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

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.