Effects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies

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dc.contributor.authorVan Deventer, Ruan
dc.contributor.supervisorAchilonu, Ikechukwu Anthony
dc.date.accessioned2024-10-26T08:24:47Z
dc.date.available2024-10-26T08:24:47Z
dc.date.issued2023-07
dc.descriptionA dissertation submitted in fulfilment of the requirements for the degree Mater of Science in Biochemistry & Cell Biology, to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023.
dc.description.abstractNNAT is an attractive target for drug development due to its crucial role in NAD+ synthesis. However, its characterisation in ESKAPE species, such as Klebsiella pneumoniae and Enterococcus faecium, remains limited. This study aimed to elucidate the binding mechanism of ATP, a pivotal substrate, to these NNAT species, focusing on the role of divalent metal ion cofactors. KpNNAT and EfNNAT enzymes were overexpressed and purified, yielding approximately 2 mg/ml for both. Various techniques were employed to investigate their properties, including far-UV CD, extrinsic ANS fluorescence, stopped-flow kinetic analyses, and MD simulations. The results revealed that KpNNAT could bind ATP independently of divalent metal ions, but catalytic activity required the presence of Mg2+. The kinetic analysis showed ka values of 5.99 μM-1 .sec-1 without divalent metal ions and 5.72 μM-1 .sec-1 in the presence of Mg2+. The "pseudo"-specific activity values were 0.005 μmol/min/mg without divalent metal ions and 0.374 μmol/min/mg in the presence of Mg2+. Conversely, recombinant EfNNAT exhibited limited ATP association, and the reasons for this remain unclear. Overall, this study shed light on the structural dynamics and functional kinetics of ATP association in both NNAT species. The findings contribute to our understanding of this druggable target and provide insights into the inactivity of EfNNAT, which warrants further investigation.
dc.description.sponsorshipNational Research Foundation (NRF) of South Africa.
dc.description.sponsorshipUniversity of the Witwatersrand, Johannesburg - Post-graduate Merit Award.
dc.description.submitterMM2024
dc.facultyFaculty of Science
dc.identifier0000-0002-1571-1978
dc.identifier.citationVan Deventer, Ruan. (2023). Effects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies. [Master's dissertation, University of the Witwatersrand, Johannesburg]. https://hdl.handle.net/10539/41973
dc.identifier.urihttps://hdl.handle.net/10539/41973
dc.language.isoen
dc.publisherUniversity of the Witwatersrand, Johannesburg
dc.rights©2023 University of the Witwatersrand, Johannesburg. All rights reserved. The copyright in this work vests in the University of the Witwatersrand, Johannesburg. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of University of the Witwatersrand, Johannesburg.
dc.rights.holderUniversity of the Witwatersrand, Johannesburg
dc.schoolSchool of Molecular and Cell Biology
dc.subjectESKAPE
dc.subjectComputational modeling
dc.subjectBiochemistry
dc.subjectRecombinant protein
dc.subjectStopped-flow kinetics
dc.subjectNNAT
dc.subjectATP
dc.subjectKlebsiella pneumoniae
dc.subjectEnterococus faecium
dc.subjectMant-ATP
dc.subjectMolecular dynamics
dc.subjectUCTD
dc.subject.otherSDG-3: Good health and well-being
dc.titleEffects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies
dc.typeDissertation
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