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Histidine 352 (His352) and tryptophan 355 (Trp355) are essential for flax UGT74S1 glucosylation activity toward secoisolariciresinol.

Ghose K, McCallum J, Sweeney-Nixon M, Fofana B - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, Prince Edward Island, C1A 4N6, Canada; University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, C1A 4P3, Canada.

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.

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Comparative metabolite profiles obtained from enzyme assays with the wild type and the mutant UGT74S1 proteins using SECO as acceptor and UDP-glucose as sugar donor.SDG and SMG lignan yield (mg/mL) were obtained from three independent replicates. Vertical bars are standard deviations of the means. Mutant proteins are indicated by their one-letter amino acid codes. W, Tryp; C, Cys; Q, Gln; A, Ala; S, Ser; H, His; D, Asp.
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pone-0116248-g003: Comparative metabolite profiles obtained from enzyme assays with the wild type and the mutant UGT74S1 proteins using SECO as acceptor and UDP-glucose as sugar donor.SDG and SMG lignan yield (mg/mL) were obtained from three independent replicates. Vertical bars are standard deviations of the means. Mutant proteins are indicated by their one-letter amino acid codes. W, Tryp; C, Cys; Q, Gln; A, Ala; S, Ser; H, His; D, Asp.

Mentions: To determine the biological effects of the predicted ligand binding site alterations induced by site-directed mutagenesis, enzyme assays were performed using the purified proteins from the wild type UGT74S1 and each of the 6 six mutant versions. A significant reduction of UGT74S1 glucosyltransferase activity towards SECO was observed in all mutants when compared to the wild type (Fig. 3). None of the UGT74S1 mutants or wild type were shown to glucosylate any of the other aglycone substrates tested, and only the substrate’s peaks were observed on chromatograms (data not shown). When Trp355 was substituted by either Ala or Gly, a complete abolition of activity was observed. No glucosylation activity was also observed when His352 was substituted by Asp352. Mutation of Cys335, Gln337 and Ser357 altered the lignan profiles in the reactions, with a significant reduction to level of SDG production, while still producing SMG intermediate. Mutant Cys335Ala produced a significantly (P<0.001) lower level of SDG compared to the wild type, but produced a significantly higher (P<0.001) amount of SMG. Mutants Gln337Ala and Ser357Ala produced only SMG which was significantly (P<0.001) less than that produced by the wild type. No SDG was produced by these two mutants (Fig. 3).


Histidine 352 (His352) and tryptophan 355 (Trp355) are essential for flax UGT74S1 glucosylation activity toward secoisolariciresinol.

Ghose K, McCallum J, Sweeney-Nixon M, Fofana B - PLoS ONE (2015)

Comparative metabolite profiles obtained from enzyme assays with the wild type and the mutant UGT74S1 proteins using SECO as acceptor and UDP-glucose as sugar donor.SDG and SMG lignan yield (mg/mL) were obtained from three independent replicates. Vertical bars are standard deviations of the means. Mutant proteins are indicated by their one-letter amino acid codes. W, Tryp; C, Cys; Q, Gln; A, Ala; S, Ser; H, His; D, Asp.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340967&req=5

pone-0116248-g003: Comparative metabolite profiles obtained from enzyme assays with the wild type and the mutant UGT74S1 proteins using SECO as acceptor and UDP-glucose as sugar donor.SDG and SMG lignan yield (mg/mL) were obtained from three independent replicates. Vertical bars are standard deviations of the means. Mutant proteins are indicated by their one-letter amino acid codes. W, Tryp; C, Cys; Q, Gln; A, Ala; S, Ser; H, His; D, Asp.
Mentions: To determine the biological effects of the predicted ligand binding site alterations induced by site-directed mutagenesis, enzyme assays were performed using the purified proteins from the wild type UGT74S1 and each of the 6 six mutant versions. A significant reduction of UGT74S1 glucosyltransferase activity towards SECO was observed in all mutants when compared to the wild type (Fig. 3). None of the UGT74S1 mutants or wild type were shown to glucosylate any of the other aglycone substrates tested, and only the substrate’s peaks were observed on chromatograms (data not shown). When Trp355 was substituted by either Ala or Gly, a complete abolition of activity was observed. No glucosylation activity was also observed when His352 was substituted by Asp352. Mutation of Cys335, Gln337 and Ser357 altered the lignan profiles in the reactions, with a significant reduction to level of SDG production, while still producing SMG intermediate. Mutant Cys335Ala produced a significantly (P<0.001) lower level of SDG compared to the wild type, but produced a significantly higher (P<0.001) amount of SMG. Mutants Gln337Ala and Ser357Ala produced only SMG which was significantly (P<0.001) less than that produced by the wild type. No SDG was produced by these two mutants (Fig. 3).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, Prince Edward Island, C1A 4N6, Canada; University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, C1A 4P3, Canada.

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.

Show MeSH
Related in: MedlinePlus