Tropinone

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Birgit Dräger - One of the best experts on this subject based on the ideXlab platform.

  • Potato plants with genetically engineered tropane alkaloid precursors
    Planta, 2017
    Co-Authors: Nadine Küster, Sabine Rosahl, Birgit Dräger
    Abstract:

    Main conclusion Solanum tuberosum Tropinone reductase I reduced Tropinone in vivo . Suppression of Tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescine N -methyltransferase did not alter calystegine accumulation. Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato ( Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N -methyltransferase (PMT) and Tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N -methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional Tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants.

  • immunolocalisation of two Tropinone reductases in potato solanum tuberosum l root stolon and tuber sprouts
    Planta, 2006
    Co-Authors: Heike Kaiser, Ute Richter, Ronald Keiner, Anja Brabant, Bettina Hause, Birgit Dräger
    Abstract:

    Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming Tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming Tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.

  • Tropinone reductases enzymes at the branch point of tropane alkaloid metabolism
    Phytochemistry, 2006
    Co-Authors: Birgit Dräger
    Abstract:

    Two stereospecific oxidoreductases constitute a branch point in tropane alkaloid metabolism. Products of tropane metabolism are the alkaloids hyoscyamine, scopolamine, cocaine, and polyhydroxylated nortropane alkaloids, the calystegines. Both Tropinone reductases reduce the precursor Tropinone to yield either tropine or pseudotropine. In Solanaceae, tropine is incorporated into hyoscyamine and scopolamine; pseudotropine is the first specific metabolite on the way to the calystegines. Isolation, cloning and heterologous expression of both Tropinone reductases enabled kinetic characterisation, protein crystallisation, and structure elucidation. Stereospecificity of reduction is achieved by binding Tropinone in the respective enzyme active centre in opposite orientation. Immunolocalisation of both enzyme proteins in cultured roots revealed a tissue-specific protein accumulation. Metabolite flux through both arms of the tropane alkaloid pathway appears to be regulated by the activity of both enzymes and by their access to the precursor Tropinone. Both Tropinone reductases are NADPH-dependent short-chain dehydrogenases with amino acid sequence similarity of more than 50% suggesting their descent from a common ancestor. Putative Tropinone reductase sequences annotated in plant genomes other that Solanaceae await functional characterisation.

  • overexpression of Tropinone reductases alters alkaloid composition in atropa belladonna root cultures
    Journal of Experimental Botany, 2005
    Co-Authors: Ute Richter, Grit Rothe, Bettina Rahfeld, Annekatrin Fabian, Birgit Dräger
    Abstract:

    The medicinally applied tropane alkaloids hyoscyamine and scopolamine are produced in Atropa belladonna L. and in a small number of other Solanaceae. Calystegines are nortropane alkaloids that derive from a branching point in the tropane alkaloid biosynthetic pathway. In A. belladonna root cultures, calystegine molar concentration is 2-fold higher than that of hyoscyamine and scopolamine. In this study, two Tropinone reductases forming a branching point in the tropane alkaloid biosynthesis were overexpressed in A. belladonna. Root culture lines with strong overexpression of the transcripts contained more enzyme activity of the respective reductase and enhanced enzyme products, tropine or pseudotropine. High pseudotropine led to an increased accumulation of calystegines in the roots. Strong expression of the tropine-forming reductase was accompanied by 3-fold more hyoscyamine and 5-fold more scopolamine compared with control roots, and calystegine levels were decreased by 30-90% of control. In some of the transformed root cultures, an increase of total tropane alkaloids was observed. Thus, transformation with cDNA of Tropinone reductases successfully altered the ratio of tropine-derived alkaloids versus pseudotropine-derived alkaloids.

  • functional expression of Tropinone reductase i tri and hyoscyamine 6β hydroxylase h6h from hyoscyamus niger in nicotiana tabacum
    Plant Science, 2002
    Co-Authors: Adrian J. Parr, Birgit Dräger, Pedro Rocha, Olaf Stenzel, Nicholas J Walton, Paul Christou, Mark Leech
    Abstract:

    The cDNAs from Hyoscyamus niger , encoding two enzymes of tropane-alkaloid biosynthesis, Tropinone reductase I (TR-I) and hyoscyamine-6b-hydroxylase (H6H), have been simultaneously introduced into Nicotiana tabacum using particle bombardment and expressed under the control of the CaMV 35S promoter. Southern and Northern analyses confirmed integration and transcript accumulation for both tr1 and h6h . Fertile tobacco plants expressing both transgenes were regenerated and detached leaves of these plants were fed with Tropinone and hyoscyamine, the substrates, respectively, of TR-I and H6H. Besides the expected TR-I and H6H reaction products, acetylated forms of tropine were also generated in these experiments, indicating that the expression of alkaloidpathway enzymes in a transgenic background can produce unexpected substances. In addition, leaves of the transgenic plants showed in most cases higher nicotine content than leaves of control plants. Nicotine levels were approximately 3- to 13-fold higher in both the parental transgenic lines and in T1 progeny expressing functional TR-I and H6H, compared to levels in wild-type plants and in transgenic plants carrying the nptII transgene alone. In addition, nicotine related compounds such as anatabine, nornicotine, bipyridine, anabasine, and myosmine were identified in transgenic tobacco lines and below detection limit in wild-type plants, suggesting changes in the activity of the enzymes in the nicotine biosynthetic pathway in the transgenic background. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Rajender Singh Sangwan - One of the best experts on this subject based on the ideXlab platform.

  • Tropine Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns
    2016
    Co-Authors: Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a Tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of Tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent Tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric,60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and Tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[14C]-sucrose to orphan shoot (twigs) and [14C]-chasing revealed substantial radiolabel incorporation in Tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that thes

  • Schematic representation of metabolic pathway of tropane alkaloid biosynthesis. TR-I, Tropinone reductase I; TR-II, Tropinone reductase II.
    2013
    Co-Authors: Amit Kumar Kushwaha, Neelam Singh Sangwan, Prabodh Kumar Trivedi, Arvind Singh Negi, Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    Schematic representation of metabolic pathway of tropane alkaloid biosynthesis. TR-I, Tropinone reductase I; TR-II, Tropinone reductase II.

  • Tropine Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns
    2013
    Co-Authors: Amit Kumar Kushwaha, Neelam Singh Sangwan, Prabodh Kumar Trivedi, Arvind Singh Negi, Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a Tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of Tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent Tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ∼60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and Tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[14C]-sucrose to orphan shoot (twigs) and [14C]-chasing revealed substantial radiolabel incorporation in Tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.

  • Identification of the reaction product of recombinant WsTR-I with Tropinone as substrate.
    2013
    Co-Authors: Amit Kumar Kushwaha, Neelam Singh Sangwan, Prabodh Kumar Trivedi, Arvind Singh Negi, Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    TLC: I, tropine standard; II, WsTR-I reaction mixture after catalytic reaction termination; III, NaBH4 reduction products (tropine and pseudotropine) of Tropinone; IV, Tropinone standard. GC: A, authentic Tropinone; B, authentic tropine; C, control (enzyme minus) assay mixture of WsTR-I; D, experimental (complete) assay mixture of WsTR-I assay; E, NaBH4 aided reduction products (tropine and pseudotropine) of Tropinone.

  • Homology based 3D model of tropine forming Tropinone reductase of W. somnifera (WsTR-I).
    2013
    Co-Authors: Amit Kumar Kushwaha, Neelam Singh Sangwan, Prabodh Kumar Trivedi, Arvind Singh Negi, Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    A, WsTR-I superimposed on to DsTR-I and DsTR-II. The model was constructed on Swiss-model workspace taking DsTRI (1ae1.pdb) as a template. Comparative representation was performed by UCSF Chimera package. WsTR-I, DsTR-I and DsTR-II are depicted as gray, purple and blue, respectively. NADPH and Tropinone are visible in cleft of active site; B, Tropinone binding pocket of WsTR-I. A model was prepared by alignment of WsTR-I, DsTR-I and DsTR-II following energy minimization in Swiss-PDBviewer. Tropinone binding site was visualized by Ligand Explorer. Amino acids close to Tropinone are labeled; C, Three dimensional (3-D) models of WsTR-I, DsTR-I and DsTR-II were aligned and analyzed in Pymol. Residues are lebelled in green (WsTR-I), cyan (DsTR-I) and magenta (DsTR-II). Tropinone is shown in orange.

Jan Jirschitzka - One of the best experts on this subject based on the ideXlab platform.

  • plant tropane alkaloid biosynthesis evolved independently in the solanaceae and erythroxylaceae
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Jan Jirschitzka, Gregor W Schmidt, Michael Reichelt, Bernd Schneider, Jonathan Gershenzon, John C Dauria
    Abstract:

    The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the Tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca Tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-Tropinone) stereospecifically to 2-carbomethoxy-3β-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.

  • Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Jan Jirschitzka, Gregor W Schmidt, Michael Reichelt, Bernd Schneider, Jonathan Gershenzon, John C. D’auria
    Abstract:

    The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the Tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca Tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-Tropinone) stereospecifically to 2-carbomethoxy-3β-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.

Amit Kumar Kushwaha - One of the best experts on this subject based on the ideXlab platform.

  • molecular cloning and catalytic characterization of a recombinant tropine biosynthetic Tropinone reductase from withania coagulans leaf
    Gene, 2013
    Co-Authors: Amit Kumar Kushwaha, Neelam S Sangwan, Sandhya Tripathi, Rajender S Sangwan
    Abstract:

    Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of Tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming Tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.

  • Schematic representation of metabolic pathway of tropane alkaloid biosynthesis. TR-I, Tropinone reductase I; TR-II, Tropinone reductase II.
    2013
    Co-Authors: Amit Kumar Kushwaha, Neelam Singh Sangwan, Prabodh Kumar Trivedi, Arvind Singh Negi, Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    Schematic representation of metabolic pathway of tropane alkaloid biosynthesis. TR-I, Tropinone reductase I; TR-II, Tropinone reductase II.

  • Tropine Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns
    2013
    Co-Authors: Amit Kumar Kushwaha, Neelam Singh Sangwan, Prabodh Kumar Trivedi, Arvind Singh Negi, Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a Tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of Tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent Tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ∼60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and Tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[14C]-sucrose to orphan shoot (twigs) and [14C]-chasing revealed substantial radiolabel incorporation in Tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.

  • Identification of the reaction product of recombinant WsTR-I with Tropinone as substrate.
    2013
    Co-Authors: Amit Kumar Kushwaha, Neelam Singh Sangwan, Prabodh Kumar Trivedi, Arvind Singh Negi, Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    TLC: I, tropine standard; II, WsTR-I reaction mixture after catalytic reaction termination; III, NaBH4 reduction products (tropine and pseudotropine) of Tropinone; IV, Tropinone standard. GC: A, authentic Tropinone; B, authentic tropine; C, control (enzyme minus) assay mixture of WsTR-I; D, experimental (complete) assay mixture of WsTR-I assay; E, NaBH4 aided reduction products (tropine and pseudotropine) of Tropinone.

  • Homology based 3D model of tropine forming Tropinone reductase of W. somnifera (WsTR-I).
    2013
    Co-Authors: Amit Kumar Kushwaha, Neelam Singh Sangwan, Prabodh Kumar Trivedi, Arvind Singh Negi, Laxminarain Misra, Rajender Singh Sangwan
    Abstract:

    A, WsTR-I superimposed on to DsTR-I and DsTR-II. The model was constructed on Swiss-model workspace taking DsTRI (1ae1.pdb) as a template. Comparative representation was performed by UCSF Chimera package. WsTR-I, DsTR-I and DsTR-II are depicted as gray, purple and blue, respectively. NADPH and Tropinone are visible in cleft of active site; B, Tropinone binding pocket of WsTR-I. A model was prepared by alignment of WsTR-I, DsTR-I and DsTR-II following energy minimization in Swiss-PDBviewer. Tropinone binding site was visualized by Ligand Explorer. Amino acids close to Tropinone are labeled; C, Three dimensional (3-D) models of WsTR-I, DsTR-I and DsTR-II were aligned and analyzed in Pymol. Residues are lebelled in green (WsTR-I), cyan (DsTR-I) and magenta (DsTR-II). Tropinone is shown in orange.

John C. D’auria - One of the best experts on this subject based on the ideXlab platform.

  • Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization
    Nature Publishing Group, 2018
    Co-Authors: Matthew A. Bedewitz, John C. D’auria, Daniel A. Jones, Cornelius S. Barry
    Abstract:

    Tropinone is an intermediate in the biosynthesis of tropane alkaloids. Here, the authors discovered the enzymes AbPYKS and AbCYP82M3, a non-canonical polyketide synthase and a cytochrome P450, that work sequentially to form Tropinone from N-methyl-Δ1-pyrrolinium cation

  • Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Jan Jirschitzka, Gregor W Schmidt, Michael Reichelt, Bernd Schneider, Jonathan Gershenzon, John C. D’auria
    Abstract:

    The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the Tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca Tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-Tropinone) stereospecifically to 2-carbomethoxy-3β-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.