Structural insights into the catalytic mechanism of the phenylethanoid glycoside rhamnosyltransferase UGT79G15 from Rehmannia glutinosa.

Phenylethanoid glycosides (PhGs) are a group of important natural products found in a wide variety of medicinal plants, and are known to possess outstanding pharmacological properties. Uridine diphosphate (UDP) glycosyltransferase 79G15 (UGT79G15) from Rehmannia glutinosa‌ catalyzes the conversion of osmanthuside A to osmanthuside B, a key intermediate in the PhG biosynthetic pathway, via the formation of a (1→3) glycosidic bond. In this study, we report the crystal structure of UGT79G15 in its apo form, UDP-bound form and, most importantly, its ternary complex form containing UDP and a mimic acceptor, forsythiaside A, in its active site. Structural and comparative analyses revealed that UGT79G15 has a unique 'funnel-shaped' acceptor-binding pocket with a small accessory cave sufficient to accommodate the 4'-hydroxycinnamoyl group of PhG, explaining the enzyme's regiospecificity for the 3'-OH of PhG. Further structural analysis and site-directed mutagenesis explored a number of variants of the enzyme and identified key residues that recognize and stabilize UDP-rhamnose and the sugar acceptor. Meanwhile, I204W, a point variant obtained in the process, was found to possess increased catalytic efficiency for osmanthuside A conversion, up to 2.2 times the efficiency of the wild type. This study provides mechanistic insights into the donor specificity and acceptor regioselectivity of PhG 1,3-rhamnosyltransferase and enriches structural information on plant UGTs.