Evidence for a Sequential Glucosylation of Secoisolariciresinol (SECO) into Secoisolariciresinol Monoglucoside (SMG) and Diglucoside (SDG) Lignans by the Flax Lu-UGT74S1 (Lu-UGTCL809) UDP-Glycosyltransferase

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural glycosylated diphenolic phytoestrogen. 1-uridine diphosphate glycosyltransferases (UGTs) catalyze sugar residue transfer from an activated donor to an acceptor for the synthesis of glycosylated metabolites like SDG. To date, not much is known about the flax SDG glycosylation enzymes. We identified and characterized six full length flax UGTs referred to as Lu-UGTCL809, Lu-UGTCL5227, Lu-UGTCL8584, Lu-UGTRP131, Lu-UGTRP250, and Lu-UGTRP033, classified into six UGT sub-families. All UGTs displayed the characteristic plant secondary product glycosyltransferase (PSPG) motif. Five of the UGTs were exclusively expressed in seed and the mRNA transcript expression of two revealed a clear correlation with SDG lignan biosynthesis in developing seed. The seed-specific UGTs were expressed in yeast and the proteins reacted with secoisolariciresinol (SECO) in presence of UDP-glucose. Only Lu-UGTCL809 used SECO as substrate, forming first secoisolariciresinol monoglucoside (SMG) and then SDG from the SMG intermediate. 3D structural modeling and ligand docking identified the amino acids within the PSPG motif that bind the phosphate and hydroxyl groups of UDP-glucose for both Lu-UGTCL809 (SER357 and Trp355) and Lu-UGTRP250 (Gln351, Ala352 and Gly261). Furthermore, whereas Lu-UGTCL809 binds to SECO by three hydrogen bonds between Cys335 and Gln337 and 4-OH and 3-OCH3 of SECO, Lu-UGTRP250 binds to SECO by a single hydrogen bond between Ala352 and the 9’hydroxyl group of SECO. Binding of Lu-UGTRP250 to the 9’-OH glycosylation site may explain the impaired SECO glucosylation by Lu-UGTRP250 for SMG synthesis. We report the first direct functional evidence for SECO glucosylation by a family 1 UGT.