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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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Related in: MedlinePlus

Western blot of HisTag-purified proteins from the wild type and six mutant UGT74S1 protein variants probed with antiXpress antibody.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. M, Western C precision plus protein marker mixed with conjugant (BioRad).
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pone-0116248-g002: Western blot of HisTag-purified proteins from the wild type and six mutant UGT74S1 protein variants probed with antiXpress antibody.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. M, Western C precision plus protein marker mixed with conjugant (BioRad).

Mentions: To assess the expression and functionality of the different mutant proteins, the full length cDNAs for the wild type UGT74S1 and that of each of the 6 UGT74S1 mutants were expressed in vitro in yeast. Similar to the wild type UGT74S1, all six mutants produced, along with the Histidine-Tag, a discrete protein band of 56.4 kDa (Fig. 2).


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)

Western blot of HisTag-purified proteins from the wild type and six mutant UGT74S1 protein variants probed with antiXpress antibody.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. M, Western C precision plus protein marker mixed with conjugant (BioRad).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0116248-g002: Western blot of HisTag-purified proteins from the wild type and six mutant UGT74S1 protein variants probed with antiXpress antibody.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. M, Western C precision plus protein marker mixed with conjugant (BioRad).
Mentions: To assess the expression and functionality of the different mutant proteins, the full length cDNAs for the wild type UGT74S1 and that of each of the 6 UGT74S1 mutants were expressed in vitro in yeast. Similar to the wild type UGT74S1, all six mutants produced, along with the Histidine-Tag, a discrete protein band of 56.4 kDa (Fig. 2).

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