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The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus
Thesis (MSc)--Stellenbosch University, 2025.
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| Language: | English |
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867614017761050624 |
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| access_status_str | Open Access |
| author | Wessels, Francel |
| author2 | Rohwer, J. M. |
| author_browse | Rohwer, J. M. Wessels, Francel |
| author_facet | Rohwer, J. M. Wessels, Francel |
| author_sort | Wessels, Francel |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/132319 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:45:21.489Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/132319 The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus Wessels, Francel Rohwer, J. M. Strauss, Erick Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Coenzymes -- Synthesis Staphylococcus aureus Enzyme kinetics -- Data processing Coenzyme A (CoA) Biosynthesis pathway UCTD Thesis (MSc)--Stellenbosch University, 2025. Wessels, F. 2025. The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/b494b9ef-2398-49ed-8755-daed429af9d4 ENGLISH ABSTRACT: Currently in enzymology research, scientists are frequently facing reproducibility issues, mainly because crucial metadata are not reported. These include the precise reaction conditions, comprehensive experimental conditions, and procedures used for data analysis. To address this crises, there is an urgent need to report both the experimental conditions and data to ensure the Findability, Accessibility, Interoperability and Reusability (FAIR) of enzymatic data. Furthermore, coenzyme A (CoA) is an essential co-factor to all living organisms since it is involved in numerous metabolic and cellular pathways. This metabolic pathway has raised strong interest as a drug target against various pathogens, including Staphylococcus aureus (S. aureus), which is considered as one of the leading causes for hospital associated infections. Currently, the bi-functional enzyme complex consisting of phosphopantothenoylcysteine synthetase (PPCS) and phophopantothenoylcysteine decarboxylase (PPCDC), also referred to as CoaBC, the second and third enzymes in the CoA biosynthesis pathway has not yet been characterised kinetically for S. aureus. Therefore, the aim of this project is to kinetically characterize this enzyme in accordance with the FAIR guidelines. To achieve this aim, CoaBC and 4’-phosphopantetheine adenylyltransferase (PPAT), which is responsible for catalyzing the reversible conversion of 4’-phosphopantetheine (PPanSH) to dephospho-CoA (DePCoA) with the release of pyrophosphate (PPi), from S. aureus were expressed and purified using Ni²+-based Immobilised metal affinity chromatography (IMAC). Followed by SDS-PAGE analysis to confirm the purity and identity of these two enzymes. Thereafter, the activity of PPCS and PPAT were assayed by measuring the produced PPi in a coupled assay, linking PPi to the oxidation of two NADH molecules, which could be measured spectrophotometrically at 340 nm. The OT-2 liquid handler was utilized to automate microtiter plate-based kinetic assays to ensure that experimental protocols can be tightly integrated with the data analysis workflows. The kinetics of PPCS and PPAT were studied using initial rate kinetics by selecting the linear section at the start of each reaction through visual inspection. Three different mechanistic scenarios for PPCS were investigated, which included the random binding mechanism, sequential binding mechanism and Bi-Uni-Uni-Bi- Ping-Pong mechanism. Among these, the Bi-Uni-Uni-Bi-Ping-Pong mechanism was identified as the best model to describe the activity for PPCS based on the different Akaike Information Criterion (AIC) values obtained for each mechanism. In addition, we successfully obtained parameters for PPCS (Bi-Uni-Uni-Bi-Ping-Pong mechanism) and PPAT. However, Kictp of PPCS for this mechanism was practically non-identifiable. Moreover, during the second step of the reaction catalysed by CoaBC, CO₂ is released; this cannot be measured spectrophotometrically. Thus, we constructed a CoaBC-PPAT combined kinetic model using PySCeS, where datasets with CoaBC and PPAT and datasets without PPAT were combined and the kinetic parameters previously obtained for the first step of CoaBC and PPAT were included, to fit the kinetic parameter values for the second step catalysed by CoaBC. We were able to parametrize kcat2 but found Km;ppancys to be structurally non-identifiable. Finally, experimental conditions, kinetic parameters and kinetic models were stored in an EnzymeML document using PyEnzyme software. Therefore, this study not only provided the starting point for the kinetic characterization of CoaBC from S. aureus using microtiter-plate based assays, but also showed how using PyEnzyme software can facilitate the capturing of metadata and data in a structured and standardized manner, in an attempt to address the reproducibility crisis. AFRIKAANSE OPSOMMING: Tans in ensiemologie-navorsing, staar wetenskaplikes reproduseerbaarheidskwessies in die gesig, hoofsaaklik omdat belangrike metadata nie gerapporteer word nie. Dit sluit in die presiese reaksietoestande, omvattende eksperimentele toestande en die prosedures wat vir data analiese gebruik word. Om hierdie krisis aan te spreek, is daar ’n dringende behoefte om beide die eksperimentele kondisies en data te rapporteer om die vindbaarheid, toeganklikheid, interoperabiliteit en herbruikbaarheid (FAIR) van ensiematiese data in ooreenstemming met die FAIR-riglyne te verseker. Verder is koënsien A (KoA)’n noodsaaklike ko-faktor vir alle lewende organismes aangesien dit betrokke is by talle metaboliese en sellulêre prosesse. Die metaboliese pad van KoA het groot belangstelling as ’n geneesmiddelteiken teen verskeie patogene gewek, insluitend S. aureus, wat beskou word as een van die hoofoorsake vir hospitaalverwante siektes. Op hierdie stadium is CoaBC van S. aureus nog nie kineties gekarakteriseer nie. Daarom is die doel van hierdie projek om CoaBC van S. aureus kineties te karakteriseer en om te verseker dat die ensiematiese data sodanig aan die FAIR-riglyne voldoen. Om hierdie doel te bereik, is CoaBC en 4’-fosfopanteïen adenieltransferase (PPAT), wat verantwoordelik is vir die kataliese van die omkeerbare omskakeling van 4’-fosfopantetien (PPanSH) na defosfo-CoA (DePCoA) met die vrystelling van pirofosfaat (PPi) van S. aureus was uitgedruk en gesuiwer deur gebruik te maak van Ni2+-gebaseerde geïmmobiliseerde metaalaffiniteitschromatografie (IMAC). Gevolg deur SDS-gelelektroforese analiese om die suiwerheid en identiteit van die twee ensieme te bevestig. Daarna is die aktiwiteit van PPCS en PPAT getoets deur die geproduseerde PPi te meet in ’n gekoppelde essai wat PPi koppel aan die oksidasie van twee NADH molekules, wat spektrofotometries by 340 nm gemeet kon word. Die OT-2-vloeistofhanteerder was gebruik om mikrotiter-plaat-gebaseerde kinetiese essais te outomatiseer om te verseker dat eksperimentele protokolle nou geïntegreer kan word met die data analiese. Die kinetika van PPCS en PPAT was bestudeer deur gebruik te maak van aanvanklike tempo deur die lineêre snit aan die begin van elke reaksie deur middel van visuele inspeksie te kies. Drie verskillende meganistiese senarios vir PPCS was geondersoek, wat die ewekansige bindingsmeganisme, opeenvolgende bindingsmeganisme en Bi-Uni-Uni-Bi-Ping-Pong meganisme ingesluit het. Onder hierdie was die Bi-Uni-Uni-Bi-Ping-Pong meganisme geïdentifiseer as die beste model om die aktiwiteit van PPCS te beskryf gebaseer op die verskillende Akaike Information Criterion (AIC) waardes. Daarna het ons suksesvol parameters vir PPCS (Bi-Uni-Uni-Bi-Ping-Pong meganisme) en PPAT verkry. Daar was egter gevind dat Kictp vir die Bi-Uni-Uni-Bi-Ping-Pong meganisme feitlik onidentifiseerbaar was. Verder, tydens die tweede stap van die reaksie wat deur CoaBC gekataliseer word, word CO₂ vrygestel wat nie spektrofotometries gemeet kan word nie. Ons het dus ’n CoaBC-PPAT gekombineerde model met behulp van PySCeS gekonstruktureer, waar datastelle met CoaBC en PPAT en datastelle sonder PPAT gekombineer word en die kinetiese parameters wat voorheen verkry was vir die eerste stap van CoaBC en PPAT ingesluit is, om die kinetiese parameterwaardes te pas vir die tweede stap wat deur CoaBC gekataliseer word. Ons kon kcₐt₂ parameteriseer, maar het gevind dat Km;ppₐncys struktureel onidentifiseerbaar was. Verder was alle eksperimentele toestande, kinetiese parameters en kinetiese modelle in ’n EnzymeML dokument gestoor met behulp van PyEnzyme sagteware. Dus, hierdie studie verskaf nie net die beginpunt vir die kinetiese karakterisering van CoaBC vanaf S. aureus met behulp van mikrotiter-plaat-gebaseerde essais nie, maar het ook gewys hoe PyEnzyme sagteware gebruik kan word om belangrike metadata en data vas te lê in ’n poging om die reproduseerbaarheidskrisis aan te spreek. Masters 2025-06-03T12:10:32Z 2025-06-03T12:10:32Z 2025-03 Thesis https://scholar.sun.ac.za/handle/10019.1/132319 en Stellenbosch University xvii, 104 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Coenzymes -- Synthesis Staphylococcus aureus Enzyme kinetics -- Data processing Coenzyme A (CoA) Biosynthesis pathway UCTD Wessels, Francel The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus |
| title | The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus
|
| title_full | The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus
|
| title_fullStr | The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus
|
| title_full_unstemmed | The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus
|
| title_short | The FAIR kinetic characterization of CoaBC from Staphyloccocus aureus
|
| title_sort | fair kinetic characterization of coabc from staphyloccocus aureus |
| topic | Coenzymes -- Synthesis Staphylococcus aureus Enzyme kinetics -- Data processing Coenzyme A (CoA) Biosynthesis pathway UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/132319 |
| work_keys_str_mv | AT wesselsfrancel thefairkineticcharacterizationofcoabcfromstaphyloccocusaureus AT wesselsfrancel fairkineticcharacterizationofcoabcfromstaphyloccocusaureus |