Histidine 352 Are Essential for Flax UGT74S1 Glucosylation Activity toward Secoisolariciresinol

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.sup.357 and Trp.sup.355 as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys.sup.335, Gln.sup.337 and Trp.sup.355 were predicted to bind the 7-OH, 2-OCH.sub.3 and 17-OCH.sub.3 of SECO. Site-directed mutagenesis of Cys.sup.335, Gln.sup.337, His.sup.352, Trp.sup.355 and Ser.sup.357 .sub., 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.sup.355 was substituted to Ala.sup.355 and Gly.sup.355 or when changing His.sup.352 to Asp.sup.352 .sub., and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp.sup.355 and His.sup.352 are critical for UGT74S1's glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.