Investigation and characterisation of dephospho-coenzyme A kinase: a potential drug target

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dc.contributor.advisorDe Villiers, Marianneen_ZA
dc.contributor.advisorStrauss, Ericken_ZA
dc.contributor.authorBouwer, Wilmarieen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Biochemistry.en_ZA
dc.date.accessioned2018-11-22T11:11:58Z
dc.date.accessioned2018-12-10T06:37:00Z
dc.date.available2018-11-22T11:11:58Z
dc.date.available2018-12-10T06:37:00Z
dc.date.issued2018-12
dc.descriptionThesis (MSc)--Stellenbosch University, 2018. en_ZA
dc.description.abstractENGLISH ABSTRACT: Due to the rise in antimicrobial resistance, the coenzyme A (CoA) biosynthesis pathway has been identified as a metabolic process of interest as a potential new antimicrobial drug target that could aid in relieving the threat to global healthcare. The CoA biosynthesis pathway consists of five enzymes, each a potential novel drug target. The focus of this study was on dephospho-Coenzyme A kinase (DPCK), the last enzyme in the pathway, as it is thought to hold the most control over the flux through the CoA pathway and is largely unexplored. Particular focus was given to DPCK from Staphylococcus aureus and Plasmodium falciparum. Both these organisms have shown resistance to current drug treatments available in a clinical setting, thus causing resistant strains to become more prevalent and pose a greater threat to global healthcare. The aims of this study were to expand current knowledge on these two organisms’ DPCK enzymes, as neither of these enzymes have been characterised previously. The first aim of this study focussed on characterising S. aureus DPCK, whilst comparing the results to previously characterised DPCK enzymes from Corynebacterium ammoniagenes, Corynebacterium glutamicum, and Escherichia coli. From this we were able to determine the quaternary structure of DPCKs from S. aureus, C. ammoniagenes and C. glutamicum to be trimers in solution, whereas DPCK from E. coli was confirmed to be a monomer as previously reported in literature. Following this, we have determined that the specific activity of S. aureus DPCK is very low, in contrary to what is found for C. ammoniagenes DPCK. Techniques to aid in measuring the low activity of the enzyme were further explored. This led to the use of high performance liquid chromatography (HPLC) assays that incorporated all the CoA biosynthesis pathway enzymes to determine specific activity. We concluded that the presence of all of the other pathway enzymes, at physiological ratios, aided the specific activity of S. aureus DPCK. In the second aim of this study we wanted to determine the activity of P. falciparum DPCK, as this enzyme is the only CoA biosynthesis pathway enzyme that does not localise to the cytosol of the parasite. Further, little information is available on the activity of the parasite’s CoA biosynthesis enzymes as they are difficult to express recombinantly using bacterial expression vectors. We successfully determined conditions for that yielded soluble expression and purification conditions of P. falciparum DPCK, however the enzyme is not very stable in solution. This unfortunately prevented us from determining the activity of the enzyme. The results from this study have shed more light on methods to determine the activity of DPCK enzymes and have established soluble expression conditions for P. falciparum DPCK from a bacterial expression vector. Therefore, it can form the basis in future investigations on DPCK as a potential drug target.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: As gevolg van die toename in weerstandbiedende mikrobes, is die koënsiem A (KoA) biosintese padweg geïdentifiseer as ‘n metaboliese proses wat ondersoek kan word as 'n potensiële nuwe antimikrobiese teenmiddelteiken. Die KoA biosintese padweg bestaan uit vyf ensieme - elk 'n potensiële nuwe teenmiddelteiken. Die fokus van hierdie studie was op defosfokoënsiem A kinase (DFKK), die laaste ensiem in die padweg, omdat dit beskou word as die ensiem met die meeste beheer oor KoA regulering. Hierdie ensiem is nog nie baie bestudeer nie. In hierdie studie is spesifieke klem gelê op DFKK van Staphylococcus aureus en Plasmodium falciparum. Beide hierdie organismes het weerstandbiedendheid getoon teen huidige kliniese, beskibare medisyne. Dit veroorsaak dat weerstandbiedende antimikrobes meer algemeen voorkom en nog meer globale gesondheid bedreig. Die doelwitte van hierdie studie was om die huidige kennis oor DFKK van die twee organismes uit te brei, aangesien nie een van die ensieme al voorheen gekarakteriseer is nie. Die eerste doelwit van hierdie studie het gefokus op die karakterisering van S. aureus DFKK. Dit is dan vergelyk met die resultate van vorige gekarakteriseerde DFKK ensieme van Corynebacterium ammoniagenes, Corynebacterium glutamicum, en Escherichia coli. Dit was moontlik om die kwaternêre struktuur van S. aureus, C. ammoniagenes en C. glutamicum DFKK te onderskeidelik te bepaal as trimere in oplossing. DFKK van E. coli is bevestig as ‘n monomeer, in lyn met wat gerapporteer is in literatuur. Hierna het ons vasgestel dat die spesikieke aktiwiteit van S. aureus DFKK baie laag is, in stryd met wat gevind is vir C. ammoniagenes DFKK. Tegnieke om te help met die bepaling van die lae aktiwiteit van die ensiem is verder ondersoek. Dit het gelei tot die gebruik van hoë-optrede vloeistofkromatografie (HOFK) toetse waarin al die KoA biosintese padweg ensieme geïnkorporeer was om spesifieke aktiwiteit te bepaal. Ons het tot die gevolgtrekking gekom dat die teenwoordigheid van al die ander padweg ensieme, by fisiologise verhoudinge, die spesifieke aktiwiteit van S. aureus DFKK ondersteun het. Die tweede doelwit van die studie het gefokus om die aktiwiteit van P. falciparum DFKK te bepaal, aangesien hierdie ensiem die enigste KoA biosintese padweg ensiem van die parasiet is wat nie na die sitosol lokaliseer nie. Verder, is daar min informasie beskikbaar rakende die aktiwiteit van enige van die parasiet se KoA biosintese padweg ensieme, aangesien dit moelik is om hulle d.m.v rekombinante bakteriële uitdrukkinsvektore te produseer. Ons het vir die eerste keer kondisies bepaal wat oplosbare uitdrukking gee. Verder, kon ons suiweringstoestande vir P. falciparum DFKK bepaal, alhoewel die ensiem nie stabiel in oplosing is nie. Dit het ons dus verhoed om die aktiwiteit van die ensiem te bepaal. Die bevindinge van hierdie studie het meer lig op metodes gewerp m.b.t. die aktiwiteit van DFKK ensieme. Verder was oplosbare uitdrukkingskondisies vir P. falciparum DFKK van 'n bakteriële uitdrukkingsvektor bepaal. Dus, kan die uitkomste van die betrokke studie die basis vorm vir toekomstige ondersoeke op DFKK as 'n moontlike teenmiddelteiken.af_ZA
dc.description.versionMastersen_ZA
dc.embargo.terms2023-12-31
dc.format.extentxiii, 77 pages : illustrations(some color), mapsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/105218
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshCoenzymes -- Inhibitorsen_ZA
dc.subject.lcshCoenzyme Aen_ZA
dc.subject.lcshDephospho-Coenzyme A kinaseen_ZA
dc.subject.lcshStaphylococcus aureusen_ZA
dc.subject.lcshPlasmodium falciparumen_ZA
dc.subject.lcshMicroorganisms -- Effect of drugs onen_ZA
dc.subject.lcshDrug resistanceen_ZA
dc.subject.lcshProtein kinases -- Regulationen_ZA
dc.titleInvestigation and characterisation of dephospho-coenzyme A kinase: a potential drug targeten_ZA
dc.typeThesisen_ZA
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