A comparative analysis of CoA biosynthesis in selected organisms: a metabolite study

Goosen, Rene (2016-03)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH SUMMARY: This study investigated the biochemical regulation of CoA production because it is an essential pathway that presents an important target for antimicrobial drug discovery studies. Currently, the specific life-sustaining functions of CoA are not clearly defined and a better understanding of the regulation of the CoA biosynthesis pathway would aid in the understanding of the relevance of maintaining specific CoA levels for survival. Regulation of CoA production was investigated on two levels. First, it was determined if production is up-regulated under conditions predicted to be associated with increased demand in S. aureus. Second, regulation of production of CoA by the salvage pathway in E. coli was investigated. In S. aureus it was found that CoA production is up-regulated under conditions of oxidative stress by an as yet unidentified mechanism. This led to an investigation of the regulation in the CoA biosynthesis pathway to understand how production is controlled. At present, the regulation of CoA production is thought to occur at a single, rate-limiting step identified as the first enzyme in the pathway, pantothenate kinase (PanK). Failure of inhibition of PanK to result in growth inhibition suggested that a re-evaluation of this premise is required. To this end, a systems analysis approach was taken in this study to elucidate the control of CoA production by the salvage pathway. Previously, a lack of analytical tools to measure the intermediates of CoA biosynthesis hampered investigations into regulation of the pathway and a holistic study has not been performed to elucidate the control profile. Consequently a method was also developed for the quantification of all the intermediates of the CoA salvage pathway based on derivatization with a fluorescent thiol probe and HPLC analysis. This method allowed for time course analysis of the reconstituted pathway to be performed to provide a holistic interpretation of CoA production. A kinetic model of the pathway was constructed from rate equations parameterized with a combination of experimentally determined values and values reported in the literature. Time course profiles were used to validate the model for subsequent control analyses. Both time course profiles and predictions made by the model indicated that PanK is unlikely to control the rate of CoA production under most conditions, and that it is in fact dephospho-CoA kinase (DPCK), the last enzyme in the pathway, that controls the rate under physiological conditions. This implies that DPCK is the best target for inhibition of the CoA biosynthetic pathway because it is far more likely to be in control of the rate of CoA production under physiological conditions. This finding is significant to antimicrobial drug development efforts because it suggests that the target focus should be shifted from PanK to DPCK. Therefore the findings of this study represent a major shift in our current understanding of the regulation of the rate of CoA production. It also highlights the importance of conducting a detailed systems analysis when studying metabolic pathways from both regulatory and drug development perspectives.

AFRIKAANSE OPSOMMING: Hierdie studie het die biochemiese regulering van KoA produksie ondersoek, want dit is 'n noodsaaklike padweg wat 'n belangrike teiken bied vir studies vir die ontdekking van antimikrobiese middels. Die spesifieke lewensonderhoudende funksies van KoA word tans nie duidelik gedefinieer nie en 'n beter begrip van die regulering van die KoA biosintese padweg sal help om die rol van die handhawing van spesifieke KoA vlakke vir oorlewing te verstaan. Regulering van KoA produksie word ondersoek op twee vlakke. Eerstens, word daar vasgestel of die produksie vermeerder word onder toestande waar ʼn verhoogde aanvraag na KoA voorspel word in S. aureus. Tweedens, word die regulering van die produksie van KoA deur die herwinningspadweg in E. Coli, ondersoek. In S. aureus is bevind dat KoA produksie wel vermeerder onder toestande van oksidatiewe stres deur 'n onbekende meganisme. Dit het gelei tot 'n ondersoek van die regulasie in die KoA biosintese pad om te verstaan hoe die produksie beheer word. Tans word dit verstaan dat die regulering van KoA produksie plaasvind by 'n enkele, koers-bepalende stap, algemeen aanvaar as die eerste ensiem in die pad, pantotenaatkinase (PanK). Die mislukking van die inhibisie van PanK om groei te inhibeer, stel voor dat 'n herevaluering van hierdie uitgangspunt vereis word. Vir hierdie doel is 'n stelselontleding benadering gevolg wat in hierdie studie lig werp op die beheer van KoA produksie in die KoA-herwinningspadweg. Voorheen, was daar 'n gebrek aan analitiese gereedskap om die intermediate van die KoA biosintese padweg te meet. Dit het die uitvoer van ʼn holistiese studie van die regulering van die padweg belemmer. Gevolglik is daar in hierdie studie 'n metode ontwikkel vir die kwantifisering van al die intermediate van die KoA herwinningspadweg, gebaseer op derivatisasie van die intermediate en hoë-prestasie vloeistof chromatografie analise. Hierdie metode het toegelaat vir tydsverloopanalise van die padweg wat uitgevoer moet word om 'n holistiese interpretasie van KoA produksie lewer. A kinetiese model van die padweg is opgebou uit snelheidsvergelykings wat geparameteriseer is met 'n kombinasie van eksperimenteel bepaalde waardes en waardes wat in die literatuur gerapporteer is. Gevolglik is tydsverloop profiele gebruik om die model vir beheer ontledings te bekragtig. Beide tydsverloop profiele en voorspellings deur die model het aangedui dat dit onwaarskynlik is vir PanK om die koers van KoA produksie onder die meeste omstandighede te beheer, en dat dit in werklikheid dephospho-KoA kinase (DPCK) is, die laaste ensiem in die pad, wat die produksie beheer onder fisiologiese toestande. Dit impliseer dat DPCK die beste teiken is vir die inhibisie van KoA biosintese, want dit is meer geneig om in beheer te wees van die koers van KoA produksie onder fisiologiese toestande. Hierdie bevinding is betekenisvol vir studies vir die ontwikkeling van antimikrobiese middels, want dit dui daarop dat die teiken fokus moet verskuif van PanK na DPCK. Dus verteenwoordig die bevindinge van hierdie studie 'n groot verskuiwing in die huidige begrip van die regulering van KoA produksie. Dit beklemtoon ook die belangrikheid van die uitvoer van 'n omvattende stelselontledingsstudie van metaboliese padweë wat ondersoek word vir die ontwikkeling van antimikrobiese middels.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98337
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