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Eric Laine - One of the best experts on this subject based on the ideXlab platform.
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characterization of luwrky36 a flax transcription factor promoting Secoisolariciresinol biosynthesis in response to fusarium oxysporum elicitors in linum usitatissimum l hairy roots
Planta, 2019Co-Authors: Lucija Markulin, Cyrielle Corbin, Christophe Hano, Stéphane Drouet, Sullivan Renouard, Tatiana Lopez, Charlene Durpoix, Charlotte Mathieu, Daniel Auguin, Eric LaineAbstract:MAIN CONCLUSION: The involvement of a WRKY transcription factor in the regulation of lignan biosynthesis in flax using a hairy root system is described. Secoisolariciresinol is the main flax lignan synthesized by action of LuPLR1 (pinoresinol–lariciresinol reductase 1). LuPLR1 gene promoter deletion experiments have revealed a promoter region containing W boxes potentially responsible for the response to Fusarium oxysporum. W boxes are bound by WRKY transcription factors that play a role in the response to stress. A candidate WRKY transcription factor, LuWRKY36, was isolated from both abscisic acid and Fusarium elicitor-treated flax cell cDNA libraries. This transcription factors contains two WRKY DNA-binding domains and is a homolog of AtWRKY33. Different approaches confirmed LuWRKY36 binding to a W box located in the LuPLR1 promoter occurring through a unique direct interaction mediated by its N-terminal WRKY domain. Our results propose that the positive regulator action of LuWRKY36 on the LuPLR1 gene regulation and lignan biosynthesis in response to biotic stress is positively mediated by abscisic acid and inhibited by ethylene. Additionally, we demonstrate a differential Fusarium elicitor response in susceptible and resistant flax cultivars, seen as a faster and stronger LuPLR1 gene expression response accompanied with higher Secoisolariciresinol accumulation in HR of the resistant cultivar.
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Pinoresinol–lariciresinol reductases, key to the lignan synthesis in plants
Planta, 2019Co-Authors: Lucija Markulin, Cyrielle Corbin, Christophe Hano, Stéphane Drouet, Sullivan Renouard, Daniel Auguin, Ivan Mateljak, Laurent Gutierrez, Elisabeth Fuss, Eric LaineAbstract:Main conclusion This paper provides an overview on activity, stereospecificity, expression and regulation of pinoresinol–lariciresinol reductases in plants. These enzymes are shared by the pathways to all 8–8′ lignans derived from pinoresinol. Pinoresinol–lariciresinol reductases (PLR) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols. They catalyze two successive reduction steps leading to the production of lariciresinol or Secoisolariciresinol from pinoresinol. Two Secoisolariciresinol enantiomers can be synthetized with different fates. Depending on the plant species, these enantiomers are either final products (e.g., in the flaxseed where it is stored after glycosylation) or are the starting point for the synthesis of a wide range of lignans, among which the aryltetralin type lignans are used to semisynthesize anticancer drugs such as Etoposide^®. Thus, the regulation of the gene expression of PLRs as well as the possible specificities of these reductases for one reduction step or one enantiomer are key factors to fine-tune the lignan synthesis. Results published in the last decade have shed light on the presence of more than one PLR in each plant and revealed various modes of action. Nevertheless, there are not many results published on the PLRs and most of them were obtained in a limited range of species. Indeed, a number of them deal with wild and cultivated flax belonging to the genus Linum . Despite the occurrence of lignans in bryophytes, pteridophytes and monocots, data on PLRs in these taxa are still missing and indeed the whole diversity of PLRs is still unknown. This review summarizes the data, published mainly in the last decade, on the PLR gene expression, enzymatic activity and biological function.
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The lignan (+)-Secoisolariciresinol extracted from flax hulls is an effective protectant of linseed oil and its emulsion against oxidative damage
European Journal of Lipid Science and Technology, 2017Co-Authors: Christophe Hano, Cyrielle Corbin, Anthony Quéro, Roland Molinié, François Mesnard, Raphaëlle Savoire, Natacha Rombaut, Brigitte Thomasset, Stéphane Drouet, Eric LaineAbstract:© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Secoisolariciresinol (SECO) is a natural antioxidant lignan accumulated in large amounts in the seedcoat of flax and retained in the flaxseed cake residue during linseed oil pressing. Here SECO was extracted and purified from flaxseed cake and assayed for its ability to prevent oxidation of linseed oil and an o/w emulsion containing linseed oil. For this purpose, an accelerated storage (Schaal oven) test was performed and SECO effectiveness was compared to that of two antioxidants commonly used in food and cosmetic products: α-tocopherol (α-TOCO) and butylated hydroxyanisole (BHA). In our hands, SECO addition, ranging from 50 to 500 μmole per kg oil, significantly decreased the production of both primary (conjugated dienes, CD) and secondary (thiobarbituric acid-reactive substances, TBARS) oxidation products. This study evidenced that SECO is an effective stabilizer of linseed oil and its o/w emulsion and this protective effect outperformed both the natural α-TOCO and the synthetic BHA antioxidants. In particular, SECO was the most effective in the protection of the o/w emulsion against secondary oxidation products, which makes it a potential alternative preservative for oily products in foods and cosmetics. Practical applications: The present study could lead to applications in the food and cosmetic industries for the stabilization of o/w emulsions. Addition of SECO to linseed oil, already rich in ω-3, could also result in a new functional food with synergistic beneficial actions on human health. Secoisolariciresinol, a natural antioxidant lignan accumulated in large amounts in the seedcoat of flax is here presented as an effective protectant of both bulk oil and o/w emulsion against secondary oxidation products, which makes it a potential alternative preservative for oily products in foods and cosmetics.
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flaxseed linum usitatissimum l extract as well as Secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α amylase activity
Bioorganic & Medicinal Chemistry Letters, 2013Co-Authors: Christophe Hano, Cyrielle Corbin, Roland Molinié, Frédéric Lamblin, Sullivan Renouard, Esmatullah Barakzoy, Joel Doussot, Eric LaineAbstract:Type 2 diabetes mellitus (T2DM) is one of the common global diseases. Flaxseed is by far the richest source of the dietary lignans (i.e., Secoisolariciresinol diglucoside) which have been shown to delay the development of T2DM in animal models. Herein, we propose the first evidences for a mechanism of action involving the inhibition of the pancreatic α-amylase (EC 3.2.1.1) by flaxseed-derived lignans that could therefore constitute a promising nutraceutical for the prevention and the treatment of T2DM.
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cellulase assisted release of Secoisolariciresinol from extracts of flax linum usitatissimum hulls and whole seeds
Food Chemistry, 2010Co-Authors: Sullivan Renouard, Cyrielle Corbin, Christophe Hano, Frédéric Lamblin, O. Fliniaux, François Mesnard, Tatiana Lopez, Josiane Montguillon, Esmatullah Barakzoy, Eric LaineAbstract:Abstract Extraction of Secoisolariciresinol from seed hulls and whole seeds of flax was improved using an enzymatic step with cellulase R10 from Trichoderma reesei that allowed better yield as compared to β-glucosidase. The cellulase assisted extraction process was further optimised for different parameters such as duration and concentration of hydromethanolic extraction, duration of alkaline hydrolysis, pH, duration and incubation temperature as well as enzyme concentration. Best results were obtained using a method including the following successive steps: 16 h of 70% hydromethanolic extraction, 6 h of 0.1 M sodium hydroxide hydrolysis followed by a 6 h incubation with 1 unit ml −1 of cellulase R10 in 0.1 M citrate–phosphate buffer pH 2.8 at 40 °C. Under these conditions, all forms of the main flax lignan were recovered as the aglycone form, i.e. Secoisolariciresinol. Highest yields in Secoisolariciresinol diglucoside (SDG) equivalent reached 7.72% of flaxseed hull (cv. Baladin) dry weight and 2.88% of whole seed (cv. Barbara) weight, thus allowing a significant improvement in comparison with published methods.
Christophe Hano - One of the best experts on this subject based on the ideXlab platform.
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characterization of luwrky36 a flax transcription factor promoting Secoisolariciresinol biosynthesis in response to fusarium oxysporum elicitors in linum usitatissimum l hairy roots
Planta, 2019Co-Authors: Lucija Markulin, Cyrielle Corbin, Christophe Hano, Stéphane Drouet, Sullivan Renouard, Tatiana Lopez, Charlene Durpoix, Charlotte Mathieu, Daniel Auguin, Eric LaineAbstract:MAIN CONCLUSION: The involvement of a WRKY transcription factor in the regulation of lignan biosynthesis in flax using a hairy root system is described. Secoisolariciresinol is the main flax lignan synthesized by action of LuPLR1 (pinoresinol–lariciresinol reductase 1). LuPLR1 gene promoter deletion experiments have revealed a promoter region containing W boxes potentially responsible for the response to Fusarium oxysporum. W boxes are bound by WRKY transcription factors that play a role in the response to stress. A candidate WRKY transcription factor, LuWRKY36, was isolated from both abscisic acid and Fusarium elicitor-treated flax cell cDNA libraries. This transcription factors contains two WRKY DNA-binding domains and is a homolog of AtWRKY33. Different approaches confirmed LuWRKY36 binding to a W box located in the LuPLR1 promoter occurring through a unique direct interaction mediated by its N-terminal WRKY domain. Our results propose that the positive regulator action of LuWRKY36 on the LuPLR1 gene regulation and lignan biosynthesis in response to biotic stress is positively mediated by abscisic acid and inhibited by ethylene. Additionally, we demonstrate a differential Fusarium elicitor response in susceptible and resistant flax cultivars, seen as a faster and stronger LuPLR1 gene expression response accompanied with higher Secoisolariciresinol accumulation in HR of the resistant cultivar.
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Pinoresinol–lariciresinol reductases, key to the lignan synthesis in plants
Planta, 2019Co-Authors: Lucija Markulin, Cyrielle Corbin, Christophe Hano, Stéphane Drouet, Sullivan Renouard, Daniel Auguin, Ivan Mateljak, Laurent Gutierrez, Elisabeth Fuss, Eric LaineAbstract:Main conclusion This paper provides an overview on activity, stereospecificity, expression and regulation of pinoresinol–lariciresinol reductases in plants. These enzymes are shared by the pathways to all 8–8′ lignans derived from pinoresinol. Pinoresinol–lariciresinol reductases (PLR) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols. They catalyze two successive reduction steps leading to the production of lariciresinol or Secoisolariciresinol from pinoresinol. Two Secoisolariciresinol enantiomers can be synthetized with different fates. Depending on the plant species, these enantiomers are either final products (e.g., in the flaxseed where it is stored after glycosylation) or are the starting point for the synthesis of a wide range of lignans, among which the aryltetralin type lignans are used to semisynthesize anticancer drugs such as Etoposide^®. Thus, the regulation of the gene expression of PLRs as well as the possible specificities of these reductases for one reduction step or one enantiomer are key factors to fine-tune the lignan synthesis. Results published in the last decade have shed light on the presence of more than one PLR in each plant and revealed various modes of action. Nevertheless, there are not many results published on the PLRs and most of them were obtained in a limited range of species. Indeed, a number of them deal with wild and cultivated flax belonging to the genus Linum . Despite the occurrence of lignans in bryophytes, pteridophytes and monocots, data on PLRs in these taxa are still missing and indeed the whole diversity of PLRs is still unknown. This review summarizes the data, published mainly in the last decade, on the PLR gene expression, enzymatic activity and biological function.
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The lignan (+)-Secoisolariciresinol extracted from flax hulls is an effective protectant of linseed oil and its emulsion against oxidative damage
European Journal of Lipid Science and Technology, 2017Co-Authors: Christophe Hano, Cyrielle Corbin, Anthony Quéro, Roland Molinié, François Mesnard, Raphaëlle Savoire, Natacha Rombaut, Brigitte Thomasset, Stéphane Drouet, Eric LaineAbstract:© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Secoisolariciresinol (SECO) is a natural antioxidant lignan accumulated in large amounts in the seedcoat of flax and retained in the flaxseed cake residue during linseed oil pressing. Here SECO was extracted and purified from flaxseed cake and assayed for its ability to prevent oxidation of linseed oil and an o/w emulsion containing linseed oil. For this purpose, an accelerated storage (Schaal oven) test was performed and SECO effectiveness was compared to that of two antioxidants commonly used in food and cosmetic products: α-tocopherol (α-TOCO) and butylated hydroxyanisole (BHA). In our hands, SECO addition, ranging from 50 to 500 μmole per kg oil, significantly decreased the production of both primary (conjugated dienes, CD) and secondary (thiobarbituric acid-reactive substances, TBARS) oxidation products. This study evidenced that SECO is an effective stabilizer of linseed oil and its o/w emulsion and this protective effect outperformed both the natural α-TOCO and the synthetic BHA antioxidants. In particular, SECO was the most effective in the protection of the o/w emulsion against secondary oxidation products, which makes it a potential alternative preservative for oily products in foods and cosmetics. Practical applications: The present study could lead to applications in the food and cosmetic industries for the stabilization of o/w emulsions. Addition of SECO to linseed oil, already rich in ω-3, could also result in a new functional food with synergistic beneficial actions on human health. Secoisolariciresinol, a natural antioxidant lignan accumulated in large amounts in the seedcoat of flax is here presented as an effective protectant of both bulk oil and o/w emulsion against secondary oxidation products, which makes it a potential alternative preservative for oily products in foods and cosmetics.
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flaxseed linum usitatissimum l extract as well as Secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α amylase activity
Bioorganic & Medicinal Chemistry Letters, 2013Co-Authors: Christophe Hano, Cyrielle Corbin, Roland Molinié, Frédéric Lamblin, Sullivan Renouard, Esmatullah Barakzoy, Joel Doussot, Eric LaineAbstract:Type 2 diabetes mellitus (T2DM) is one of the common global diseases. Flaxseed is by far the richest source of the dietary lignans (i.e., Secoisolariciresinol diglucoside) which have been shown to delay the development of T2DM in animal models. Herein, we propose the first evidences for a mechanism of action involving the inhibition of the pancreatic α-amylase (EC 3.2.1.1) by flaxseed-derived lignans that could therefore constitute a promising nutraceutical for the prevention and the treatment of T2DM.
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concentration kinetics of Secoisolariciresinol diglucoside and its biosynthetic precursor coniferin in developing flaxseed
Phytochemical Analysis, 2013Co-Authors: Jingjing Fang, Christophe Hano, O. Fliniaux, Sullivan Renouard, Aina Ramsay, Christian Paetz, Evangelos C Tatsis, Eric Grand, Albrecht Roscher, François MesnardAbstract:ABSTRACT:Introduction – In the plant kingdom, flaxseed (Linum usitatissimum L.) is the richest source of Secoisolariciresinol diglucoside(SDG), which is of great interest because of its potential health benefits for human beings. The information about the kineticsof SDG formation during flaxseed development is rare and incomplete.Objective – In this study, a reversed-phase high-performance liquid chromatography–diode array detection (HPLC-DAD)method was developed to quantify SDG and coniferin, a key biosynthetic precursor of SDG in flaxseed.Methodology – Seeds from different developmental stages, which were scaled by days after flowering (DAF), were harvested.After alkaline hydrolysis, the validated HPLC method was applied to determine SDG and coniferin concentrations of flaxseedfrom different developing stages.Results – Coniferin was found in the entire capsule as soon asflowering started and became undetectable 20 DAF. SDGwas detected 6 DAF, and the concentration increased until maturity. On the other hand, the SDG amount in a singleflaxseed approached the maximum around 25 DAF, before desiccation started. Concentration increase between 25DAF and 35 DAF can be attributed to corresponding seed weight decrease.Conclusion – The biosynthesis of coniferin is not synchronous with that of SDG. Hence, the concentrations of SDG and con-iferin change during flaxseed development. Copyright © 2012 John Wiley & Sons, Ltd.Keywords: HPLC-DAD; coniferin; flaxseed; Secoisolariciresinol diglucoside; Linum usitatissimum
Norman G Lewis - One of the best experts on this subject based on the ideXlab platform.
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Secoisolariciresinol dehydrogenase mode of catalysis and stereospecificity of hydride transfer in podophyllum peltatum
Organic and Biomolecular Chemistry, 2006Co-Authors: Syed G A Moinuddin, Laurence B Davin, Michael A Costa, Buhyun Youn, Diana L Bedgar, Gregory L Helms, Chulhee Kang, Norman G LewisAbstract:Secoisolariciresinol dehydrogenase (SDH) catalyzes the NAD+ dependent enantiospecific conversion of Secoisolariciresinol into matairesinol. In Podophyllum species, (−)-matairesinol is metabolized into the antiviral compound, podophyllotoxin, which can be semi-synthetically converted into the anticancer agents, etoposide, teniposide and Etopophos®. Matairesinol is also a precursor of the cancer-preventative “mammalian” lignan, enterolactone, formed in the gut following ingestion of, for example, various high fiber dietary foods, as well as being an intermediate to numerous defense compounds in vascular plants. This study investigated the mode of enantiospecific Podophyllum SDH catalysis, the order of binding, and the stereospecificity of hydride abstraction/transfer from Secoisolariciresinol to NAD+. SDH contains a highly conserved catalytic triad (Ser153, Tyr167 and Lys171), whose activity was abolished with site-directed mutagenesis of Tyr167Ala and Lys171Ala, whereas mutagenesis of Ser153Ala only resulted in a much reduced catalytic activity. Isothermal titration calorimetry measurements indicated that NAD+ binds first followed by the substrate, (−)-Secoisolariciresinol. Additionally, for hydride transfer, the incoming hydride abstracted from the substrate takes up the pro-S position in the NADH formed. Taken together, a catalytic mechanism for the overall enantiospecific conversion of (−)-Secoisolariciresinol into (−)-matairesinol is proposed.
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crystal structures of apo form and binary ternary complexes of podophyllum Secoisolariciresinol dehydrogenase an enzyme involved in formation of health protecting and plant defense lignans
Journal of Biological Chemistry, 2005Co-Authors: Buhyun Youn, Laurence B Davin, Norman G Lewis, Syed G A Moinuddin, Chulhee KangAbstract:Abstract (-)-Matairesinol is a central biosynthetic intermediate to numerous 8–8′-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos®. It is formed by action of an enantiospecific Secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-Secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative “mammalian” lignan or “phytoestrogen” enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, Secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum Secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 A resolution, respectively. The enzyme is a homotetramer, consisting of an α/β single domain monomer containing seven parallel β-strands flanked by eight α-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD+ and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of Secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.
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Crystal structures of apo-form and binary/ternary complexes of Podophyllum Secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans
Journal of Biological Chemistry, 2005Co-Authors: Buhyun Youn, Laurence B Davin, Norman G Lewis, Syed G A Moinuddin, Chulhee KangAbstract:Abstract (-)-Matairesinol is a central biosynthetic intermediate to numerous 8–8′-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos®. It is formed by action of an enantiospecific Secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-Secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative “mammalian” lignan or “phytoestrogen” enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, Secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum Secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 A resolution, respectively. The enzyme is a homotetramer, consisting of an α/β single domain monomer containing seven parallel β-strands flanked by eight α-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD+ and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of Secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.
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An historical perspective on lignan biosynthesis: Monolignol, allylphenol and hydroxycinnamic acid coupling and downstream metabolism
Phytochemistry Reviews, 2003Co-Authors: Laurence B Davin, Norman G LewisAbstract:This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-pinoresinol. In a species dependent manner, (+)- or (−)-pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then Secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific Secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords Secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [^13C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling. Abbreviations: CD – circular dichroism; e.e. – enantiomeric excess; DP – dirigent protein; ESI-MS – electrospray ionization mass spectrometry; MALDI -TOF – matrix assisted laser desorption ionization-time of flight; MALLS – multiangle laser light scattering; PLR – pinoresinol lariciresinol reductase; SDH – Secoisolariciresinol dehydrogenase.
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Secoisolariciresinol dehydrogenase purification cloning and functional expression implications for human health protection
Journal of Biological Chemistry, 2001Co-Authors: Michael A Costa, Laurence B Davin, Helene C Pelissier, Norman G LewisAbstract:Abstract Matairesinol is a central precursor in planta in the biosynthesis of numerous lignans, including that of the important antiviral and anticancer agent, podophyllotoxin. In this study, the ∼32-kDa NAD-dependent Secoisolariciresinol dehydrogenase, which catalyzes the enantiospecific conversion of (−)-Secoisolariciresinol into (−)-matairesinol in Forsythia intermedia, was purified >6,000-fold to apparent homogeneity. The 831-base pair cDNA clone encoding this 277-amino acid protein was next obtained from a library constructed from F. intermedia stem tissue, whose fully functional recombinant protein, produced by expression of this cDNA in Escherichia coli, catalyzed the same enantiospecific conversion via the corresponding lactol intermediate. A homologous Secoisolariciresinol dehydrogenase gene was also isolated from a Podophyllum peltatum rhizome cDNA library, whose 834-base pair cDNA clone encoded a 278-amino acid protein with a calculated molecular mass of ∼32 kDa. Expression of this protein in E. coli produced a fully functional recombinant protein that also catalyzed the enantiospecific conversion of (−)-Secoisolariciresinol into (−)-matairesinol via the intermediary lactol. Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting. With this overall enzymatic conversion now unambiguously defined, the entire biochemical pathway to the lignans, Secoisolariciresinol and matairesinol, has been elucidated. Last, both Secoisolariciresinol and matairesinol are metabolized in the gut of mammals, following digestion of high fiber dietary grains, seeds, and berries, into the so-called “mammalian” lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the onset of breast and prostate cancers.
Laurence B Davin - One of the best experts on this subject based on the ideXlab platform.
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Secoisolariciresinol dehydrogenase mode of catalysis and stereospecificity of hydride transfer in podophyllum peltatum
Organic and Biomolecular Chemistry, 2006Co-Authors: Syed G A Moinuddin, Laurence B Davin, Michael A Costa, Buhyun Youn, Diana L Bedgar, Gregory L Helms, Chulhee Kang, Norman G LewisAbstract:Secoisolariciresinol dehydrogenase (SDH) catalyzes the NAD+ dependent enantiospecific conversion of Secoisolariciresinol into matairesinol. In Podophyllum species, (−)-matairesinol is metabolized into the antiviral compound, podophyllotoxin, which can be semi-synthetically converted into the anticancer agents, etoposide, teniposide and Etopophos®. Matairesinol is also a precursor of the cancer-preventative “mammalian” lignan, enterolactone, formed in the gut following ingestion of, for example, various high fiber dietary foods, as well as being an intermediate to numerous defense compounds in vascular plants. This study investigated the mode of enantiospecific Podophyllum SDH catalysis, the order of binding, and the stereospecificity of hydride abstraction/transfer from Secoisolariciresinol to NAD+. SDH contains a highly conserved catalytic triad (Ser153, Tyr167 and Lys171), whose activity was abolished with site-directed mutagenesis of Tyr167Ala and Lys171Ala, whereas mutagenesis of Ser153Ala only resulted in a much reduced catalytic activity. Isothermal titration calorimetry measurements indicated that NAD+ binds first followed by the substrate, (−)-Secoisolariciresinol. Additionally, for hydride transfer, the incoming hydride abstracted from the substrate takes up the pro-S position in the NADH formed. Taken together, a catalytic mechanism for the overall enantiospecific conversion of (−)-Secoisolariciresinol into (−)-matairesinol is proposed.
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crystal structures of apo form and binary ternary complexes of podophyllum Secoisolariciresinol dehydrogenase an enzyme involved in formation of health protecting and plant defense lignans
Journal of Biological Chemistry, 2005Co-Authors: Buhyun Youn, Laurence B Davin, Norman G Lewis, Syed G A Moinuddin, Chulhee KangAbstract:Abstract (-)-Matairesinol is a central biosynthetic intermediate to numerous 8–8′-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos®. It is formed by action of an enantiospecific Secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-Secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative “mammalian” lignan or “phytoestrogen” enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, Secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum Secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 A resolution, respectively. The enzyme is a homotetramer, consisting of an α/β single domain monomer containing seven parallel β-strands flanked by eight α-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD+ and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of Secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.
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Crystal structures of apo-form and binary/ternary complexes of Podophyllum Secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans
Journal of Biological Chemistry, 2005Co-Authors: Buhyun Youn, Laurence B Davin, Norman G Lewis, Syed G A Moinuddin, Chulhee KangAbstract:Abstract (-)-Matairesinol is a central biosynthetic intermediate to numerous 8–8′-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos®. It is formed by action of an enantiospecific Secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-Secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative “mammalian” lignan or “phytoestrogen” enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, Secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum Secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 A resolution, respectively. The enzyme is a homotetramer, consisting of an α/β single domain monomer containing seven parallel β-strands flanked by eight α-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD+ and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of Secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.
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An historical perspective on lignan biosynthesis: Monolignol, allylphenol and hydroxycinnamic acid coupling and downstream metabolism
Phytochemistry Reviews, 2003Co-Authors: Laurence B Davin, Norman G LewisAbstract:This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-pinoresinol. In a species dependent manner, (+)- or (−)-pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then Secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific Secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords Secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [^13C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling. Abbreviations: CD – circular dichroism; e.e. – enantiomeric excess; DP – dirigent protein; ESI-MS – electrospray ionization mass spectrometry; MALDI -TOF – matrix assisted laser desorption ionization-time of flight; MALLS – multiangle laser light scattering; PLR – pinoresinol lariciresinol reductase; SDH – Secoisolariciresinol dehydrogenase.
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Secoisolariciresinol dehydrogenase purification cloning and functional expression implications for human health protection
Journal of Biological Chemistry, 2001Co-Authors: Michael A Costa, Laurence B Davin, Helene C Pelissier, Norman G LewisAbstract:Abstract Matairesinol is a central precursor in planta in the biosynthesis of numerous lignans, including that of the important antiviral and anticancer agent, podophyllotoxin. In this study, the ∼32-kDa NAD-dependent Secoisolariciresinol dehydrogenase, which catalyzes the enantiospecific conversion of (−)-Secoisolariciresinol into (−)-matairesinol in Forsythia intermedia, was purified >6,000-fold to apparent homogeneity. The 831-base pair cDNA clone encoding this 277-amino acid protein was next obtained from a library constructed from F. intermedia stem tissue, whose fully functional recombinant protein, produced by expression of this cDNA in Escherichia coli, catalyzed the same enantiospecific conversion via the corresponding lactol intermediate. A homologous Secoisolariciresinol dehydrogenase gene was also isolated from a Podophyllum peltatum rhizome cDNA library, whose 834-base pair cDNA clone encoded a 278-amino acid protein with a calculated molecular mass of ∼32 kDa. Expression of this protein in E. coli produced a fully functional recombinant protein that also catalyzed the enantiospecific conversion of (−)-Secoisolariciresinol into (−)-matairesinol via the intermediary lactol. Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting. With this overall enzymatic conversion now unambiguously defined, the entire biochemical pathway to the lignans, Secoisolariciresinol and matairesinol, has been elucidated. Last, both Secoisolariciresinol and matairesinol are metabolized in the gut of mammals, following digestion of high fiber dietary grains, seeds, and berries, into the so-called “mammalian” lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the onset of breast and prostate cancers.
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Functional characterization of the pinoresinol–lariciresinol reductase-2 gene reveals its roles in yatein biosynthesis and flax defense response
Planta, 2017Co-Authors: Cyrielle Corbin, Frédéric Lamblin, Stéphane Drouet, Sullivan Renouard, Tatiana Lopez, Lucija Markulin, Joel Doussot, Ivan Mateljak, Cédric Decourtil, Daniel AuguinAbstract:Main conclusion This study provides new insights into the biosynthesis regulation and in planta function of the lignan yatein in flax leaves. Pinoresinol–lariciresinol reductases (PLR) catalyze the conversion of pinoresinol into Secoisolariciresinol (SECO) in lignan biosynthesis. Several lignans are accumulated in high concentrations, such as SECO accumulated as Secoisolariciresinol diglucoside (SDG) in seeds and yatein in aerial parts, in the flax plant ( Linum usitatissimum L.) from which two PLR enzymes of opposite enantioselectivity have been isolated. While LuPLR1 catalyzes the biosynthesis of (+)-SECO leading to (+)-SDG in seeds, the role(s) of the second PLR (LuPLR2) is not completely elucidated. This study provides new insights into the in planta regulation and function of the lignan yatein in flax leaves: its biosynthesis relies on a different PLR with opposite stereospecificity but also on a distinct expression regulation. RNAi technology provided evidence for the in vivo involvement of the LuPLR2 gene in the biosynthesis of (−)-yatein accumulated in flax leaves. LuPLR2 expression in different tissues and in response to stress was studied by RT-qPCR and promoter-reporter transgenesis showing that the spatio-temporal expression of the LuPLR2 gene in leaves perfectly matches the (−)-yatein accumulation and that LuPLR2 expression and yatein production are increased by methyl jasmonate and wounding. A promoter deletion approach yielded putative regulatory elements. This expression pattern in relation to a possible role for this lignan in flax defense is discussed.
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flaxseed linum usitatissimum l extract as well as Secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α amylase activity
Bioorganic & Medicinal Chemistry Letters, 2013Co-Authors: Christophe Hano, Cyrielle Corbin, Roland Molinié, Frédéric Lamblin, Sullivan Renouard, Esmatullah Barakzoy, Joel Doussot, Eric LaineAbstract:Type 2 diabetes mellitus (T2DM) is one of the common global diseases. Flaxseed is by far the richest source of the dietary lignans (i.e., Secoisolariciresinol diglucoside) which have been shown to delay the development of T2DM in animal models. Herein, we propose the first evidences for a mechanism of action involving the inhibition of the pancreatic α-amylase (EC 3.2.1.1) by flaxseed-derived lignans that could therefore constitute a promising nutraceutical for the prevention and the treatment of T2DM.
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cellulase assisted release of Secoisolariciresinol from extracts of flax linum usitatissimum hulls and whole seeds
Food Chemistry, 2010Co-Authors: Sullivan Renouard, Cyrielle Corbin, Christophe Hano, Frédéric Lamblin, O. Fliniaux, François Mesnard, Tatiana Lopez, Josiane Montguillon, Esmatullah Barakzoy, Eric LaineAbstract:Abstract Extraction of Secoisolariciresinol from seed hulls and whole seeds of flax was improved using an enzymatic step with cellulase R10 from Trichoderma reesei that allowed better yield as compared to β-glucosidase. The cellulase assisted extraction process was further optimised for different parameters such as duration and concentration of hydromethanolic extraction, duration of alkaline hydrolysis, pH, duration and incubation temperature as well as enzyme concentration. Best results were obtained using a method including the following successive steps: 16 h of 70% hydromethanolic extraction, 6 h of 0.1 M sodium hydroxide hydrolysis followed by a 6 h incubation with 1 unit ml −1 of cellulase R10 in 0.1 M citrate–phosphate buffer pH 2.8 at 40 °C. Under these conditions, all forms of the main flax lignan were recovered as the aglycone form, i.e. Secoisolariciresinol. Highest yields in Secoisolariciresinol diglucoside (SDG) equivalent reached 7.72% of flaxseed hull (cv. Baladin) dry weight and 2.88% of whole seed (cv. Barbara) weight, thus allowing a significant improvement in comparison with published methods.
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Pinoresinol–lariciresinol reductase gene expression and Secoisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds
Planta, 2006Co-Authors: Christophe Hano, Frédéric Lamblin, O. Fliniaux, B. Legrand, R. R. J. Arroo, François Mesnard, L. Gutiérrez, I. Martin, Eric LaineAbstract:The transcription activity of the pinoresinol–lariciresinol reductase (PLR) gene of Linum usitatissimum (so-called LuPLR), a key gene in lignan synthesis, was studied by RT-PCR and promoter–reporter transgenesis. The promoter was found to drive transcription of a GUSint reporter gene in the seed coats during the flax seed development. This fitted well with the tissue localization monitored by semi-quantitative RT-PCR of LuPLR expression. Accumulation of the main flax lignan Secoisolariciresinol diglucoside was coherent with LuPLR expression during seed development. This three-way approach demonstrated that the LuPLR gene is expressed in the seed coat of flax seeds, and that the synthesis of PLR enzyme occurs where flax main lignan is found stored in mature seeds, confirming its involvement in SDG synthesis.
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pinoresinol lariciresinol reductase gene expression and Secoisolariciresinol diglucoside accumulation in developing flax linum usitatissimum seeds
Planta, 2006Co-Authors: Christophe Hano, Frédéric Lamblin, O. Fliniaux, B. Legrand, R. R. J. Arroo, François Mesnard, I. Martin, Laurent Gutierrez, Eric LaineAbstract:The transcription activity of the pinoresinol–lariciresinol reductase (PLR) gene of Linum usitatissimum (so-called LuPLR), a key gene in lignan synthesis, was studied by RT-PCR and promoter–reporter transgenesis. The promoter was found to drive transcription of a GUSint reporter gene in the seed coats during the flax seed development. This fitted well with the tissue localization monitored by semi-quantitative RT-PCR of LuPLR expression. Accumulation of the main flax lignan Secoisolariciresinol diglucoside was coherent with LuPLR expression during seed development. This three-way approach demonstrated that the LuPLR gene is expressed in the seed coat of flax seeds, and that the synthesis of PLR enzyme occurs where flax main lignan is found stored in mature seeds, confirming its involvement in SDG synthesis.
