The expression, purification and characterization of escherichia coli NudL, a putative coenzyme A hydrolase

Thesis (MSc)--Stellenbosch University, 2021.ENGLISH ABSTRACT: Coenzyme A (CoA) is an essential cofactor that is synthesized by a conserved five-step pathway from pantothenic acid (vitamin B5). The regulation of CoA levels is vital in supporting normal cellular function. In higher order organisms, CoA degradation and the recycling of CoA have proven efficient for the dynamic control of CoA levels, but in prokaryotes the contribution of CoA degradation to the regulation of CoA levels remains largely unstudied. Escherichia coli is known to make significant quantities of 4'-phosphopantetheine (PPanSH), a CoA biosynthesis intermediate, the excess of which is irreversibly exported to the extracellular environment. Early studies revealed that the source of the high PPanSH levels is both from the biosynthesis and the degradation of CoA. In E. coli, CoA degradation can be mediated via an indirect mechanism through acyl carrier protein (ACP) prosthetic group turnover, where AcpH releases the PPanSH moiety from the holo-ACP, or via a direct mechanism by an unidentified enzyme. One possible mechanism for direct degradation of CoA is by the action of a CoA hydrolase, a Nudix hydrolase subfamily member. Nudix hydrolases are a superfamily of housekeeping enzymes that regulate metabolic intermediates or remove toxic nucleotide metabolites. Nudix hydrolases specific for CoA are able to degrade CoA at the pyrophosphate moiety into 3',5'-ADP and PPanSH. E. coli has 13 predicted Nudix enzymes of which NudL is the only one yet to be characterized. NudL is encoded by the yeaB gene and contains both the Nudix box and NuCoA motif that points to its selectivity for CoA and strongly suggests that it is a CoA hydrolase. The first part of this study focused on optimizing the production of pure, soluble NudL protein for testing its putative CoA hydrolase activity. The protein was found to be unstable and significant challenges were experienced with soluble recombinant protein expression. NudL could therefore only be partially purified for the purpose of activity testing. The second part of this study focussed on characterizing NudL’s activity. Both the partially purified enzyme and E. coli lysate containing overexpressed NudL was found to be able to degrade CoA and form PPanSH. These results provide strong evidence that CoA hydrolysis into PPansH is mediated by NudL and confirms the presence of CoA hydrolase activity in E. coli lysate. CoA hydrolase activity was dependent on the presence of a metal cofactor and NudL appeared to prefer Mn²⁺ over Mg²⁺. Following this, the physiological relevance of NudL was investigated. The results of metabolomic studies revealed that NudL contributes significantly to the intracellular PPanSH levels as a knockout mutant lacking the enzyme had considerably lower intracellular PPanSH levels. An ∆acpH mutant also showed a similar trend, confirming that CoA degradation contributes to the intracellular PPanSH pool. Conditions which promote CoA degradation were also investigated. Transcription of the NudL- encoding gene yeaB did not appear to be influenced by oxidative stress; however, a significant growth defect was observed for the mutant strains when acetate was the carbon source, suggesting that CoA degradation by NudL is important during growth on acetate. The results of this study support a direct mechanism of CoA degradation in E. coli, specifically by the NudL enzyme, being the last E. coli Nudix hydrolase to be experimentally characterized. Further investigations are needed to understand how this enzyme is regulated. This may provide insight into how CoA levels are regulated in E. coli in general.AFRIKAANSE OPSOMMING: Koënsiem A (KoA) is 'n noodsaaklike ko-faktor wat deur middel van 'n gekonserveerde vyf-stap- padweg vanaf pantoteensuur (vitamien B5) gesintetiseer word. Die regulering van KoA-vlakke is noodsaaklik om normale sellulêre funksie te ondersteun. In hoër orde organismes het KoA-afbraak en die herwinning van KoA effektief blyk te wees vir die dinamiese beheer van KoA-vlakke, maar in prokariote bly die bydrae van CoA-afrbaak tot die regulering van KoA-vlakke grotendeels onbestudeer. Dit is wel bekend dat Escherichia coli beduidende hoeveelhede maak van 4'-fosfopanteteïne (PPanSH), 'n KoA-biosintese-tussenproduk, waarvan die oormaat onomkeerbaar na die ekstrasellulêre omgewing uitgevoer word. Vroeë studies het uitgewys dat die bron van die hoë PPanSH-vlakke beide die gevolg is van die biosintese en die afbraak van KoA. In E. coli kan KoA-afbraak plaasvind deur 'n indirekte meganisme deur die omset van die prostetiese groep van die asiel-draerproteïen (ACP), deurdat AcpH die PPanSH-groep van die holo-ACP vrystel, of via 'n direkte meganisme gekataliseer deur 'n ongeïdentifiseerde ensiem. Een moontlike meganisme vir direkte afbraak van KoA is deur die werking van 'n KoA-hidrolase, spesifiek ‘n lid van die Nudix-hidrolase-subfamilie. Nudix-hidrolases is 'n superfamilie van huishoudingsensieme wat metaboliese tussenprodukte reguleer of toksiese nukleotiedmetaboliete verwyder. Nudix-hidrolases wat spesifiek vir KoA is kan KoA in die pirofosfaatgedeelte hidroliseer om 3',5'-ADP en PPanSH te vorm. E. coli het 13 voorspelde Nudix- ensieme waarvan NudL die enigste is wat nog gekarakteriseer moet word. NudL word gekodeer deur die yeaB-geen en bevat beide die Nudix-Koks en die NuKoA-motief, waarvan laasgenoemde die ensiem se selektiwiteit vir KoA aandui en dus sterk daarop wys dat dit 'n KoA-hidrolase is. Die eerste gedeelte van hierdie studie het gefokus op die optimisering van die produksie van suiwer, oplosbare NudL-proteïene om die vermeende CoA-hidrolase-aktiwiteit daarvan te toets. Daar is gevind dat die proteïen onstabiel is en dat die oplosbare rekombinante proteïenuitdrukwing beduidende uitdagings ondervind. NudL kon dus slegs gedeeltelik gesuiwer word om die aktiwiteit daarvan te toets. Die tweede deel van hierdie studie het gefokus op die karakterisering van NudL se aktiwiteit. Daar is gevind dat beide die gedeeltelik gesuiwerde ensiem en E. coli lisaat wat ooruitdruklike NudL bevat, KoA kon afbreek en PPanSH kon vorm. Hierdie resultate lewer sterk bewyse dat KoA-hidrolise in PPansH deur NudL bemiddel word en bevestig die teenwoordigheid van KoA-hidrolase-aktiwiteit in E. coli lisaat. CoA-hidrolase-aktiwiteit was afhanklik van die teenwoordigheid van 'n metaal ko-faktor, en NudL het blykbaar Mn²⁺ bo Mg²⁺ verkies. Hierna is die fisiologiese relevansie van NudL ondersoek. Die resultate van metabolomiese studies het aan die lig gebring dat NudL aansienlik bydra tot die intrasellulêre PPanSH-vlakke, aangesien 'n uitklop-mutant sonder die ensiem aansienlik laer intrasellulêre PPanSH-vlakke gehad het. 'N ∆acpH-mutant het ook 'n soortgelyke neiging getoon, wat bevestig dat KoA-afbraak bydra tot die intrasellulêre PPanSH-poel. Toestande wat CoA-afbraak bevorder is ook ondersoek. Transkripsie van die NudL-koderende geen yeaB blyk nie deur oksidatiewe stress beïnvloed te word nie; 'n beduidende groeifout is egter waargeneem vir die mutante stamme wanneer asetaat die koolstofbron was, wat daarop dui dat KoA-afbraak deur NudL belangrik is tydens die groei op asetaat. Die resultate van hierdie studie ondersteun 'n direkte meganisme van CoA-afbraak in E. coli, spesifiek deur die NudL-ensiem, wat die laaste E. coli Nudix-hidrolase is wat eksperimenteel gekarakteriseer is. Verdere ondersoeke is nodig om te verstaan hoe hierdie ensiem gereguleer word. Dit kan insig gee in hoe KoA-vlakke in E. coli in die algemeen gereguleer word.Master