Functional characterization and engineering of the sugar donor specificity of a 2″-O-xylosyltransferase from Panax notoginseng for rare saponins biosynthesis.

2″-O-xylosylation is a rare glycosylation pattern conferring unique bioactivities to triterpenoid saponins in Panax species. Here, we identify a novel 2″-O-xylosyltransferase, PnUGT94X1, from P. notoginseng, which regioselectively catalyzes 2″-O-xylosylation at the C3-linked sugar of diverse saponins. PnUGT94X1 exhibits a pronounced preference for UDP-xylose and a broad specificity tolerance for sugar acceptors including protopanaxadiol-, cycloartenol-, and oleanane-type saponins. Site-directed mutagenesis revealed that the residues C140 and T141, with a distance of 22 residues downstream of the Asp in the catalytic His-Asp dyad, synergistically govern substrate preference between UDP-xylose and UDP-glucose, which is defined as an important part of sugar donor selectivity-determining region (SDSR). Using PnUGT94X1 and its engineered mutants, eleven rare saponins including six new compounds were biosynthesized by 2″-O-xylosylation or 2″-O-glucosylation, three of them exhibited significant effects (at 5 mg·kg-1) against LPS-induced acute lung injury in mice. The elucidated SDSR module provides a strategic framework for engineering UGTs to alter their sugar donors, thereby enabling the synthesis structurally diverse glycosides.