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Joachim Stockigt - One of the best experts on this subject based on the ideXlab platform.

  • Molecular Architecture of Strictosidine Glucosidase - the Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family
    The Plant cell, 2007
    Co-Authors: Leif Barleben, Martin Ruppert, Santosh Panjikar, Juergen A Koepke, Joachim Stockigt
    Abstract:

    Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of Monoterpenoid Indole Alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple Indole Alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids that occupy the active site surface of the enzyme. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-207, Glu-416, His-161, and Trp-388 in catalysis. Comparison of the catalytic pocket of SG with that of other plant glucosidases demonstrates the structural importance of Trp-388. Compared with all other glucosidases of plant, bacterial, and archaeal origin, SG9s residue Trp-388 is present in a unique structural conformation that is specific to the SG enzyme. In addition to STR1 and vinorine synthase, SG represents the third structural example of enzymes participating in the biosynthetic pathway of the Rauvolfia Alkaloid ajmaline. The data presented here will contribute to deciphering the structure and reaction mechanism of other higher plant glucosidases.

  • the structure of rauvolfia serpentina strictosidine synthase is a novel six bladed β propeller fold in plant proteins
    The Plant Cell, 2006
    Co-Authors: Santosh Panjikar, Elke A Loris, Juergen Koepke, Joachim Stockigt
    Abstract:

    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.

  • 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, 2002
    Co-Authors: Y. V. Sheludko, Irina Gerasimenko, Heinz Kolshorn, Joachim Stockigt
    Abstract:

    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.

  • 3 oxo rhazinilam a new Indole Alkaloid from rauvolfia serpentina rhazya stricta hybrid plant cell cultures
    Journal of Natural Products, 2001
    Co-Authors: Irina Gerasimenko, Y. V. Sheludko, Joachim Stockigt
    Abstract:

    A new Monoterpenoid Indole Alkaloid, 3-oxo-rhazinilam (1), was isolated from intergeneric somatic hybrid cell cultures of Rauvolfia serpentina and Rhazya stricta, and the structure was determined by detailed 1D and 2D NMR analysis. It was also proved that 3-oxo-rhazinilam (1) is a natural constituent of the hybrid cells.

  • the gene encoding polyneuridine aldehyde esterase of Monoterpenoid Indole Alkaloid biosynthesis in plants is an ortholog of the alpha betahydrolase super family
    FEBS Journal, 2000
    Co-Authors: Emine Dogru, Heribert Warzecha, Frank Seibel, Sophie Haebel, Friedrich Lottspeich, Joachim Stockigt
    Abstract:

    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.

Xiao-dong Luo - One of the best experts on this subject based on the ideXlab platform.

  • rapid and unambiguous assignment of proton deficient n acyl amidine Indole Alkaloid by a combination of calculation methods
    Tetrahedron Letters, 2021
    Co-Authors: Rongping Zhang, Xu-jie Qin, Cai-feng Ding, Xiaolong Shi, Guy Sedar Singor Njateng, Zhi Dai, Hongbin Zhang, Xiao-dong Luo
    Abstract:

    Abstract The unprecedently rearranged 6/5/8/6/5 pentacyclic Monoterpenoid Indole Alkaloid voafriamidine from Voacanga africana contains an uncommon N-acyl amidine structural motif that forms a unique 2,4-diazatricycle-[6,5,11,8,03,7] tetradecane skeleton. Despite the lack of proper NMR correlations for the relayed quaternary carbon and nitrogen, the complex structure was finally elucidated by the combination of ACD/Structure Elucidator, density functional theory (DFT) calculations of the NMR spectra and electronic circular dichroism (ECD) analyses.

  • melokhanines a j bioactive Monoterpenoid Indole Alkaloids with diverse skeletons from melodinus khasianus
    Journal of Natural Products, 2016
    Co-Authors: Gui-guang Cheng, Afsar Khan, Lu Liu, Ya-ping Liu, Xiao-dong Luo, Paul-keilah Lunga, Bo Hou, Ying-ying Chen, Zhili Zuo
    Abstract:

    The new melokhanines A–J (1–10) and 22 known (11–32) Alkaloids were isolated from the twigs and leaves of Melodinus khasianus. The new compounds and their absolute configurations were elucidated by extensive analysis of spectroscopic, X-ray diffraction, and computational data. Melokhanine A (1), composed of a hydroxyindolinone linked to an octahydrofuro[2,3-b]pyridine moiety, is an unprecedented Monoterpenoid Indole Alkaloid. Melokhanines B–H (2–8) possess a new 6/5/5/6/6 pentacyclic Indole Alkaloid skeleton. Alkaloids 1–16, 25–27, 31, and 32 showed the best antibacterial activity against Pseudomonas aeruginosa (MIC range 2–22 μM). Among the seven dermatophytes tested, compound 1 showed significant inhibitory activity against Microsporum canis, M. ferrugineum, and Trichophyton ajelloi (MIC range 38–150 μM), i.e., half the efficacy of the positive control, griseofulvin.

  • alstoscholarisines f and g two unusual Monoterpenoid Indole Alkaloids from the leaves of alstonia scholaris
    Tetrahedron Letters, 2015
    Co-Authors: Xingwei Yang, Afsar Khan, Ya-ping Liu, Yu Zhang, Changwei Song, Liping Jiang, Yongbin Chen, Xiao-dong Luo
    Abstract:

    Alstoscholarisine F (1), a Monoterpenoid Indole Alkaloid pigment with unprecedented carbon skeleton, and alstoscholarisine G (2) incorporated with a third nitrogen atom were isolated from the long-term stored leaves of Alstonia scholaris. Their structures were established by extensive MS and NMR spectroscopic analysis and the absolute configuration of 1 was defined by comparison of experimental and calculated ECDs. Compounds 1 and 2 were subjected to hippocampal neuronal stem cells (NSCs) proliferation evaluation, but they did not show significant effect. (C) 2015 Elsevier Ltd. All rights reserved.

  • A New Type of Monoterpenoid Indole Alkaloid Precursor from Alstonia rostrata
    Organic letters, 2011
    Co-Authors: Xiang-hai Cai, Ya-ping Liu, Mei-fen Bao, Yu Zhang, Chun-xia Zeng, Xiao-dong Luo
    Abstract:

    Currently, all Monoterpenoid Indole Alkaloids (MIAs) have been derived from strictosidine, which originates from the condensation of tryptophan with secologanin in a 1:1 ratio. However, our phytochemical research on Alstonia rostrata revealed a potential new precursor for these compounds. We isolated the alstrostines A and B, and it was determined that they were derived from tryptophan and secologanin in a 1:2 ratio, which supported the presence of a new type of MIA precursor.

  • Melotenine A, a Cytotoxic Monoterpenoid Indole Alkaloid from Melodinus tenuicaudatus
    Organic Letters, 2010
    Co-Authors: Tao Feng, Xiang-hai Cai, Ya-ping Liu, Yuan-yuan Wang, Xiao-dong Luo
    Abstract:

    Melotenine A (1), an unprecedented skeleton with a 6/5/5/6/7 pentacyclic rearranged ring system, was isolated from Melodinus tenuicaudatus. The structure was elucidated by means of spectroscopic methods and further confirmed by the single-crystal X-ray diffraction analysis. A possible biogenesis was also proposed. Melotenine A exhibited potential inhibition against five human cancer cell lines.

Vincenzo De Luca - One of the best experts on this subject based on the ideXlab platform.

  • the atp binding cassette transporter vmtpt2 vmabcg1 is involved in export of the Monoterpenoid Indole Alkaloid vincamine in vinca minor leaves
    Phytochemistry, 2017
    Co-Authors: Zerihun Demessie, Kathlyn N Woolfson, Vincenzo De Luca
    Abstract:

    Vinca minor is a herbaceous plant from the Apocynaceae family known to produce over 50 monoterpene Indole Alkaloids (MIAs). These include several biologically active MIAs that have a range of pharmaceutical activities. The present study shows that the MIAs, vincamine, akuammicine, minovincinine, lochnericine and vincadifformine tend to be secreted on V. minor leaf surfaces. A secretion mechanism of MIAs, previously described for Catharanthus roseus, appears to be mediated by a member (CrTPT2) of the pleiotropic drug resistance ABC transporter subfamily. The molecular cloning of an MIA transporter (VmTPT2/VmABCG1) that is predominantly expressed in V. minor leaves was functionally characterized in yeast and established it as an MIA efflux transporter. The similar function of VmTPT2/VmABCG1 to CrTPT2 increases the likelihood that this MIA transporter family may have co-evolved within members of Apocynaceae family to secrete selected MIAs and to regulate leaf MIA surface chemistry.

  • rauvolfia serpentina n methyltransferases involved in ajmaline and nβ methylajmaline biosynthesis belong to a gene family derived from γ tocopherol c methyltransferase
    Plant Journal, 2016
    Co-Authors: Paulo Cazaresflores, Dylan Levac, Vincenzo De Luca
    Abstract:

    Summary Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied Monoterpenoid Indole Alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in Indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline Alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ-methylajmaline, an unusual charged MIA found in R. serpentina.

  • discovery and metabolic engineering of iridoid secoiridoid and Monoterpenoid Indole Alkaloid biosynthesis
    Phytochemistry Reviews, 2016
    Co-Authors: Yang Qu, Antje M. K. Thamm, Vincenzo De Luca
    Abstract:

    There has been remarkable progress in the discovery of specialized metabolism pathways in the past few years. This has largely been due to the advent of inexpensive high throughput sequencing technologies, improved gene annotation methods and the development of tools for testing candidate genes for their involvement in particular biosynthetic pathways. This review describes the recent discoveries made on new steps in Monoterpenoid Indole Alkaloid (MIA) biosynthesis within Catharanthus roseus. The review also places these discoveries in context of the existing literature on regulation and production of MIAs in whole plants compared to cell and organ cultures.

  • Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Michael L. A. E. Easson, Jordan Froese, Razvan Simionescu, Tomas Hudlicky, Vincenzo De Luca
    Abstract:

    Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the Monoterpenoid Indole Alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine.

  • opium poppy and madagascar periwinkle model non model systems to investigate Alkaloid biosynthesis in plants
    Plant Journal, 2008
    Co-Authors: Peter J Facchini, Vincenzo De Luca
    Abstract:

    Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of Alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy (Papaver somniferum), which produces several benzylisoquinoline Alkaloids, and Madagascar periwinkle (Catharanthus roseus), which accumulates an array of Monoterpenoid Indole Alkaloids, have emerged as the premier organisms used to study plant Alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical Alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and Monoterpenoid Indole Alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of Alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the Alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and Monoterpenoid Indole Alkaloid biosynthesis in opium poppy and C. roseus is presented.

Santosh Panjikar - One of the best experts on this subject based on the ideXlab platform.

  • The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed β-propeller fold in plant proteins. Plant Cell 2006
    2015
    Co-Authors: Santosh Panjikar, Juergen Koepke, Elke C Loris
    Abstract:

    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 b-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 b-propeller structures. Structural analysis and site-directed mutagenesis experi-ments 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

  • Stockigt J: Molecular architecture of strictosidine glucosidase: The gateway to the biosynthesis of the Monoterpenoid Indole Alkaloid family. Plant Cell 2007
    2013
    Co-Authors: Leif Barleben, Santosh Panjikar, Juergen A Koepke, Joachim Stöckigta
    Abstract:

    Strictosidine b-D-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of Monoterpenoid Indole Alkaloids in plants. This family is composed of;2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple Indole Alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids that occupy the active site surface of the enzyme. Structural analysis an

  • The Structure of Rauvolfia serpentina Strictosidine Synthase Is a Novel Six-Bladed b-Propeller Fold in Plant Proteins W
    2013
    Co-Authors: Santosh Panjikar, Juergen Koepke, Elke C Loris, Joachim Stöckigt A
    Abstract:

    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 b-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 b-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

  • Molecular Architecture of Strictosidine Glucosidase - the Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family
    The Plant cell, 2007
    Co-Authors: Leif Barleben, Martin Ruppert, Santosh Panjikar, Juergen A Koepke, Joachim Stockigt
    Abstract:

    Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of Monoterpenoid Indole Alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple Indole Alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids that occupy the active site surface of the enzyme. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-207, Glu-416, His-161, and Trp-388 in catalysis. Comparison of the catalytic pocket of SG with that of other plant glucosidases demonstrates the structural importance of Trp-388. Compared with all other glucosidases of plant, bacterial, and archaeal origin, SG9s residue Trp-388 is present in a unique structural conformation that is specific to the SG enzyme. In addition to STR1 and vinorine synthase, SG represents the third structural example of enzymes participating in the biosynthetic pathway of the Rauvolfia Alkaloid ajmaline. The data presented here will contribute to deciphering the structure and reaction mechanism of other higher plant glucosidases.

  • the molecular architecture of major enzymes from ajmaline biosynthetic pathway
    Phytochemistry Reviews, 2007
    Co-Authors: Santosh Panjikar, Martin Ruppert, Leif Barleben, Elke A Loris, Marco Hill
    Abstract:

    The biosynthetic pathway leading to the Monoterpenoid Indole Alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)8 barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new Alkaloid.

Alex Van Moerkercke - One of the best experts on this subject based on the ideXlab platform.

  • the basic helix loop helix transcription factor bis2 is essential for Monoterpenoid Indole Alkaloid production in the medicinal plant catharanthus roseus
    Plant Journal, 2016
    Co-Authors: Alex Van Moerkercke, Priscille Steensma, Fabian Schweizer, Ivo Gariboldi, Robin Vanden Bossche, Javiera Espoz, Purin Candra Purnama, Karel Miettinen, Rebecca De Clercq, Johan Memelink
    Abstract:

    Monoterpenoid Indole Alkaloids (MIAs) are produced as plant defence compounds. In the medicinal plant Catharanthus roseus, they comprise the anticancer compounds vinblastine and vincristine. The iridoid (Monoterpenoid) pathway forms one of the two branches that feed MIA biosynthesis and its activation is regulated by the transcription factor (TF) basic helix-loop-helix (bHLH) iridoid synthesis 1 (BIS1). Here, we describe the identification and characterisation of BIS2, a jasmonate (JA)-responsive bHLH TF expressed preferentially in internal phloem-associated parenchyma cells, which transactivates promoters of iridoid biosynthesis genes and can homodimerise or form heterodimers with BIS1. Stable overexpression of BIS2 in C. roseus suspension cells and transient ectopic expression of BIS2 in C. roseus petal limbs resulted in increased transcript accumulation of methylerythritol-4-phosphate and iridoid pathway genes, but not of other MIA genes or triterpenoid genes. Transcript profiling also indicated that BIS2 expression is part of an amplification loop, as it is induced by overexpression of either BIS1 or BIS2. Accordingly, silencing of BIS2 in C. roseus suspension cells completely abolished the JA-induced upregulation of the iridoid pathway genes and subsequent MIA accumulation, despite the presence of induced BIS1, indicating that BIS2 is essential for MIA production in C. roseus.

  • the bhlh transcription factor bis1 controls the iridoid branch of the Monoterpenoid Indole Alkaloid pathway in catharanthus roseus
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Alex Van Moerkercke, Priscille Steensma, Fabian Schweizer, Jacob Pollier, Ivo Gariboldi, Richard J Payne, Robin Vanden Bossche
    Abstract:

    Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the Monoterpenoid Indole Alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix-loop-helix (bHLH) transcription factor from clade IVa inducing the Monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures.