A Second-Shell Mutation Preorganizes HpnG for Broad-Scope Nucleoside Transglycosylation.

Nucleoside analogues are important therapeutic scaffolds, but their synthesis is often protecting-group-intensive. Purine nucleoside phosphorylases offer a mild enzymatic alternative, yet broad substrate accommodation does not necessarily lead to productive glycosyl transfer. Here, we repurposed HpnG, a hopanoid-associated purine nucleoside phosphorylase, through second-shell engineering. Structure-guided substitution of D124 with glycine increased 2-fluoroadenosine formation from 11% to 91% yield and accelerated inosine phosphorolysis by more than 100-fold with little change in the apparent Km for inosine. HpnG-D124G also accepted diverse purine nucleobases and ribose, 2'-deoxyribose, and arabinose donors, enabling access to drug-relevant nucleosides. Molecular dynamics simulations and electronic-structure calculations suggest that D124G enriches donor-acceptor conformations that are properly aligned for glycosyl transfer. These findings establish productive active-site preorganization as a practical strategy for engineering broad-scope nucleoside biocatalysts.