Characterization of a Purine Nucleoside Phosphorylase From Colwellia 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.