Functional characterization and protein engineering of glycosyltransferase for 2"-O-xylosylation of ginsenoside Rg3.

The limited abundance of xylosylated ginsenosides and the lack of efficient biocatalysts hinder their pharmacological exploration. This study identified a 2"-O-xylosyltransferase (PnUGT57) from Panax notoginseng that catalyzes the conversion of ginsenoside Rg3 to notoginsenoside ST4. Wild-type PnUGT57 preferred UDP-xylose over UDP-glucose and UDP-rhamnose and displayed limited thermostability (t = 6.73 h at 30 °C). To enhance UDP-xylose specificity, sequence-guided mutagenesis generated the C140A variant, which achieved remarkable UDP-xylose specificity (100 % conversion) with a 1.34-fold increase in catalytic efficiency while showing weak activity toward UDP-glucose (8.7 %) and UDP-rhamnose (5.2 %) activity. The F367A mutant possesses only xylosyltransferase activity but with reduced catalytic efficiency (0.3-fold of the WT). Molecular docking revealed that the enhanced UDP-xylose specificity in C140A and F367A resulted from the loss of key hydrogen bonding and hydrophobic interactions. To improve thermostability, computational design produced a triple mutant (P101S/L200C/G255D) with an 8.58-fold longer half-life (57.76 h), attributed to optimized surface charge distribution and improved hydration layer formation, as confirmed by molecular dynamics simulation. The combinatorial mutant C140A/P101S/L200C/G255D synergistically improved UDP-xylose specificity, thermostability, and catalytic efficiency, enabling efficient ST4 biosynthesis. This study elucidates the catalytic mechanism of PnUGT57 and presents engineered variants as promising biocatalysts for sustainable ginsenoside production.