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dc.contributor.authorTang, Peijun
dc.contributor.authorHarding, Christopher John
dc.contributor.authorDickson, Alison
dc.contributor.authorda Silva, R.G.
dc.contributor.authorHarrison, David James
dc.contributor.authorMelo Czekster, Clarissa
dc.date.accessioned2024-02-15T16:30:01Z
dc.date.available2024-02-15T16:30:01Z
dc.date.issued2024-03-01
dc.identifier298523907
dc.identifier043244d9-3e17-4782-b1d7-43612741722f
dc.identifier85185611057
dc.identifier.citationTang , P , Harding , C J , Dickson , A , da Silva , R G , Harrison , D J & Melo Czekster , C 2024 , ' Snapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferase ' , ACS Catalysis , vol. 14 , no. 5 , pp. 3090-3102 . https://doi.org/10.1021/acscatal.3c06260en
dc.identifier.issn2155-5435
dc.identifier.otherORCID: /0000-0002-7163-4057/work/153451504
dc.identifier.otherORCID: /0000-0001-9041-9988/work/153451546
dc.identifier.otherORCID: /0000-0002-4150-2467/work/153451748
dc.identifier.otherORCID: /0000-0002-1308-8190/work/153451959
dc.identifier.urihttps://hdl.handle.net/10023/29265
dc.descriptionFunding: P.T. is funded by IBioIC (IBioIC 2020-2-1), and C.M.C. is funded by the Wellcome Trust (217078/Z/19/Z). C.M.C. and D.H. are funded by research grants from NuCana plc..en
dc.description.abstractNucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2′-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2′-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2′-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2′-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5–9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2′-fluoro-2′-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2′-difluoro-2′-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2′-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.
dc.format.extent13
dc.format.extent5376511
dc.language.isoeng
dc.relation.ispartofACS Catalysisen
dc.subjectDeoxyribonucleoside transferaseen
dc.subjectNucleosidesen
dc.subjectBiocatalysisen
dc.subjectProtein engineeringen
dc.subjectThermophilicen
dc.subjectQH301 Biologyen
dc.subjectDASen
dc.subject.lccQH301en
dc.titleSnapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferaseen
dc.typeJournal articleen
dc.contributor.institutionUniversity of St Andrews. School of Biologyen
dc.contributor.institutionUniversity of St Andrews. Biomedical Sciences Research Complexen
dc.contributor.institutionUniversity of St Andrews. School of Medicineen
dc.contributor.institutionUniversity of St Andrews. Sir James Mackenzie Institute for Early Diagnosisen
dc.contributor.institutionUniversity of St Andrews. Cellular Medicine Divisionen
dc.identifier.doi10.1021/acscatal.3c06260
dc.description.statusPeer revieweden


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