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Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Pantothenate kinase (PanK) enzymes catalyze the first reaction in the five step biosynthesis of the essential cofactor coenzyme A. Enzymes representing each of the three identified PanK types have been studied and characterized and these PanK types exhibits a unique diversity between different organisms, therefore highlighting them as potential drug targets. In this study the type III PanK of specifically pathogenic bacteria were characterized with the goal of developing type-specific inhibitors. Several questions about the activity of the Mycobacterium tuberculosis enzyme was answered, which addresses the contradicting results achieved in related PanK studies performed to date. Furthermore the first inhibitors, that are competitive to the pantothenate binding site, were designed, synthesized and tested against the Pseudomonas aeruginosa enzyme. This resulted in the discovery of the most potent inhibitors of the type III PanKs to date. / AFRIKAANSE OPSOMMING: Pantoteensuurkinase-ensiem (PanK) kataliseer die eerste stap in die vyf stap biosintese van die lewens belangrike en essensiële kofaktor, koënsiem A (KoA). Die meerderheid patogeniese bakterieë, waaronder die organisme wat tuberkulose veroorsaak, besit ‘n unieke vorm van die PanK-ensiem. Gevolglik word hierdie ensieme as belangrike teikens vir die ontwikkeling van antibakteriële middels beskou. In hierdie studie is die aktiwiteit van die Mycobacterium tuberculosis ensiem gekarakteriseer wat verskeie teenstrydige bevindings oor hierdie ensiem beantwoord het. Verder is nuwe inhibitore vir die Pseudomonas aeruginosa ensiem ontwerp, gesintetiseer en getoets. Die beste inhibitore van hierdie tipe ensiem tot op hede is sodoende geïdentifiseer.

This thesis is an investigation of novel mono-ADP-ribosylating toxins. In the current data-rich era, making the leap from sequence data to knowledge is a task that requires an elegant bioinformatics toolset to pinpoint questions. A strategy to expand important protein-family knowledge is required, particularly in cases in which primary sequence identity is low but structural conservation is high. For example, the mono-ADP-ribosylating toxins fit these criteria and several approaches have been used to accelerate the discovery of new family members. A newly developed tactic for detecting remote members of this family -- in which fold recognition dominates -- reduces reliance on sequence similarity and advances us toward a true structure-based protein-family expansion methodology. Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins identified and characterized using in silico and cell-based techniques. Medically relevant toxins from Mycobacterium avium and Enterococcus faecalis were also uncovered. Agriculturally relevant toxins were found in Photorhabdus luminescens and Vibrio splendidus. Computer software was used to build models and analyze each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. Yeast-based activity tests have since confirmed activity. Vibrio cholerae produces cholix – a potent protein toxin of particular interest that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 2.1Å apo X-ray structure as well as a 1.8Å X-ray structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD+). Hallmark catalytic residues were substituted and analyzed both for NAD+ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These new toxins serve as a reference for ongoing inhibitor development for this important class of virulence factors. In addition to using toxins as targets for antivirulence compounds, they can be used to make vaccines and new cancer therapies. / Natural Sciences and Engineering Research Council (CGS-D), Canadian Institutes of Health Research, Cystic Fibrosis Canada, Human Frontier Science Program, Ontario government (OGSST), University of Guelph (Graduate Research Scholarship)