Snapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferase

Peijun Tang, Christopher John Harding, Alison Dickson, R.G. da Silva, David James Harrison, Clarissa Melo Czekster*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)
10 Downloads (Pure)

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.
Original languageEnglish
Pages (from-to)3090-3102
Number of pages13
JournalACS Catalysis
Volume14
Issue number5
Early online date13 Feb 2024
DOIs
Publication statusPublished - 1 Mar 2024

Keywords

  • Deoxyribonucleoside transferase
  • Nucleosides
  • Biocatalysis
  • Protein engineering
  • Thermophilic

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