Identification and functional characterization of UDP-glycosyltransferases catalyzing the glycosylation of oleoside 11-methyl ester in Olea europaea

Main conclusion Two UDP-glycosyltransferases from Olea europaea were identified and characterized. Both catalyze the glycosylation of oleoside 11-methyl ester to form 7-β-1-D-glucopyranosyl 11-methyl oleoside, a putative intermediate in the oleuropein biosynthetic pathway. Introduction Oleuropein, a secoiridoid glycoside abundant in Oleaceae plants, is extensively utilized in the pharmaceutical, nutraceutical, and cosmetic industries due to its diverse pharmacological activities. The biosynthetic pathway from oleoside 11-methyl ester (OME) to oleuropein remains incompletely elucidated. In this study, two UDP-glycosyltransferases (UGTs), OeUGT1and OeUGT2, were identified in O. europaea. First, based on differential expression analysis and the characteristics of enzymes catalyzing carboxyl glycosylation, 51 differentially expressed UGTs were narrowed to 6 candidates. Both in vivo and in vitro enzyme activity assays demonstrated that OeUGT1 could catalyze OME to 7-β-1-D-glucopyranosyl 11-methyl oleoside (OME-Glc). The enzymatic properties of OeUGT1 were then characterized. The optimal reaction temperature was found to be 30 °C, and the optimal pH was 6.0. OeUGT1 exhibited a Michaelis constant ( K m ) of 1.69 ± 0.37 mM and a catalytic efficiency ( k cat / K m ) of 3.57 ± 0.84 mM⁻ 1  s⁻ 1 . OeUGT1 showed strict substrate selectivity, acting on only sinapinic acid out of other nine carboxyl-containing substrates tested. A local BLAST search of the O. europaea transcriptome using the OeUGT1 amino acid sequence was conducted and its isoenzyme was identified, OeUGT2. OeUGT2 exhibits 87.3% sequence identity to OeUGT1. Phylogenetic analysis placed OeUGT1 (designated as UGT75L98 by the UGT Nomenclature Committee) and OeUGT2 (designated UGT75L99) within the UGT75 family, a clade known to catalyze the glycosylation of carboxyl groups. OeUGT1 had a specific activity of 0.2719 μmol min −1  mg −1 , approximately 2.78-fold higher than OeUGT2 (0.0977 μmol min −1  mg −1 ), indicating that OeUGT1 is the major enzyme responsible for OME glycosylation in olive. This work advances our understanding of oleuropein biosynthesis.