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Lutz Heide - One of the best experts on this subject based on the ideXlab platform.

  • a membrane bound Prenyltransferase catalyzes the o prenylation of 1 6 dihydroxyphenazine in the marine bacterium streptomyces sp cnq 509
    ChemBioChem, 2014
    Co-Authors: Philipp Zeyhle, Judith S Bauer, Marco Steimle, Franziska Leipoldt, Manuela Rosch, Jorn Kalinowski, Harald Gross, Lutz Heide
    Abstract:

    Streptomyces sp. CNQ-509 produces the rare O-prenylated phenazines marinophenazines A and B. To identify the enzyme catalyzing the O-prenyl transfer in marinophenazine biosynthesis, we sequenced the genome of S. sp. CNQ-509. This led to the identification of two genomic loci harboring putative phenazine biosynthesis genes. The first locus contains orthologues for all seven genes involved in phenazine-1-carboxylic acid biosynthesis in pseudomonads. The second locus contains two known phenazine biosynthesis genes and a putative Prenyltransferase gene termed cnqPT1. cnqPT1 codes for a membrane protein with sequence similarity to the Prenyltransferase UbiA of ubiquinone biosynthesis. The enzyme CnqPT1 was identified as a 1,6-dihydroxyphenazine geranyltransferase, which catalyzes the CO bond formation between C-1 of the geranyl moiety and O-6 of the phenazine scaffold. CnqPT1 is the first example of a Prenyltransferase catalyzing O-prenyl transfer to a phenazine.

  • structure and mechanism of the magnesium independent aromatic Prenyltransferase cloq from the clorobiocin biosynthetic pathway
    Journal of Molecular Biology, 2010
    Co-Authors: Ute Metzger, Lutz Heide, Sascha Keller, Clare E M Stevenson, David M Lawson
    Abstract:

    Abstract CloQ is an aromatic Prenyltransferase from the clorobiocin biosynthetic pathway of Streptomyces roseochromogenes var. oscitans . It is involved in the synthesis of the prenylated hydroxybenzoate moiety of the antibiotic, specifically catalyzing the attachment of a dimethylallyl moiety to 4-hydroxyphenylpyruvate. Herein, we report the crystal structure of CloQ and use it as a framework for interpreting biochemical data from both wild-type and variant proteins. CloQ belongs to the aromatic Prenyltransferase family, which is characterized by an unusual core fold comprising five consecutive ααββ elements that form a central 10-stranded anti-parallel β-barrel. The latter delineates a solvent-accessible cavity where substrates bind and catalysis takes place. This cavity has well-defined polar and nonpolar regions, which have distinct roles in substrate binding and facilitate a Friedel–Crafts-type mechanism. We propose that the juxtaposition of five positively charged residues in the polar region circumvents the necessity for a Mg 2+ , which, by contrast, is a strict requirement for the majority of Prenyltransferases characterized to date. Our structure of CloQ complexed with 4-hydroxyphenylpyruvate reveals the formation of a covalent link between the substrate and Cys215 to yield a thiohemiketal species. Through site-directed mutagenesis, we show that this link is not essential for enzyme activity in vitro . Furthermore, we demonstrate that CloQ will accept alternative substrates and, therefore, has the capacity to generate a range of prenylated compounds. Since prenylation is thought to enhance the bioactivity of many natural products, CloQ offers considerable promise as a biocatalyst for the chemoenzymatic synthesis of novel compounds with therapeutic potential.

  • Prenyl transfer to aromatic substrates in the biosynthesis of aminocoumarins, meroterpenoids and phenazines: the ABBA Prenyltransferase family.
    Phytochemistry, 2009
    Co-Authors: Orwah Saleh, Yvonne Haagen, Kerstin Seeger, Lutz Heide
    Abstract:

    Aromatic Prenyltransferases transfer prenyl moieties onto aromatic acceptor molecules, catalyzing an electrophilic substitution of the aromatic ring under formation of carbon-carbon bonds. They give rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria. This review describes a recently discovered family of aromatic Prenyltransferases. The structure of these enyzmes shows a type of beta/alpha fold with antiparallel beta strands. Due to the alpha-beta-beta-alpha architecture of this fold, this group of enzymes was designated as ABBA Prenyltransferases. They lack the (N/D)DxxD motif which is characteristic for many other Prenyltransferases. At present, 14 genes with sequence similarity to ABBA Prenyltransferases can be identified in the database. A phylogenetic analysis of these genes separates them into two clades. One of them comprises the 4-hydroxyphenylpyruvate 3-dimethylallyltransferases CloQ and NovQ involved in aminocoumarin antibiotic biosynthesis in Streptomyces strains, as well as four genes of unknown function from fungal genomes. The other clade comprises genes involved in the biosynthesis of prenylated naphthoquinones and prenylated phenazines in different streptomycetes. ABBA Prenyltransferases are soluble biocatalysts which can easily be obtained as homogeneous proteins in significant amounts. Their substrates are accommodated in a surprisingly spacious central cavity which explains their promiscuity for different aromatic substrates. Therefore, the enzymes of this family represent attractive tools for the chemoenzymatic synthesis of bioactive molecules.

  • prenyl transfer to aromatic substrates genetics and enzymology
    Current Opinion in Chemical Biology, 2009
    Co-Authors: Lutz Heide
    Abstract:

    Aromatic Prenyltransferases catalyze the transfer of prenyl moieties to aromatic acceptor molecules and give rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria. Significant progress has been made in the biochemistry and genetics of this heterogeneous group of enzymes in the past years. After 30 years of extensive research on plant prenylflavonoid biosynthesis, finally the first aromatic Prenyltransferases involved in the formation of these compounds have been cloned. In bacteria, investigations of the newly discovered family of ABBA Prenyltransferases revealed a novel type of protein fold, the PT barrel. In fungi, a group of closely related indole Prenyltransferase was found to carry out aromatic prenylations with different substrate specificity and regiospecificity, and to catalyze both regular and reverse prenylations.

  • CloQ, a Prenyltransferase involved in clorobiocin biosynthesis
    Proceedings of the National Academy of Sciences of the United States of America, 2003
    Co-Authors: Florence Pojer, Emmanuel Wemakor, Bernd Kammerer, Huawei Chen, Christopher T. Walsh, Lutz Heide
    Abstract:

    Ring A (3-dimethylallyl-4-hydroxybenzoic acid) is a structural moiety of the aminocoumarin antibiotics novobiocin and clorobiocin. In the present study, the Prenyltransferase involved in the biosynthesis of this moiety was identified from the clorobiocin producer (Streptomyces roseochromogenes), overexpressed, and purified. It is a soluble, monomeric 35-kDa protein, encoded by the structural gene cloQ. 4-Hydroxyphenylpyruvate and dimethylallyl diphosphate were identified as the substrates of this enzyme, with Km values determined as 25 and 35 μM, respectively. A gene inactivation experiment confirmed that cloQ is essential for ring A biosynthesis. Database searches did not reveal any similarity of CloQ to known Prenyltransferases, and the enzyme did not contain the typical prenyl diphosphate binding site (N/D)DXXD. In contrast to most of the known Prenyltransferases, the enzymatic activity was not dependent on the presence of magnesium, and in contrast to the membrane-bound polyPrenyltransferases involved in ubiquinone biosynthesis, CloQ did not accept 4-hydroxybenzoic acid as substrate. CloQ and the similar NovQ from the novobiocin producer seem to belong to a new class of Prenyltransferases.

Karamat Fazeelat - One of the best experts on this subject based on the ideXlab platform.

  • Molecular evolution of parsnip (Pastinaca sativa) membrane-bound Prenyltransferases for linear and/or angular furanocoumarin biosynthesis
    'Wiley', 2016
    Co-Authors: Munakata Ryosuke, Olry Alexandre, Karamat Fazeelat, Courdavault Vincent, Sugiyama Akifumi, Date Yoshiaki, Krieger Celia, Silie Prisca, Foureau Emilien, Papon Nicolas
    Abstract:

    International audienceIn Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different Prenyltransferases, Pastinaca sativa Prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary Prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin

  • Identification et caractérisation fonctionnelle des deux premiers prényltransférases aromatiques impliqués dans la biosynthèse de furanocoumarines et des coumarines prénylés chez deux familles de plantes : Rutaceae et Apiaceae
    2013
    Co-Authors: Karamat Fazeelat
    Abstract:

    Les furocoumarines constituent l'une des classes de métabolites secondaires dérivant de la voie de biosynthèse des phénylpropanoïdes. Elles ont été décrites comme étant des phytoaléxines mais sont également très largement utilisées par l'Homme pour leurs propriétés thérapeutiques. Un certain nombre d'études biochimiques ont été réalisées afin d'en comprendre la biosynthèse mais peu de choses sont connues concernant leur déterminisme moléculaire. Dans cette étude, nous nous sommes concentrés sur la caractérisation fonctionnelle de gènes appartenant aux prényltransférases aromatiques potentiellement impliquées dans cette voie de biosynthèse. Les prényltransférases catalysent la première étape de la voie de biosynthèse des furocoumarines linéaires ou angulaires. Elles permettent l'addition d'un groupement dimethylally pyrophosphate (DMAPP) sur l'umbelliférone. En utilisant SfN8DT-1, une prényltransférase aromatique récemment caractérisée, comme sonde, nous avons identifié 7 gènes candidats chez deux familles de plantes (Rutaceae et Apiaceae). Dans la mesure où il a été décrit que ces enzymes étaient des protéines membranaires, nous avons adapté un système d'expression hétérologue basé sur l'utilisation de N. benthamiana. Ce système a été validé par l'expression de deux protéines membranaires : un cytochrome P450 CYP98A22 et une prényltransférase déjà caractérisée SfN8DT-1. Nous avons ensuite utilisé ce système d'expression pour réaliser l'étude des 7 gènes nouvellement isolés. Ces travaux nous ont permis de caractériser la première umbelliferone Prenyltransferase de Petroselinum crispum capable de catalyser in vitro et in vivo la prénylation du carbone 6 ou 8 de l'umbelliférone en présence de DMAPP permettant ainsi la synthèse respectivement de demethylsuberosin et d'osthenol. Par ailleurs, une étude réalisée in planta chez le persil a permis de mettre en évidence une relation positive entre le niveau d'expression du gène et la teneur en umbelliférone prénylée. L'étude de la surexpression du gène chez Ruta graveolens a permis de mettre en évidence un lien entre l'expression du gène et la disparition de l'umbelliférone. Enfin nous avons identifié la même activité pour une prényltransférase de Pastinaca sativa, ce qui nous amène à émettre l'hypothèse que l'étape de prénylation n'est pas une étape limitante dans la biosynthèse des furocoumarines angulaires, étant donné que le persil ne produit que des furocoumarines linéaires, tandis que le panais produit des furocoumarines linéaires et angulaires. L'utilisation de ces mêmes systèmes d'expression hétérologue de N. benthamiana et R. graveolens nous a également permis d'identifier une seconde prényltransférase aromatique capable de catalyser l'addition de géranylpyrophosphate (GPP) sur l'umbelliférone et sur l'esculétineFuranocoumarins constitute one of the classes of secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These molecules are described as phytoalexins in plants but are also used by humans for their pharmaceutical properties. A large number of biochemical studies were carried out to understand their biosynthetic pathway but little information was available concerning the genes involved in the pathway. In this study, we focused on the characterization of genes encoding for aromatic Prenyltransferases which were described to be involved in this pathway. Prenlyltransferases catalyze the entry step to the linear or angular furanocoumarin pathway. Hence they catalyze the addition of a dimethylallyl pyrophosphate (DMAPP) prenyl moiety to umbelliferone. Using a recently characterized aromatic Prenyltransferase (SfN8DT-1) as a probe, we isolated 7 different candidate genes from two plant families (Rutaceae and Apiaceae). As these enzymes were described as membrane bound proteins, we adapted a heterologous expression system made up of Nicotiana benthamian aand we validated its efficiency by using two membrane-associated enzymes: a cytochrome P450 (CYP98A22) and the already described Prenyltransferase SfN8DT-1. Subsequently, this system was used to perform the functional characterization of the 7 newly identified proteins. This way we succeeded to characterize the first umbelliferone Prenyltransferase of Petroselinum crispum that was able to catalyze both the 6-C and 8-C prenylation of umbelliferone with DMAPP producing demethylsuberosin and osthenol respectively. We made evidence that these reactions occurred both in vitro and in vivo. In addition, in planta studies performed in P.crispum showed a positive relationship between the gene expression level and the content of prenylated umbelliferone. The overexpression of this gene was investigated in Ruta graveolens and we could provide evidences of a link between the enzymatic activity and the disappearance of umbelliferone. We also reported a similar activity for a Prenyltransferase isolated from Pastinaca sativa, which makes us assume that the prenylation step is not a rate limiting step in the biosynthetic pathway of angular furanocoumarins since parsley is producing only linear furanocoumarins whereas parsnip is producing both linear and angular furanocoumarins. In addition, using the same N. benthamiana and R. graveolens heterologous expression systems, we identified a second aromatic Prenyltransferase able to catalyze the addition of geranyl pyrophosphate (GPP) both to umbelliferone and esculeti

  • Identification et caractérisation fonctionnelle des deux premiers prényltransférases aromatiques impliqués dans la biosynthèse de furanocoumarines et des coumarines prénylés chez deux familles de plantes : Rutaceae et Apiaceae
    HAL CCSD, 2013
    Co-Authors: Karamat Fazeelat
    Abstract:

    Furanocoumarins constitute one of the classes of secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These molecules are described as phytoalexins in plants but are also used by humans for their pharmaceutical properties. A large number of biochemical studies were carried out to understand their biosynthetic pathway but little information was available concerning the genes involved in the pathway. In this study, we focused on the characterization of genes encoding for aromatic Prenyltransferases which were described to be involved in this pathway. Prenlyltransferases catalyze the entry step to the linear or angular furanocoumarin pathway. Hence they catalyze the addition of a dimethylallyl pyrophosphate (DMAPP) prenyl moiety to umbelliferone. Using a recently characterized aromatic Prenyltransferase (SfN8DT-1) as a probe, we isolated 7 different candidate genes from two plant families (Rutaceae and Apiaceae). As these enzymes were described as membrane bound proteins, we adapted a heterologous expression system made up of Nicotiana benthamian aand we validated its efficiency by using two membrane-associated enzymes: a cytochrome P450 (CYP98A22) and the already described Prenyltransferase SfN8DT-1. Subsequently, this system was used to perform the functional characterization of the 7 newly identified proteins. This way we succeeded to characterize the first umbelliferone Prenyltransferase of Petroselinum crispum that was able to catalyze both the 6-C and 8-C prenylation of umbelliferone with DMAPP producing demethylsuberosin and osthenol respectively. We made evidence that these reactions occurred both in vitro and in vivo. In addition, in planta studies performed in P.crispum showed a positive relationship between the gene expression level and the content of prenylated umbelliferone. The overexpression of this gene was investigated in Ruta graveolens and we could provide evidences of a link between the enzymatic activity and the disappearance of umbelliferone. We also reported a similar activity for a Prenyltransferase isolated from Pastinaca sativa, which makes us assume that the prenylation step is not a rate limiting step in the biosynthetic pathway of angular furanocoumarins since parsley is producing only linear furanocoumarins whereas parsnip is producing both linear and angular furanocoumarins. In addition, using the same N. benthamiana and R. graveolens heterologous expression systems, we identified a second aromatic Prenyltransferase able to catalyze the addition of geranyl pyrophosphate (GPP) both to umbelliferone and esculetinLes furocoumarines constituent l'une des classes de métabolites secondaires dérivant de la voie de biosynthèse des phénylpropanoïdes. Elles ont été décrites comme étant des phytoaléxines mais sont également très largement utilisées par l'Homme pour leurs propriétés thérapeutiques. Un certain nombre d'études biochimiques ont été réalisées afin d'en comprendre la biosynthèse mais peu de choses sont connues concernant leur déterminisme moléculaire. Dans cette étude, nous nous sommes concentrés sur la caractérisation fonctionnelle de gènes appartenant aux prényltransférases aromatiques potentiellement impliquées dans cette voie de biosynthèse. Les prényltransférases catalysent la première étape de la voie de biosynthèse des furocoumarines linéaires ou angulaires. Elles permettent l'addition d'un groupement dimethylally pyrophosphate (DMAPP) sur l'umbelliférone. En utilisant SfN8DT-1, une prényltransférase aromatique récemment caractérisée, comme sonde, nous avons identifié 7 gènes candidats chez deux familles de plantes (Rutaceae et Apiaceae). Dans la mesure où il a été décrit que ces enzymes étaient des protéines membranaires, nous avons adapté un système d'expression hétérologue basé sur l'utilisation de N. benthamiana. Ce système a été validé par l'expression de deux protéines membranaires : un cytochrome P450 CYP98A22 et une prényltransférase déjà caractérisée SfN8DT-1. Nous avons ensuite utilisé ce système d'expression pour réaliser l'étude des 7 gènes nouvellement isolés. Ces travaux nous ont permis de caractériser la première umbelliferone Prenyltransferase de Petroselinum crispum capable de catalyser in vitro et in vivo la prénylation du carbone 6 ou 8 de l'umbelliférone en présence de DMAPP permettant ainsi la synthèse respectivement de demethylsuberosin et d'osthenol. Par ailleurs, une étude réalisée in planta chez le persil a permis de mettre en évidence une relation positive entre le niveau d'expression du gène et la teneur en umbelliférone prénylée. L'étude de la surexpression du gène chez Ruta graveolens a permis de mettre en évidence un lien entre l'expression du gène et la disparition de l'umbelliférone. Enfin nous avons identifié la même activité pour une prényltransférase de Pastinaca sativa, ce qui nous amène à émettre l'hypothèse que l'étape de prénylation n'est pas une étape limitante dans la biosynthèse des furocoumarines angulaires, étant donné que le persil ne produit que des furocoumarines linéaires, tandis que le panais produit des furocoumarines linéaires et angulaires. L'utilisation de ces mêmes systèmes d'expression hétérologue de N. benthamiana et R. graveolens nous a également permis d'identifier une seconde prényltransférase aromatique capable de catalyser l'addition de géranylpyrophosphate (GPP) sur l'umbelliférone et sur l'esculétin

  • Identification et caractérisation fonctionnelle des deux premiers prényltransférases aromatiques impliqués dans la biosynthèse de furanocoumarines et des coumarines prénylés chez deux familles de plantes (Rutaceae et Apiaceae)
    2013
    Co-Authors: Karamat Fazeelat, Hehn Alain, Bourgaud Frédéric
    Abstract:

    Les furocoumarines constituent l'une des classes de métabolites secondaires dérivant de la voie de biosynthèse des phénylpropanoïdes. Elles ont été décrites comme étant des phytoaléxines mais sont également très largement utilisées par l'Homme pour leurs propriétés thérapeutiques. Un certain nombre d'études biochimiques ont été réalisées afin d'en comprendre la biosynthèse mais peu de choses sont connues concernant leur déterminisme moléculaire. Dans cette étude, nous nous sommes concentrés sur la caractérisation fonctionnelle de gènes appartenant aux prényltransférases aromatiques potentiellement impliquées dans cette voie de biosynthèse. Les prényltransférases catalysent la première étape de la voie de biosynthèse des furocoumarines linéaires ou angulaires. Elles permettent l'addition d'un groupement dimethylally pyrophosphate (DMAPP) sur l'umbelliférone. En utilisant SfN8DT-1, une prényltransférase aromatique récemment caractérisée, comme sonde, nous avons identifié 7 gènes candidats chez deux familles de plantes (Rutaceae et Apiaceae). Dans la mesure où il a été décrit que ces enzymes étaient des protéines membranaires, nous avons adapté un système d'expression hétérologue basé sur l'utilisation de N. benthamiana. Ce système a été validé par l'expression de deux protéines membranaires : un cytochrome P450 CYP98A22 et une prényltransférase déjà caractérisée SfN8DT-1. Nous avons ensuite utilisé ce système d'expression pour réaliser l'étude des 7 gènes nouvellement isolés. Ces travaux nous ont permis de caractériser la première umbelliferone Prenyltransferase de Petroselinum crispum capable de catalyser in vitro et in vivo la prénylation du carbone 6 ou 8 de l'umbelliférone en présence de DMAPP permettant ainsi la synthèse respectivement de demethylsuberosin et d'osthenol. Par ailleurs, une étude réalisée in planta chez le persil a permis de mettre en évidence une relation positive entre le niveau d'expression du gène et la teneur en umbelliférone prénylée. L'étude de la surexpression du gène chez Ruta graveolens a permis de mettre en évidence un lien entre l'expression du gène et la disparition de l'umbelliférone. Enfin nous avons identifié la même activité pour une prényltransférase de Pastinaca sativa, ce qui nous amène à émettre l'hypothèse que l'étape de prénylation n'est pas une étape limitante dans la biosynthèse des furocoumarines angulaires, étant donné que le persil ne produit que des furocoumarines linéaires, tandis que le panais produit des furocoumarines linéaires et angulaires. L'utilisation de ces mêmes systèmes d'expression hétérologue de N. benthamiana et R. graveolens nous a également permis d'identifier une seconde prényltransférase aromatique capable de catalyser l'addition de géranylpyrophosphate (GPP) sur l'umbelliférone et sur l'esculétineFuranocoumarins constitute one of the classes of secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These molecules are described as phytoalexins in plants but are also used by humans for their pharmaceutical properties. A large number of biochemical studies were carried out to understand their biosynthetic pathway but little information was available concerning the genes involved in the pathway. In this study, we focused on the characterization of genes encoding for aromatic Prenyltransferases which were described to be involved in this pathway. Prenlyltransferases catalyze the entry step to the linear or angular furanocoumarin pathway. Hence they catalyze the addition of a dimethylallyl pyrophosphate (DMAPP) prenyl moiety to umbelliferone. Using a recently characterized aromatic Prenyltransferase (SfN8DT-1) as a probe, we isolated 7 different candidate genes from two plant families (Rutaceae and Apiaceae). As these enzymes were described as membrane bound proteins, we adapted a heterologous expression system made up of Nicotiana benthamian aand we validated its efficiency by using two membrane-associated enzymes: a cytochrome P450 (CYP98A22) and the already described Prenyltransferase SfN8DT-1. Subsequently, this system was used to perform the functional characterization of the 7 newly identified proteins. This way we succeeded to characterize the first umbelliferone Prenyltransferase of Petroselinum crispum that was able to catalyze both the 6-C and 8-C prenylation of umbelliferone with DMAPP producing demethylsuberosin and osthenol respectively. We made evidence that these reactions occurred both in vitro and in vivo. In addition, in planta studies performed in P.crispum showed a positive relationship between the gene expression level and the content of prenylated umbelliferone. The overexpression of this gene was investigated in Ruta graveolens and we could provide evidences of a link between the enzymatic activity and the disappearance of umbelliferone. We also reported a similar activity for a Prenyltransferase isolated from Pastinaca sativa, which makes us assume that the prenylation step is not a rate limiting step in the biosynthetic pathway of angular furanocoumarins since parsley is producing only linear furanocoumarins whereas parsnip is producing both linear and angular furanocoumarins. In addition, using the same N. benthamiana and R. graveolens heterologous expression systems, we identified a second aromatic Prenyltransferase able to catalyze the addition of geranyl pyrophosphate (GPP) both to umbelliferone and esculetinMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Kazufumi Yazaki - One of the best experts on this subject based on the ideXlab platform.

  • homogeneous purification and characterization of lepgt1 a membrane bound aromatic substrate Prenyltransferase involved in secondary metabolism of lithospermum erythrorhizon
    FEBS Journal, 2013
    Co-Authors: Kazuaki Ohara, Koji Mito, Kazufumi Yazaki
    Abstract:

    Membrane-bound type Prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate Prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 μg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic Prenyltransferases as well as for crystallographic analysis of this novel enzyme family.

  • hlpt 1 a membrane bound Prenyltransferase responsible for the biosynthesis of bitter acids in hops
    Biochemical and Biophysical Research Communications, 2012
    Co-Authors: Yusuke Tsurumaru, Kanako Sasaki, Tatsuya Miyawaki, Takayuki Momma, Naoyuki Umemoto, Yoshihiro Uto, Masaki Momose, Kazufumi Yazaki
    Abstract:

    Female flowers of hop (Humulus lupulus L.) develop a large number of glandular trichomes called lupulin glands that contain a variety of prenylated compounds such as α- and β-acid (humulone and lupulone, respectively), as well as xanthohumol, a chalcone derivative. These prenylated compounds are biosynthesized by Prenyltransferases catalyzing the transfer of dimethylallyl moiety to aromatic substances. In our previous work, we found HlPT-1 a candidate gene for such a Prenyltransferase in a cDNA library constructed from lupulin-enriched flower tissues. In this study, we have characterized the enzymatic properties of HlPT-1 using a recombinant protein expressed in baculovirus-infected insect cells. HlPT-1 catalyzed the first transfer of dimethylallyl moiety to phloroglucinol derivatives, phlorisovalerophenone, phlorisobutyrophenone and phlormethylbutanophenone, leading to the formation of humulone and lupulone derivatives. HlPT-1 also recognized naringenin chalcone as a flavonoid substrate to yield xanthohumol, and this broad substrate specificity is a unique character of HlPT-1 that is not seen in other reported flavonoid Prenyltransferases, all of which show strict specificity for their aromatic substrates. Moreover, unlike other aromatic substrate Prenyltransferases, HlPT-1 revealed an exclusive requirement for Mg(2+) as a divalent cation for its enzymatic activity and also showed exceptionally narrow optimum pH at around pH 7.0.

  • molecular characterization of a membrane bound Prenyltransferase specific for isoflavone from sophora flavescens
    Journal of Biological Chemistry, 2011
    Co-Authors: Kanako Sasaki, Yusuke Tsurumaru, Hirobumi Yamamoto, Kazufumi Yazaki
    Abstract:

    Prenylated isoflavones are secondary metabolites that are mainly distributed in legume plants. They often possess divergent biological activities such as anti-bacterial, anti-fungal, and anti-oxidant activities and thus attract much attention in food, medicinal, and agricultural research fields. Prenyltransferase is the key enzyme in the biosynthesis of prenylated flavonoids by catalyzing a rate-limiting step, i.e. the coupling process of two major metabolic pathways, the isoprenoid pathway and shikimate/polyketide pathway. However, so far only two genes have been isolated as Prenyltransferases involved in the biosynthesis of prenylated flavonoids, namely naringenin 8-dimethylallyltransferase from Sophora flavescens (SfN8DT-1) specific for some limited flavanones and glycinol 4-dimethylallyltransferase from Glycine max (G4DT), specific for pterocarpan substrate. We have in this study isolated two novel genes coding for membrane-bound flavonoid Prenyltransferases from S. flavescens, an isoflavone-specific Prenyltransferase (SfG6DT) responsible for the prenylation of the genistein at the 6-position and a chalcone-specific Prenyltransferase designated as isoliquiritigenin dimethylallyltransferase (SfiLDT). These Prenyltransferases were enzymatically characterized using a yeast expression system. Analysis on the substrate specificity of chimeric enzymes between SfN8DT-1 and SfG6DT suggested that the determinant region for the specificity of the flavonoids was the domain neighboring the fifth transmembrane α-helix of the Prenyltransferases.

  • an aromatic Prenyltransferase like gene hlpt 1 preferentially expressed in lupulin glands of hop
    Plant Biotechnology, 2010
    Co-Authors: Yusuke Tsurumaru, Kanako Sasaki, Tatsuya Miyawaki, Takayuki Momma, Naoyuki Umemoto, Kazufumi Yazaki
    Abstract:

    Prenylated aromatic compounds represent the chemical components in the glandular trichomes (lupulin glands) of hops (Humulus lupulus, Cannabinaceae), which give the characteristic flavor and taste of beer. To isolate cDNAs for Prenyltransferase recognizing aromatic substrates in hops, we constructed a cDNA library from the lupulin glands and randomly sequenced 11,233 EST clones, to obtain 6,613 non-redundant EST sequence information. Among them, we found an aromatic Prenyltransferase-like gene (HlPT-1), which possessed three features of the plant aromatic Prenyltransferase family, i.e., a D-rich motif, membrane-spanning domains, and a transit peptide. The tissue-specific expression study of HlPT-1 in the intact plant revealed this gene to be highly expressed in hop corns (female flowers), especially in the lupulin glands. Subcellular localization analysis using GFP fusion proteins suggested that HlPT-1 was localized to plastids. Phylogenetic analysis predicted that the HlPT-1 gene evolved from homogentisate Prenyltransferases involved with vitamin E and plastoquinone biosynthesis.

  • prenylation of aromatic compounds a key diversification of plant secondary metabolites
    Phytochemistry, 2009
    Co-Authors: Kazufumi Yazaki, Kanako Sasaki, Yusuke Tsurumaru
    Abstract:

    Prenylation plays a major role in the diversification of aromatic natural products, such as phenylpropanoids, flavonoids, and coumarins. This biosynthetic reaction represents the crucial coupling process of the shikimate or polyketide pathway providing an aromatic moiety and the isoprenoid pathway derived from the mevalonate or methyl erythritol phosphate (MEP) pathway, which provides the prenyl (isoprenoid) chain. In particular, prenylation contributes strongly to the diversification of flavonoids, due to differences in the prenylation position on the aromatic rings, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g., cyclization and hydroxylation, resulting in the occurrence of ca. 1000 prenylated flavonoids in plants. Many prenylated flavonoids have been identified as active components in medicinal plants with biological activities, such as anti-cancer, anti-androgen, anti-leishmania, and anti-nitric oxide production. Due to their beneficial effects on human health, prenylated flavonoids are of particular interest as lead compounds for producing drugs and functional foods. However, the gene coding for Prenyltransferases that catalyze the key step of flavonoid prenylation have remained unidentified for more than three decades, because of the membrane-bound nature of these enzymes. Recently, we have succeeded in identifying the first Prenyltransferase gene SfN8DT-1 from Sophora flavescens, which is responsible for the prenylation of the flavonoid naringenin at the 8-position, and is specific for flavanones and dimethylallyl diphosphate (DMAPP) as substrates. Phylogenetic analysis showed that SfN8DT-1 has the same evolutionary origin as Prenyltransferases for vitamin E and plastoquinone. A Prenyltransferase GmG4DT from soybean, which is involved in the formation of glyceollin, was also identified recently. This enzyme was specific for pterocarpan as its aromatic substrate, and (-)-glycinol was the native substrate yielding the direct precursor of glyceollin I. These enzymes are localized to plastids and the prenyl chain is derived from the MEP pathway. Further relevant genes involved in the prenylation of other types of polyphenol are expected to be cloned by utilizing the sequence information provided by the above studies.

Célia Krieger - One of the best experts on this subject based on the ideXlab platform.

  • molecular evolution of parsnip pastinaca sativa membrane bound Prenyltransferases for linear and or angular furanocoumarin biosynthesis
    New Phytologist, 2016
    Co-Authors: Ryosuke Munakata, Célia Krieger, Alexandre Olry, Fazeelat Karamat, Vincent Courdavault, Akifumi Sugiyama, Yoshiaki Date
    Abstract:

    In Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different Prenyltransferases, Pastinaca sativa Prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary Prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin.

Bradley S Moore - One of the best experts on this subject based on the ideXlab platform.

  • algal neurotoxin biosynthesis repurposes the terpene cyclase structural fold into an n Prenyltransferase
    Proceedings of the National Academy of Sciences of the United States of America, 2020
    Co-Authors: Jonathan R Chekan, Shaun M K Mckinnie, Joseph P Noel, Bradley S Moore
    Abstract:

    Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of Prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the Prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.

  • algal neurotoxin biosynthesis repurposes the terpene cyclase structural fold into an n Prenyltransferase
    bioRxiv, 2020
    Co-Authors: Jonathan R Chekan, Shaun M K Mckinnie, Joseph P Noel, Bradley S Moore
    Abstract:

    Prenylation is a common biological reaction in all domains of life whereupon prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these biotransformations are structurally distinct from ubiquitous terpene cyclases that instead assemble terpene molecules via intramolecular rearrangements. Herein we report the structure and molecular details of a new family of Prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the Nprenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the Prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical Mg2+-binding motif. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to alter their isoprene donor specificities (geranyl versus dimethylallyl diphosphate) by a single amino acid switch. While the diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor.

  • functional characterization of the cyclomarin cyclomarazine Prenyltransferase cymd directs the biosynthesis of unnatural cyclic peptides
    Journal of Natural Products, 2010
    Co-Authors: Andrew W Schultz, Chad A Lewis, Michael R Luzung, Phil S Baran, Bradley S Moore
    Abstract:

    In vitro and in vivo characterization of the cyclomarin/cyclomarazine Prenyltransferase CymD revealed its ability to prenylate tryptophan prior to incorporation into both cyclic peptides by the nonribosomal peptide synthetase CymA. This knowledge was utilized to bioengineer novel derivatives of these marine bacterial natural products by providing synthetic N-alkyl tryptophans to a Prenyltransferase-deficient mutant of Salinispora arenicola CNS-205.