Browsing by Author "Muneri, Ndivhuwo Olga"
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- ItemBiocatalytic preparation and characterization of alternative substrate of MshB, a mycothiol pathway enzyme(Stellenbosch : Stellenbosch University, 2012-12) Muneri, Ndivhuwo Olga; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, utilizes mycothiol (MSH) as the major low molecular weight thiol to protect itself against oxidative stress and thereby to ensure its growth and survival. MSH is a pseudo-disaccharide molecule that contains an α(1→1) glycosidic bond, and is biosynthesised in five enzymatic steps involving the enzymes MshA, MshA2, MshB, MshC and MshD. Owing to the essentiality of MSH to M. tuberculosis, various studies have focused on the MSH biosynthetic and other MSH-dependent enzymes viewed as potential drug targets for the development of antituberculosis agents. In the course of this study two practical challenges affecting the development of inhibitors of one the MSH biosynthesis pathway enzyme, MshB, were addressed. These challenges entail the lack of a high-throughput continuous assay to determine MshB activity, and the poor availability of the natural and alternative MshB substrates. In this study an alternate MshB substrate was characterized and shown to undergo a rearrangement reaction upon deactylation, which allowed the development of a new continuous assay for MshB activity that uses DNTB (Ellman’s reagent). In addition, three new α-thioglycoligases were created from the α-Nacetylglucosaminidase of Clostridium perfringens. These enzymes showed potential as biocatalysts that can be used for the enzymatic synthesis of thioglycoside-based alternative substrates of MshB.
- ItemEngineering and application of glycosidase derived biocatalysts in the study of mycothiol pathway enzymes(Stellenbosch : Stellenbosch University, 2017-03) Muneri, Ndivhuwo Olga; Strauss, Erick; Moracci, Marco; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Glycosides are complex carbohydrates that are involved in essential cellular and molecular biological processes within all living organisms. In addition, certain glycosides have anti-cancer, antioxidant, anti-inflammatory, antibacterial, antiviral, antiparasitic and antifungal activities. The functions of glycosides in biological processes and in biomedicine have led to a high demand for these organic molecules. However, the study of glycosides is hindered by the practical challenges in generating these compounds synthetically. This is even more true in the case of glycosides bound by means of α-glycosidic bonds, as most of the available synthetic methods promote the formation of β-glycosides. Methods that form α-glycosides are either low yielding or also promotes β-glycoside formation, resulting in the formation of mixtures that are challenging to separate. During the course of this study novel α- thioglycoligases derived from a CAZy family GH89 α-N-acetylglucosaminidase were prepared and characterized for their ability to form α-glycosides through biotransformation. The utility of the new biocatalysts was showcased by preparing several α-GlcNAc-based glycosides of biomedical and chemical interest. The products were purified or modified further through chemical transformations (such as “Click”-cycloaddition) and subsequently tested as potential small molecules chaperones for the treatment of Sanfilippo syndrome and/or as alternative substrates for the MshB, a mycothiol biosynthetic enzyme that has been identified as a potential target for development of new antituberculosis agents. Through the discovery of these molecules and the development of a new continuous deacetylase enzyme assay the findings of this study will significantly strengthen our ability to mobilise α-GlcNAc-based glycosides as part of ongoing research efforts in these fields.