Characterization of a Purine Nucleoside Phosphorylase From <i>Colwellia</i> sp. WH041 for Multienzyme Synthesis of Nucleoside Analogs

Multienzyme cascades provide an efficient biocatalytic strategy for synthesizing nucleoside analogs from simple sugar donors and nucleobases. In pentose-to-nucleoside cascades, purine nucleoside phosphorylase (PNP) catalyzes the terminal N-glycosylation step and strongly influences product formation and substrate scope. Here, seven putative PNP homologs from microorganisms isolated from low-temperature environments were evaluated under EcRK-EcPPM-PNP cascade conditions. A PNP from Colwellia sp. WH041, designated ColPNP, showed the highest arabinofuranosylation activity and was selected for further characterization. ColPNP displayed optimal activity at 45 °C and exhibited a 12.7-fold higher catalytic efficiency than Escherichia coli PNP at the same temperature. Molecular dynamics simulations supported the favorable catalytic performance of ColPNP under moderate-temperature conditions. After optimization of the EcRK-EcPPM-ColPNP cascade, vidarabine conversion reached 58.2% within 12 h. The optimized system was further applied to synthesize diverse nucleoside analogs with conversions of 45.8%-89.8%, and guanosine analogs were obtained through sequential EcADA-mediated deamination. This study expands the PNP toolbox and demonstrates ColPNP as an efficient module for enzymatic nucleoside analog synthesis.