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Thesis (Phd (Chemistry and Polymer Science))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy (Chemistry) at Stellenbosch University. / ENGLISH ABSTRACT: The human pathogen Staphylococcus aureus is a major cause of hospital-, and more recently, community-acquired infections. The rate at which this organism is acquiring resistance to antibiotics is increasing while the development of new antibiotics is slowing down. There is therefore a desperate need for new antistaphylococcal agents, and in particular ones with novel mechanisms of action that can be used to circumvent established resistance pathways. Unlike humans, S. aureus employs the essential cofactor coenzyme A (CoA) as its major low molecular weight thiol. Together, CoA and the enzyme CoA disulfide reductase (CoADR) are responsible for maintaining the internal redox homeostasis in this organism, and disruption of this balance (or reduction of CoA levels) may therefore be potential mechanisms by which new antistaphylococcal agents may act. In this study we set out to achieve this by direct inhibition of CoADR, and by inhibition of one or more of the CoA biosynthetic enzymes. For the inhibition of CoADR CoA analogues containing Michael acceptors were designed and prepared by employing a chemo-enzymatic approach. This strategy involved the chemical synthesis of pantothenamides containing α,β-unsaturated ester, ketone and sulfone moieties as Michael acceptors, followed by their biotransformation into the corresponding CoA analogues by three CoA biosynthetic enzymes. The compounds prepared in this manner all inhibited CoADR potently. A full kinetic evaluation of the inhibition by these compounds suggested that these compounds act by alkylation of the single active site cysteine of CoADR in an irreversible fashion. In this study we also set out to determine the mechanism of action of the antistaphylococcal compound CJ-15,801, which is structurally similar to pantothenic acid, the biosynthetic precursor of CoA. Due to this similarity we proposed that the antibiotic properties of CJ-15,801 are based on the inhibition of enzymes involved in CoA biosynthesis and metabolism. Our investigations confirmed that the second enzyme of the CoA pathway, phosphopantothenoylcysteine synthetase (PPCS), acts as the main target of CJ-15,801. These studies were followed by an investigation into alternative synthetic methodologies for the preparation of CJ-15,801 and its analogues. As a result an established Pd-catalyzed coupling reaction was modified and applied in the third known total synthesis of CJ-15,801, as well as of several of its analogues. This protocol has several advantages over its predecessors, most importantly its suitability for preparing these compounds on large (up to one gram) scale. / AFRIKAANSE OPSOMMING: Die menslike patogeen Staphylococcus aureus is 'n wesenlike oorsaak van hospitaal- en meer onlangs gemeenskap-verworwe infeksies. Terwyl die tempo waarteen hierdie organisme weerstandbiedig teenoor antibiotika raak toeneem, neem die ontwikkeling van nuwe antibiotiese middels af. Dit is dus van kardinale belang dat nuwe antistafilokokale middels ontwikkel word, en meer spesifiek antibiotika met 'n nuwe meganisme van aksie wat gebruik kan word om huidige weerstandbiedende padweë te ontwyk. In teenstelling met mense, gebruik S. aureus die essensiele kofaktor koënsiem A (KoA) as sy vernaamste lae molekulere gewig tiol. Die ensiem KoA disulfied reduktase (KoADR) en KoA is saam verantwoordelik om die interne redoks homeostase in hierdie organisme te handhaaf, en ontwrigting van die balans (of vermindering van KoA vlakke) kan dus potensieel 'n meganisme van aksie wees waardeur nuwe antistafilokokale middels kan optree. In hierdie studie het ons gepoog om dit te bewerkstellig deur KoADR direk te inhibeer, asook deur inhibisie van een of meer van die KoA biosintetiese ensieme. Vir die inhibisie van KoADR is KoA-analoë wat Michael-akseptor groepe bevat ontwerp en berei deur van 'n chemo-ensiematiese benadering gebruik te maak. Met hierdie strategie is pantoteenamiede gesintetiseer wat α,β-onversadigde ester, ketoon en vinielsulfoon funksionaliteite as Michael-akseptore bevat, gevolg deur biotransformasie na die ooreenstemmende KoA-analoë met behulp van drie CoA biosintetiese ensieme. Die verbindings gesintetiseer met hierdie metode het almal KoADR potent geinhibeer. 'n Omvattende kinetiese evaluasie het voorgestel dat al hierdie verbindings funksioneer deur alkielering van die enkele aktiewe setel sisteïen van KoADR op 'n onomkeerbare wyse. In die studie het ons ook gepoog om die meganisme van aksie van die antistafilokokale verbinding CJ-15,801 te bepaal. Hierdie verbinding is struktureel soortgelyk aan pantoteensuur, die biosintetiese voorganer van KoA. As gevolg van hierdie ooreenkomste het ons voorgestel dat die antibiotiese aktiwiteit van CJ-15,801 die gevolg is van die inhibisie van een of meer van die ensieme wat verantwoordelik is vir KoA biosintese en metabolisme. Ons ondersoeke het bevestig dat die tweede ensiem in die KoA biosintetiese padweg, naamlik fosfopantotenoïelsisteïensintetase, die hoofteiken van CJ-15,801 is. Hierdie studies is gevolg deur die ondersoek van alternatiewe metodologieë vir die sintese van CJ-15,801 en analoë daarvan. 'n Gevestigde Pd-gekataliseerde koppelings reaksie was gevolglik gemodifiseer en toegepas om slegs die derde totale sintese van CJ-15,801 te bewerkstelling, asook die sintese van verskeie analoë daarvan. Hierdie protokol hou verskeie voordele in vergelyking met sy voorgangers, waarvan die mees belangrikste die bereiding van hierdie verbindings op groot (tot een gram) skaal is.

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Call number: LD2668 .T4 1959 R36

Gentamicin C complex is a mixture of five structurally similar aminoglycoside antibiotics, gentamicins C1, C1a, C2, C2a, and C2b, produced by the actinomycete bacterium Micromonospora echinospora. It is established in clinical use and despite significant toxicity remains valuable to treat severe Gram-negative bacterial infections. There is a pressing need to develop novel versions of such antibiotics to combat the rise of resistance among pathogens. Engineering of the pathway requires a detailed knowledge of the genes, enzymes, and intermediates involved. The final steps of gentamicin biosynthesis begin at gentamicin X2, the last common intermediate of the C complex. 6'-C-Methylation generates two branches, with analogous reactions happening in both. Candidate genes and enzymes for the steps from the first 6'-C-methylated intermediate, G418, to an aminated metabolite JI-20B have already been described, but none for the subsequent loss of two hydroxyl groups from Ring II, or the N-methylation that then occurs. A novel separation method using dynamic countercurrent chromatography was successfully applied to the difficult purification of gentamicin metabolites. The results described here allowed a detailed mechanism to be proposed for almost the entire pathway from G418 to the C complex, and by analogy for the unbranched pathway, too. The last step of the pathway is 6'-N-methylation of gentamicins C1a and C2. Genome mining and cell-free assays were used by the group of Professor Yuhui Sun (Wuhan University) to identify genL, a methyltransferase gene encoded elsewhere on the M. echinospora genome and capable of this catalysis. Here, in vitro reactions with recombinant GenL confirmed its function, and its kinetic parameters were measured with its substrates. The full mechanistic pathway for the late stages of gentamicin C complex biosynthesis has therefore now been elucidated.

L'amidon est un agrégat insoluble de granules semi-cristallines, contrairement aux particules hydrosolubles de glycogènes. Chez les eucaryotes photosynthétiques, le passage à l'accumulation de l'amidon est survenu après l'endosymbiose du plaste, à partir d'un réseau métabolique synthétisant du glycogène cytosolique chez l'hôte. Cela a impliqué le recrutement d'une enzyme de débranchement d'origine Chlamydienne, un parasite intracellulaire obligatoire. Cette enzyme de débranchement est considérée comme responsable de l'enlèvement des branches mal placées au niveau d’un précurseur qui, autrement, donne un polysaccharide soluble dans l'eau. Désormais, nous présentons l'implication de l'enzyme de débranchement de l'amidon dans l'agrégation de granules semi-cristallines de cyanobactéries unicellulaires qui accumulent à la fois du glycogène et des grains d'amidon semi-cristallins. Nous montrons, à travers cette thèse, qu'une enzyme de nature analogue à l'enzyme de débranchement de la plante, mais d'une origine bactérienne différente, a été recrutée pour la même fonction dans ces organismes. Nous remarquons que les deux enzymes de cyanobactéries et de plantes ont évolué séparément pour donner de l'amidon à partir d'une enzyme impliquée dans le catabolisme du glycogène. La nature polyphylétique de la transition à partir du glycogène à l'amidon, dans les Archaeplastida et les cyanobactéries, ainsi que l'évolution convergente d'un mécanisme biochimique commun, suggèrent que le débranchement des polysaccharides au cours de leur synthèse définit une exigence universelle de l'évolution du métabolisme de l'amidon dans les cellules vivantes. / Starch, unlike hydrosoluble glycogen particles aggregates into insoluble, semi-crystalline granules. In photosynthetic eukaryotes, the transition to starch accumulation occurred after plastid endosymbiosis from a pre-existing cytosolic host glycogen metabolism network. This involved the recruitment of a debranching enzyme of chlamydial pathogen origin. The latter is thought to be responsible for removing misplaced branches that would otherwise yield a water-soluble polysaccharide. We now report the implication of starch debranching enzyme in the aggregation of semi-crystalline granules of single-cell cyanobacteria that accumulate both glycogen and starch-like polymers. We show that an enzyme of analogous nature to the plant debranching enzyme but of a different bacterial origin was recruited for the same purpose in these organisms. Remarkably both the plant and cyanobacterial enzymes have evolved a novel yet identical substrate specificity from a preexisting enzyme which originally displayed the much narrower substrate preferences required for glycogen catabolism. The polyphyletic nature of the transition from glycogen to starch in Archaeplastida and cyanobacteria together with the convergent evolution of a common biochemical mechanism suggest that polysaccharide debranching defines a universal requirement for the evolution of starch metabolism in living cells.