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Joachim Stockigt - One of the best experts on this subject based on the ideXlab platform.
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the structure of Rauvolfia serpentina strictosidine synthase is a novel six bladed β propeller fold in plant proteins
The Plant Cell, 2006Co-Authors: Santosh Panjikar, Elke A Loris, Juergen Koepke, Joachim StockigtAbstract:The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ∼2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler–type reaction and represents a novel six-bladed β-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed β-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.
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functional expression of an ajmaline pathway specific esterase from Rauvolfia in a novel plant virus expression system
Planta, 2005Co-Authors: Martin Ruppert, Jorn Woll, Anatoli Giritch, Ezzat Genady, Joachim StockigtAbstract:Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.
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vomilenine reductase a novel enzyme catalyzing a crucial step in the biosynthesis of the therapeutically applied antiarrhythmic alkaloid ajmaline
Bioorganic & Medicinal Chemistry, 2002Co-Authors: Gerald Von Schumann, Joachim StockigtAbstract:Abstract Delineation of the biochemical pathway leading to the antiarrhythmic Rauvolfia alkaloid ajmaline has been an important target in biosynthetic research for many years. The biosynthetic sequence starting with tryptamine and the monoterpene secologanin consists of about 10 different steps. Most of the participating enzymes have been detected and characterized previously, except those catalyzing the reduction of the intermediate vomilenine. A novel NADPH-dependent enzyme that reduces the intermediate has been isolated from Rauvolfia serpentina cell suspension cultures. Vomilenine reductase (M r 43 kDa, temp opt 30 °C, pH opt 5.7–6.2), saturates the indolenine double bond of vomilenine with stereospecific formation of 2β( R )-1,2-dihydrovomilenine. The described detection, enrichment and properties of the reductase not only closes a gap in ajmaline biosynthesis but is also a prerequisite for overexpressing the protein heterologously for final clarification of its molecular properties.
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arbutin synthase a novel member of the nrd1β glycosyltransferase family is a unique multifunctional enzyme converting various natural products and xenobiotics
Bioorganic & Medicinal Chemistry, 2002Co-Authors: Tobias Hefner, Heribert Warzecha, Joachim Arend, Karsten Siems, Joachim StockigtAbstract:Plant glucosyltransferases (GTs) play a crucial role in natural product biosynthesis and metabolization of xenobiotics. We expressed the arbutin synthase (AS) cDNA from Rauvolfia serpentina cell suspension cultures in Escherichia coli with a 6 x His tag and purified the active enzyme to homogeneity. The recombinant enzyme had a temperature optimum of 50 degrees C and showed two different pH optima (4.5 and 6.8 or 7.5, depending on the buffer). Out of 74 natural and synthetic phenols and two cinnamyl alcohols tested as substrates for the AS, 45 were accepted, covering a broad range of structural features. Converting rates comparable to hydroquinone were not achieved. In contrast to this broad acceptor substrate specificity, only pyrimidine nucleotide activated glucose was tolerated as a donor substrate. Nucleotide and amino acid sequence analysis revealed AS to be a new member of the NRD1beta family of glycosyl transferases and placed the enzyme into the group of plant secondary product GTs. Arbutin synthase is therefore the first example of a broad spectrum multifunctional glucosyltransferase.
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isolation and structure elucidation of a new indole alkaloid from Rauvolfia serpentina hairy root culture the first naturally occurring alkaloid of the raumacline group
Planta Medica, 2002Co-Authors: Y. V. Sheludko, Irina Gerasimenko, Heinz Kolshorn, Joachim StockigtAbstract:A new monoterpenoid indole alkaloid, 10-hydroxy- N(alpha)-demethyl-19,20-dehydroraumacline ( 1), was isolated as a mixture of E- and Z-isomers from hairy root culture of Rauvolfia serpentina Benth. ex Kurz (Apocynaceae) and the structure was determined by 1D and 2D NMR analyses. The new indole alkaloid represents the first naturally occurring alkaloid of the raumacline group and its putative biosynthetical pathway is discussed.
Heribert Warzecha - One of the best experts on this subject based on the ideXlab platform.
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ligand structures of synthetic deoxa pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding and inhibitory behaviours
Journal of Enzyme Inhibition and Medicinal Chemistry, 2015Co-Authors: Liqun Xia, Meitian Wang, Haili Lin, Chitra Rajendran, Agata Staniek, Petra Hilgers, Jorg Williardt, Heribert WarzechaAbstract:Insight into the structure and inhibition mechanism of O-β-d-glucosidases by deoxa-pyranosylamine type inhibitors is provided by X-ray analysis of complexes between raucaffricine and strictosidine glucosidases and N-(cyclohexylmethyl)-, N-(cyclohexyl)- and N-(bromobenzyl)-β-d-gluco-1,5-deoxa-pyranosylamine. All inhibitors anchored exclusively in the catalytic active site by competition with appropriate enzyme substrates. Thus facilitated prospective elucidation of the binding networks with residues located at <3.9 A distance will enable the development of potent inhibitors suitable for the production of valuable alkaloid glucosides, raucaffricine and strictosidine, by means of synthesis in Rauvolfia serpentina cell suspension cultures.
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arbutin synthase a novel member of the nrd1β glycosyltransferase family is a unique multifunctional enzyme converting various natural products and xenobiotics
Bioorganic & Medicinal Chemistry, 2002Co-Authors: Tobias Hefner, Heribert Warzecha, Joachim Arend, Karsten Siems, Joachim StockigtAbstract:Plant glucosyltransferases (GTs) play a crucial role in natural product biosynthesis and metabolization of xenobiotics. We expressed the arbutin synthase (AS) cDNA from Rauvolfia serpentina cell suspension cultures in Escherichia coli with a 6 x His tag and purified the active enzyme to homogeneity. The recombinant enzyme had a temperature optimum of 50 degrees C and showed two different pH optima (4.5 and 6.8 or 7.5, depending on the buffer). Out of 74 natural and synthetic phenols and two cinnamyl alcohols tested as substrates for the AS, 45 were accepted, covering a broad range of structural features. Converting rates comparable to hydroquinone were not achieved. In contrast to this broad acceptor substrate specificity, only pyrimidine nucleotide activated glucose was tolerated as a donor substrate. Nucleotide and amino acid sequence analysis revealed AS to be a new member of the NRD1beta family of glycosyl transferases and placed the enzyme into the group of plant secondary product GTs. Arbutin synthase is therefore the first example of a broad spectrum multifunctional glucosyltransferase.
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the gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the alpha betahydrolase super family
FEBS Journal, 2000Co-Authors: Emine Dogru, Heribert Warzecha, Frank Seibel, Sophie Haebel, Friedrich Lottspeich, Joachim StockigtAbstract:The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family.
Emine Dogru - One of the best experts on this subject based on the ideXlab platform.
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the gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the alpha betahydrolase super family
FEBS Journal, 2000Co-Authors: Emine Dogru, Heribert Warzecha, Frank Seibel, Sophie Haebel, Friedrich Lottspeich, Joachim StockigtAbstract:The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family.
Brieanne Vaillancourt - One of the best experts on this subject based on the ideXlab platform.
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Phylogenetic relationships.
2012Co-Authors: Elsa Góngora-castillo, Kevin L. Childs, Greg Fedewa, John P. Hamilton, David K. Liscombe, Maria Magallanes-lundback, Kranthi K. Mandadi, Ezekiel Nims, Weerawat Runguphan, Brieanne VaillancourtAbstract:Camptotheca acuminata (Nyssaceae) is in the order Cornales within the asterid superorder of core eudicots, and Catharanthus roseus and Rauvolfia serpentina (both Apocynaceae) are in Gentainales, also within the asterids. Arabidopsis thaliana is in the family Brassicales within the rosid superorder. Fabales and Astrales are shown for orientation. Redrawn and greatly simplified from APG III [19].
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Summary of statistics of the transcriptome de novo assemblies of Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina.
2012Co-Authors: Elsa Góngora-castillo, Kevin L. Childs, Greg Fedewa, John P. Hamilton, David K. Liscombe, Maria Magallanes-lundback, Kranthi K. Mandadi, Ezekiel Nims, Weerawat Runguphan, Brieanne VaillancourtAbstract:Summary of statistics of the transcriptome de novo assemblies of Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina.
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Functional annotation results.
2012Co-Authors: Elsa Góngora-castillo, Kevin L. Childs, Greg Fedewa, John P. Hamilton, David K. Liscombe, Maria Magallanes-lundback, Kranthi K. Mandadi, Ezekiel Nims, Weerawat Runguphan, Brieanne VaillancourtAbstract:Proportion of Camptotheca acuminata, Catharanthus roseus and Rauvolfia serpentina transcripts with sequence similarity to the UniRef100 database [17], Arabidopsis thaliana proteome (http://arabidopsis.org), and Pfam domain database [18].
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Cluster of orthologous and paralogous genes families in Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina species as identified by OrthoMCL.
2012Co-Authors: Elsa Góngora-castillo, Kevin L. Childs, Greg Fedewa, John P. Hamilton, David K. Liscombe, Maria Magallanes-lundback, Kranthi K. Mandadi, Ezekiel Nims, Weerawat Runguphan, Brieanne VaillancourtAbstract:Predicted peptides from the Camptotheca acuminata, Catharanthus roseus and Rauvolfia serpentina transcriptomes were clustered using OrthoMCL [23]. A) Number of clusters (c) and genes (g) for each orthologous group. B) Number of genes in the different clusters for each species. The number of clusters and genes for each OrthoMCL group are shown. Group 1: Clusters (blue) and genes shared among C. acuminata (red), C. roseus (green) and R. serpentina (purple). Group 2: Clusters (blue) and genes shared among C. acuminata (red) and C. roseus (green). Group 3: Clusters (blue) and genes shared among C. roseus (green) and R. serpentina (purple). Group 4: Clusters (blue) and genes shared among C. acuminata (red) and R. serpentina (purple).
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Expression patterns of known genes in monoterpene indole alkaloid biosynthesis across different tissues and treatments.
2012Co-Authors: Elsa Góngora-castillo, Kevin L. Childs, Greg Fedewa, John P. Hamilton, David K. Liscombe, Maria Magallanes-lundback, Kranthi K. Mandadi, Ezekiel Nims, Weerawat Runguphan, Brieanne VaillancourtAbstract:Expression values in log2 FPKM (fragments per Kilobase of transcript per million fragments mapped) were calculated, negative values were set to zero and then were clustered using R [39]. A) Catharanthus roseus: Expression values were sorted in three major groups: Developmental tissues, Yeast extract (YE) treatment of suspension cells (SC), and Methyl jasmonate (MJ) treatment of sterile seedlings (SS) and hairy roots (HR). B) Rauvolfia serpentina. Expression values shown represent the different developmental tissues.
Friedrich Lottspeich - One of the best experts on this subject based on the ideXlab platform.
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the gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the alpha betahydrolase super family
FEBS Journal, 2000Co-Authors: Emine Dogru, Heribert Warzecha, Frank Seibel, Sophie Haebel, Friedrich Lottspeich, Joachim StockigtAbstract:The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family.
