Engineering Substrate Promiscuity of Nucleoside Phosphorylase Via an Insertions–Deletions Strategy

Nucleoside phosphorylases (NPs) are the key enzymes in the nucleoside metabolism pathway and are widely employed for the synthesis of nucleoside analogs, which are difficult to access via conventional synthetic methods. NPs are generally classified as purine nucleoside phosphorylase (PNP) and pyrimidine or uridine nucleoside phosphorylase (PyNP/UP), based on their substrate preference. Here, based on the evolutionary information on the NP-I family, we adopted an insertions-deletions (InDels) strategy to engineer the substrate promiscuity of nucleoside phosphorylase AmPNPΔS2_V102 K, which exhibits both PNP and UP activities from a trimeric PNP (AmPNP) of Aneurinibacillus migulanus. Furthermore, the AmPNPΔS2_V102 K exerted phosphorylation activities toward arabinose nucleoside, fluorosyl nucleoside, and dideoxyribose, thereby broadening the unnatural-ribose nucleoside substrate spectrum of AmPNP. Finally, six purine nucleoside analogues were successfully synthesized, using the engineered AmPNPΔS2_V102 K instead of the traditional "two-enzymes PNP/UP" approach. These results provide deep insights into the catalytic mechanisms of the PNP and demonstrate the benefits of using the InDels strategy to achieve substrate promiscuity in an enzyme, as well as broadening the substrate spectrum of the enzyme.