Cloning and functional characterization of three key glycosylation genes involved in dioscin biosynthesis

The sterol glycosyltransferase gene functions as the final key structural gene in dioscin biosynthetic pathway, catalyzing the glycosylation of 3-OH position of diosgenin to produce dioscin. In this study, two UDP-glucosyltransferase genes DoUGT80A2 and DoUGT80B1 and one UDP-rhamnosyltransferase gene ( DoURT1 ) from Dioscorea opposita were cloned. DoUGT80A2, DoUGT80B1and DoURT1 were all localized on cytoplasm. All three target proteins could be successfully induced in vitro and in vivo . Using UDP-glucose as the glycosyl donor and diosgenin as the substrate, both DoUGT80A2 and DoUGT80B1 were identified to catalyze the glycosylation of C-3 hydroxyl group of diosgenin to form trillin. Notably, DoUGT80A2 exhibited significantly higher catalytic activity than DoUGT80B1. When UDP-rhamnose (UDP-Rha) was employed as glycosyl donors with trillin as the substrate, DoURT1 was found to catalyze the conversion of trillin to dioscin. All three target proteins could form high-affinity complexes with their respective ligands. It further computationally identified that key amino acid residues critically contributed to ligand binding within these complexes. Specifically, residues Thr134 and Tyr352 exhibited significant binding contributions in the DoUGT80A2-diosgenin complex, while His505 demonstrated high contribution values in the DoUGT80B1-diosgenin complex. For the DoURT1-trillin complex, residue Ile253 was identified as pivotal contributors. Subsequent site-directed alanine substitution of these nine residues abolished or drastically reduced the production of target products in in vitro enzymatic assays, unequivocally underscoring their essential roles in stabilizing the ligand-protein complexes and maintaining their functional integrity. This study lays a critical foundation for the comprehensive elucidation of the diosgenin biosynthetic pathway and the structural characterization of steroid glycosyltransferases. • Three glycosyltransferase genes involved in dioscin biosynthesis from yam were first cloned. • DoUGT80A2 and DoUGT80B1 catalyze diosgenin 3-O-glucosylation to form trillin. • DoUGT80A2 exhibited significantly higher catalytic activity than DoUGT80B1. • DoURT catalyzes the rhamnosylation of trillin to produce the bioactive dioscin. • Four sites Thr134, Tyr352, His505 and Ile253 were essential residues for recognizing ligands.