Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides

Zayats, Evgeniy A.; Abramchik, Yulia A.; Kostromina, Maria A.; Fateev, Ilya V.; Karanov, Andrey A.; Sharafutdinova, Alexandra R.; Ptushkin, Vasiliy A.; Konstantinova, Irina D.; Golovin, Andrey V.; Esipov, Roman S.

doi:10.3390/ijms27198911

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Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides

by

Evgeniy A. Zayats

^1,*

,

Yulia A. Abramchik

¹

,

Maria A. Kostromina

¹

,

Ilya V. Fateev

¹

,

Andrey A. Karanov

^1,2,

Alexandra R. Sharafutdinova

¹,

Vasiliy A. Ptushkin

¹

,

Irina D. Konstantinova

¹

,

Andrey V. Golovin

^1,2

and

Roman S. Esipov

^1,2,*

¹

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia

²

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119991, Russia

^*

Authors to whom correspondence should be addressed.

Int. J. Mol. Sci. 2026, 27(19), 8911; https://doi.org/10.3390/ijms27198911 (registering DOI)

Submission received: 8 August 2026 / Revised: 26 September 2026 / Accepted: 28 September 2026 / Published: 7 October 2026

(This article belongs to the Special Issue Design and Application of Biocatalysts: Emerging Advances in Enzyme Technology)

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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 (k_cat). 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.

Keywords: cascade synthesis; enzyme design; boltz-2; ribokinase; modified nucleosides; modified nucleotides

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MDPI and ACS Style

Zayats, E.A.; Abramchik, Y.A.; Kostromina, M.A.; Fateev, I.V.; Karanov, A.A.; Sharafutdinova, A.R.; Ptushkin, V.A.; Konstantinova, I.D.; Golovin, A.V.; Esipov, R.S. Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides. Int. J. Mol. Sci. 2026, 27, 8911. https://doi.org/10.3390/ijms27198911

AMA Style

Zayats EA, Abramchik YA, Kostromina MA, Fateev IV, Karanov AA, Sharafutdinova AR, Ptushkin VA, Konstantinova ID, Golovin AV, Esipov RS. Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides. International Journal of Molecular Sciences. 2026; 27(19):8911. https://doi.org/10.3390/ijms27198911

Chicago/Turabian Style

Zayats, Evgeniy A., Yulia A. Abramchik, Maria A. Kostromina, Ilya V. Fateev, Andrey A. Karanov, Alexandra R. Sharafutdinova, Vasiliy A. Ptushkin, Irina D. Konstantinova, Andrey V. Golovin, and Roman S. Esipov. 2026. "Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides" International Journal of Molecular Sciences 27, no. 19: 8911. https://doi.org/10.3390/ijms27198911

APA Style

Zayats, E. A., Abramchik, Y. A., Kostromina, M. A., Fateev, I. V., Karanov, A. A., Sharafutdinova, A. R., Ptushkin, V. A., Konstantinova, I. D., Golovin, A. V., & Esipov, R. S. (2026). Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides. International Journal of Molecular Sciences, 27(19), 8911. https://doi.org/10.3390/ijms27198911

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Enhancing the Activity of Ribokinase from Thermus sp. 2.9 Through Rational Design for the Cascade Synthesis of Modified Nucleotides and Nucleosides

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