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Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the  Arabidopsis thaliana  UGT72B1 model. The ligand docking predicted Ser 357  and Trp 355  as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys 335 , Gln 337  and Trp 355  were predicted to bind the 7-OH, 2-OCH 3  and 17-OCH 3  of SECO. Site-directed mutagenesis of Cys 335 , Gln 337 , His 352 , Trp 355  and Ser 357   ,  and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp 355  was substituted to Ala 355  and Gly 355  or when changing His 352  to Asp 352   ,  and an altered metabolite profile was observed in  Cys335Ala ,  Gln337Ala , and  Ser357Ala  mutants. This study provided for the first time evidence that Trp 355  and His 352  are critical for UGT74S1’s glucosylation activity toward SECO and suggested the possibility for SMG production  in vitro .

Histidine 352 (His352) and Tryptophan 355 (Trp355) Are Essential for Flax UGT74S1 Glucosylation Activity toward Secoisolariciresinol