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Snapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferase
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dc.contributor.author | Tang, Peijun | |
dc.contributor.author | Harding, Christopher John | |
dc.contributor.author | Dickson, Alison | |
dc.contributor.author | da Silva, R.G. | |
dc.contributor.author | Harrison, David James | |
dc.contributor.author | Melo Czekster, Clarissa | |
dc.date.accessioned | 2024-02-15T16:30:01Z | |
dc.date.available | 2024-02-15T16:30:01Z | |
dc.date.issued | 2024-03-01 | |
dc.identifier | 298523907 | |
dc.identifier | 043244d9-3e17-4782-b1d7-43612741722f | |
dc.identifier | 85185611057 | |
dc.identifier.citation | Tang , 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.3c06260 | en |
dc.identifier.issn | 2155-5435 | |
dc.identifier.other | ORCID: /0000-0002-7163-4057/work/153451504 | |
dc.identifier.other | ORCID: /0000-0001-9041-9988/work/153451546 | |
dc.identifier.other | ORCID: /0000-0002-4150-2467/work/153451748 | |
dc.identifier.other | ORCID: /0000-0002-1308-8190/work/153451959 | |
dc.identifier.uri | https://hdl.handle.net/10023/29265 | |
dc.description | Funding: 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.abstract | Nucleosides 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.extent | 13 | |
dc.format.extent | 5376511 | |
dc.language.iso | eng | |
dc.relation.ispartof | ACS Catalysis | en |
dc.subject | Deoxyribonucleoside transferase | en |
dc.subject | Nucleosides | en |
dc.subject | Biocatalysis | en |
dc.subject | Protein engineering | en |
dc.subject | Thermophilic | en |
dc.subject | QH301 Biology | en |
dc.subject | DAS | en |
dc.subject.lcc | QH301 | en |
dc.title | Snapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferase | en |
dc.type | Journal article | en |
dc.contributor.institution | University of St Andrews. School of Biology | en |
dc.contributor.institution | University of St Andrews. Biomedical Sciences Research Complex | en |
dc.contributor.institution | University of St Andrews. School of Medicine | en |
dc.contributor.institution | University of St Andrews. Sir James Mackenzie Institute for Early Diagnosis | en |
dc.contributor.institution | University of St Andrews. Cellular Medicine Division | en |
dc.identifier.doi | 10.1021/acscatal.3c06260 | |
dc.description.status | Peer reviewed | en |
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