Abstract
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 Ser357 and Trp355 as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys335, Gln337 and Trp355 were predicted to bind the 7-OH, 2-OCH3 and 17-OCH3 of SECO. Site-directed mutagenesis of Cys335, Gln337, His352, Trp355 and Ser357 , 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 Trp355 was substituted to Ala355 and Gly355 or when changing His352 to Asp352 , and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp355 and His352 are critical for UGT74S1’s glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.
Highlights
Lignans are a class of diphenolic nonsteroidal phytoestrogens with a wide variety of purported health benefits [1,2,3,4,5,6,7,8]
Using 3D structural modeling of UGT74S1 protein, ligand docking, site-directed mutagenesis, heterologous expression and enzyme assays, we showed that glutamine at position 337 (Gln337) and serine at position 357 (Ser357) are essential for SECO monoglucoside (SMG) conversion to secoisolariciresinol diglucoside (SDG) and that tryptophan at position 355 (Trp355) and His352 are key critical amino acids within the plant secondary product glycosyltransferase (PSPG) motif and are determinant for UGT74S1 glucosylation activity toward SECO in vitro
The first bond occurred between the serine at position 357 (Ser357) and the oxygen atom 8 located on the phosphate group 2 within the UDP-glucose moiety, forming a 2.207 Ahydrogen bond (Table 1; Fig. 1)
Summary
Lignans are a class of diphenolic nonsteroidal phytoestrogens with a wide variety of purported health benefits [1,2,3,4,5,6,7,8]. Different types of lignans have been reported in various plant species and include secoisolariciresinol diglucoside (SDG) encountered mainly in flax (Linum usitatissimum L.) seed [9,10,11,12,13,14]. Flax lignans are usually found glycosylated in oligomeric chains [15]; its aglycone (SECO, MW 5362.4 g/mol) and monoglucoside (SMG) forms not being naturally accumulated in planta. During et al [16] reported a linearly increased uptake of lignan aglycone forms (pinolariciresinol – PINO, SECO, and enterolactone – ENL) by human intestinal Caco-2 cells through simple diffusion or by low affinity transporter. The functional characterization of the five UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and SDG in a sequential manner [29]. Its strict substrate specificity is unknown and its regiospecificity cannot be ruled out
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