Characterization of the nucleoside monophosphate kinase from Thermus thermophilus phage p23-45 and its application in the biosynthesis of deoxynucleoside triphosphates.

This article focuses on the characterization of a nucleoside monophosphate kinase (NMK) from Thermus thermophilus phage p23-45 (ϕNMK) and its application in enzymatic deoxyribonucleoside triphosphate (dNTP) synthesis. The ϕNMK enzyme exhibits broad substrate specificity for all four deoxynucleoside monophosphates (dNMPs), optimal activity at 70 °C, and notable thermostability, retaining significant activity after prolonged incubation at moderate temperatures. Structural modeling and site-directed mutagenesis identified key catalytic residues, while a one-pot dNTP synthesis system combining ϕNMK with Thermus thermophilus pyruvate kinase (TtPYK) demonstrated efficient ATP regeneration and achieved over 85% conversion of dNMPs to dNTPs. This enzymatic approach offers a simplified, environmentally friendly alternative to chemical dNTP synthesis, with potential industrial applications due to the thermostability and efficiency of the enzymes involved.