance of levels of this cofactor through the exchange of P- PantSH as a likely nexus metabolite in CoA biosynthesis. In this study we used a systematic biophysical approach to investigate the determinants of P- PantSH permeability, and whether this relates to the structure of the molecule, or a certain specific membrane composition. This was achieved by devising a new assay specific for determining the membrane permeability of thiolated metabolites (such as P-PantSH), and subsequently applying this assay in studying the permeability of P-PantSH and its structural analogues in model membranes. Our results indicate that the permeability of P-PantSH is dependent on the fluidity of the membranes in question, and that manipulation of membrane composition to affect membrane fluidity offers a new mechanism to regulate the maintenance of CoA levels from exogenous sources. Using the same assay, we were also able to study a mitochondrial solute carrier protein that is apparently involved in the regulation of CoA levels in the mitochondria. ADP-dependent efflux of P-PantSH was observed for the transporter, and this constitutes preliminary evidence of the implied role of P-PantSH as a nexus metabolite of CoA metabolism. These findings have important implications for the development of drugs that target CoA biosynthesis and utilization, whether in the context of human diseases for the treatment or delay of neurodegenerative diseases, or alternatively as antimicrobials for the treatment of infectious diseases.
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    Synthesis, profiling and mode of action studies of PanSulfAms as inhibitors of coenzyme a biosynthesis and utilization
    (Stellenbosch : Stellenbosch University, 2019-12) Jana, Collins Edward; Strauss, Erick; De Villiers, Marianne; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Though tremendous progress has been made in the development of antibacterial drugs, the evolution of antimicrobial resistance is a serious problem that is affecting the successful prevention and treatment of various infections caused by microbial pathogens. Taking this into consideration, the need for new antimicrobials is of paramount importance. Since the B-vitamin pantothenate promotes the growth of microbes, analogues of this compound that may act as antimetabolites have been synthesized and tested for their inhibitory properties against bacterial growth. N-substituted pantoyltauramides (PanSulfAms) are a class of pantothenate analogues previously synthesized by various research groups in the 1940s. These analogues demonstrated promising inhibition of the proliferation of avian malaria parasites as well as showing good antibacterial activities against Streptococcus pyogenes. In this study, the chemical and structural diversity of these analogues was expanded by preparing PanSulfAms in which various amide moieties were introduced to the sulfonic acid group of taurine. These compounds were subsequently used to produce fourteen PanSulfAms that were investigated for their potential as antibacterial agents. The growth inhibitory activities of the synthesized PanSulfAms were first investigated against Escherichia coli and Staphylococcus aureus both as models of Gram-negative and Gram-positive bacteria respectively, and because these compounds have never been investigated on these microbes. We also investigated the link between the organisms’ pantothenate kinase (PanK) types and the observed growth inhibition to ascertain whether this relates to the compounds’ mode of action. In this study, we have shown for the first time that PanSulfAms have the potential to inhibit S. aureus growth, but not that of E. coli. In addition, we have also performed the first detailed comparative kinetic analysis of PanSulfAms that cause S. aureus growth inhibition, demonstrating that PanK type directs the mode of action of these compounds. Finally, the PanSulfAms were studied for their ability to inhibit CoA biosynthesis. We show that their impact is compound-specific and relates to their interaction with the various CoA biosynthetic enzymes. We demonstrate for the first time that certain phosphorylated PanSulfAms act as inhibitors of the PPCS activity of the bifunctional CoaBC protein, and that, in combination with a specific interaction with the S. aureus PanK, has strong negative impact on the rate of CoA biosynthesis that likely contributes to their overall mode of action.