Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides
- 1Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia
- 2Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119991, Russia
Abstract
The pursuit of efficient approaches for the production of promising antiviral and cytostatic pharmaceuticals is a crucial direction in medical biotechnology, especially after the COVID-19 pandemic. Modified nucleosides and nucleotides represent a highly promising class of chemicals for the development of drugs against cancer and viral infections. Being an alternative to traditional chemical synthesis, cascades of nucleic exchange enzymes offer single-step synthesis of such compounds with exceptional stereo- and regioselectivity. Furthermore, employing thermostable enzymes for such cascades offers the advantages of increased substrate solubility, lower solution viscosity, prevention of contamination, and simplified biocatalyst purification via thermal denaturation. The first biocatalyst for two distinct cascade pathways of nucleotide and nucleoside synthesis is ribokinase. In this article, we report improving the activity of ribokinase from Thermus sp. 2.9 (TspRK) for the cascade synthesis of modified nucleosides and nucleotides. We have employed a rational design approach, including Boltz-2 modeling and sequence conservation analysis. We have engineered two mutants, T244I and N272H, achieving a 3-fold increase in ribose phosphorylation rate (kcat). Improving the enzymatic activity of a thermostable ribokinase will serve as the basis for further optimization of cascade synthesis of pharmaceutical modified nucleosides and nucleotides.
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BibTeX
@article{zayats2026enhancing,
title = {{Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides}},
author = {Evgeniy A. Zayats and Yulia A. Abramchik and Maria A. Kostromina and Ilya V. Fateev and Andrey A. Karanov and Alexandra R. Sharafutdinova and Vasiliy A. Ptushkin and Irina D. Konstantinova and Andrey V. Golovin and Roman S. Esipov},
journal = {International journal of molecular sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27198911},
url = {https://europepmc.org/article/PMC/PMC13665051},
note = {CC BY},
}
RIS
TY - JOUR
TI - Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides
AU - Evgeniy A. Zayats
AU - Yulia A. Abramchik
AU - Maria A. Kostromina
AU - Ilya V. Fateev
AU - Andrey A. Karanov
AU - Alexandra R. Sharafutdinova
AU - Vasiliy A. Ptushkin
AU - Irina D. Konstantinova
AU - Andrey V. Golovin
AU - Roman S. Esipov
PY - 2026
DA - 2026/10/01
JO - International journal of molecular sciences
DO - 10.3390/ijms27198911
UR - https://europepmc.org/article/PMC/PMC13665051
AB - The pursuit of efficient approaches for the production of promising antiviral and cytostatic pharmaceuticals is a crucial direction in medical biotechnology, especially after the COVID-19 pandemic. Modified nucleosides and nucleotides represent a highly promising class of chemicals for the development of drugs against cancer and viral infections. Being an alternative to traditional chemical synthesis, cascades of nucleic exchange enzymes offer single-step synthesis of such compounds with exceptional stereo- and regioselectivity. Furthermore, employing thermostable enzymes for such cascades offers the advantages of increased substrate solubility, lower solution viscosity, prevention of contamination, and simplified biocatalyst purification via thermal denaturation. The first biocatalyst for two distinct cascade pathways of nucleotide and nucleoside synthesis is ribokinase. In this article, we report improving the activity of ribokinase from Thermus sp. 2.9 (TspRK) for the cascade synthesis of modified nucleosides and nucleotides. We have employed a rational design approach, including Boltz-2 modeling and sequence conservation analysis. We have engineered two mutants, T244I and N272H, achieving a 3-fold increase in ribose phosphorylation rate (kcat). Improving the enzymatic activity of a thermostable ribokinase will serve as the basis for further optimization of cascade synthesis of pharmaceutical modified nucleosides and nucleotides.
SN - 1422-0067
ER -