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Takashi Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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Molecular evolution of N-Methylputrescine oxidase in tobacco.
Plant & cell physiology, 2013Co-Authors: Maliwan Naconsie, Tsubasa Shoji, Keita Kato, Takashi HashimotoAbstract:Biosynthesis of nicotine in tobacco requires N-Methylputrescine oxidase (MPO), which belongs to the copper-containing amine oxidase superfamily. Previous studies identified tobacco MPO1 and its close homolog NtDAO1 (formerly called MPO2), of which MPO1 has been shown preferentially to oxidize N-methylated amines. We show here that NtDAO1, as well as a homologous Arabidopsis diamine oxidase (DAO), accept non-N-methylated amines more efficiently than their corresponding N-methylated amines. MPO1 is coordinately regulated with other nicotine biosynthesis genes with regard to COI1-MYC2-dependent jasmonate induction and its dependence on nicotine-specific ERF transcription factors, whereas NtDAO1 is constitutively expressed at low basal levels in tobacco plants. Both MPO1 and NtDAO1 are targeted to peroxisomes by their C-terminal motifs, and the peroxisomal localization of MPO1 is required for it to function in nicotine biosynthesis in jasmonate-elicited cultured tobacco cells. Restricted occurrence of the MPO subfamily in Nicotiana and Solanum indicates that, during the formation of the Solanaceae, MPO has evolved from a DAO, which functions in polyamine catabolism within peroxisomes, by optimizing substrate preference and gene expression patterns to be suitable for alkaloid formation.
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Why does anatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells?
Plant & cell physiology, 2008Co-Authors: Tsubasa Shoji, Takashi HashimotoAbstract:Suspension-cultured cells of Nicotiana tabacum cv. Bright Yellow-2 (BY-2) grow rapidly in a highly homogenous population and still exhibit the general behavior of plant cells, and thus are often used as model systems in several areas of plant molecular and cellular biology, including secondary metabolism. While the parental tobacco variety synthesizes nicotine as a major alkaloid, the cultured tobacco cells mainly produce a related alkaloid anatabine, instead of nicotine, when elicited with jasmonates. We report here that cultured BY-2 cells scarcely express N-Methylputrescine oxidase (MPO) genes even after jasmonate elicitation. MPO is the second enzyme in the biosynthetic pathway that supplies the pyrrolidine moiety of nicotine and nornicotine, but is predicted to be dispensable for the biosynthesis of anatabine, anabasine and anatalline, which do not contain the pyrrolidine moiety. When MPO was overexpressed in tobacco BY-2 cells, nicotine synthesis was dramatically enhanced while anatabine formation was effectively suppressed. As a complementary approach, we suppressed MPO expression by RNA interference in tobacco hairy roots that normally accumulate nicotine. In the MPO-suppressed roots, the contents of anatabine, anabasine and anatalline, as well as N-Methylputrescine and putrescine, markedly increased to compensate for suppressed formation of nicotine and nornicotine. These results identify the transcriptional regulation of MPO as a critical rate-limiting step that restricts nicotine formation in cultured tobacco BY-2 cells.
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Molecular cloning of N-Methylputrescine oxidase from tobacco.
Plant & cell physiology, 2007Co-Authors: Akira Katoh, Tsubasa Shoji, Takashi HashimotoAbstract:Nicotine biosynthesis in Nicotiana species requires an oxidative deamination of N-Methylputrescine, catalyzed by N-Methylputrescine oxidase (MPO). In a screen for tobacco genes that were down-regulated in a tobacco mutant with altered regulation of nicotine biosynthesis, we identified two homologous MPO cDNAs which encode diamine oxidases of a particular subclass. Tobacco MPO genes were expressed specifically in the root, and up-regulated by jasmonate treatment. Recombinant MPO protein expressed in Escherichia coli formed a homodimer and deaminated N-Methylputrescine more efficiently than symmetrical diamines. These results indicate that MPO evolved from general diamine oxidases to function effectively in nicotine biosynthesis.
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Running title: Tobacco N-Methylputrescine oxidase
2007Co-Authors: Takashi HashimotoAbstract:Nicotine biosynthesis in Nicotiana species requires an oxidative deamination of N-Methylputrescine, catalyzed by N-Methylputrescine oxidase (MPO). In a screen for tobacco genes that were down-regulated in a tobacco mutant with altered regulation of nicotine biosynthesis, we identified two homologous MPO cDNAs which encode diamine oxidases of a particular subclass. Tobacco MPO genes were expressed specifically in the root, and up-regulated by jasmonate treatment. Recombinant MPO protein expressed in E. coli formed a homo-dimer and deaminated N-Methylputrescine more efficiently than symmetrical diamines. These results indicate that MPO evolved from general diamine oxidases to function effectively in nicotine biosynthesis.
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INAUGURAL ARTICLE by a Recently Elected Academy Member:Metabolic engineering of plant alkaloid biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Fumihiko Sato, Takashi Hashimoto, Akira Hachiya, Ken-ichi Tamura, Kum-boo Choi, Takashi Morishige, Hideki Fujimoto, Yasuyuki YamadaAbstract:Higher plants constitute one of our most important natural resources. They provide not only foodstuffs, fibers, and woods, but many chemicals, such as oils, flavorings, dyes, and pharmaceuticals. Although plants are renewable resources, some species are becoming more difficult to obtain in sufficient amounts to meet increasing demands. Destruction of natural habitats and technical difficulties in cultivation also are driving the drastic reductions in plant availability. For example, it is claimed that the demand for paclitaxel, a potent anticancer compound, could endanger forests of Taxus brevifolia (Pacific yew) because of the low paclitaxel content (40–100 mg/kg of bark) in and slow growth of the trees (1). For many natural chemicals it is possible to synthesize alternatives from petroleum, coal, or both. The economic limitations of chemical synthesis and the pollution that accompanies this type of chemical synthesis, however, have led to the development of cell culture and molecular engineering of plants for the production of important and commodity chemicals. Plant cell and organ culture offer promising alternatives for the production of chemicals because totipotency enables plant cells and organs to produce useful secondary metabolites in vitro (2). Cell culture is also advantageous in that useful metabolites are obtained under a controlled environment, independent of climatic changes and soil conditions. In addition, the products are free of microbe and insect contamination. Fermentation technology also can be used to produce desired metabolites and can be optimized to maintain high and stable yields of known quality by cellular and molecular breeding techniques to further improve productivity and quality. After extensive empirical trials, some metabolites are now being produced by large-scale cell culture (e.g., shikonin and berberine; ref. 2), but the numbers of compounds that are producible commercially by cell culture technology are still very few. The main limitations are low productivity and the necessity of the down-stream processing of the desired compounds. Molecular engineering of secondary metabolites has the potential to increase productivity and improve product composition. The Solanaceae produce a range of biologically active alkaloids that include nicotine and the tropane alkaloids (3). Tropane alkaloids, such as hyoscyamine (atropine) and scopolamine (hyoscine), which are found mainly in Hyoscyamus, Duboisia, Atropa, and Scopolia species, together with their semisynthetic derivatives, are used as parasympatholytics that competitively antagonize acetylcholine. Both the tropane ring moiety of the tropane alkaloids and the pyrrolidine ring of nicotine are derived from putrescine by way of N-Methylputrescine (MP) (Fig. (Fig.1).1). Because putrescine is metabolized to polyamines such as spermidine and spermine, the N-methylation of putrescine catalyzed by putrescine N-methyltransferase (PMT) is the first committed step in the biosynthesis of these alkaloids. Figure 1 Biosynthetic pathways of tropane alkaloids and nicotine. Tropane alkaloids and nicotine are derived from diamine putrescine produced from ornithine by ornithine decarboxylase (ODC), arginine, or both (28, 29). Putrescine is N-methylated by PMT then ... Isoquinoline alkaloids are some of the major metabolites successfully produced by plant cell culture. So far, about 60 have been isolated from plant cell cultures (ref. 4 and references therein). One example is berberine, a benzylisoquinoline alkaloid obtained from Coptis (Ranunculaceae) that is used as an antibacterial agent. Berberine biosynthesis in plant cells has been well investigated at the enzyme level (5–7). The biosynthetic pathway leading from l-tyrosine to berberine has 13 different enzymatic reactions that involve a norcoclaurine synthase, an N-methyltransferase, three O-methyltransferases (OMTs), a hydroxylase, a berberine bridge enzyme, a methylenedioxy ring-forming enzyme, and a tetrahydro protoberberine oxidase (Fig. (Fig.2).2). cDNAs of several enzymes in this pathway have been isolated and characterized: norcoclaurine 6OMT (8), a hydroxylase (9), 3′-hydroxy-N-methyl-coclaurine 4′OMT (8), a berberine bridge enzyme (10), and (S)-scoulerine 9-O-methyltransferase (SMT) (11). Figure 2 Schematic biosynthetic pathway for a variety of isoquinoline alkaloids. 1, l-tyrosine decarboxylase; 2, phenolase; 3, l-tyrosine transaminase; 4, p-hydroxyphenylpyruvate decarboxylase; 5, (S)-norcoclaurine synthase; 6, (S)-adenosyl-l-methionine:norcoclaurine ... We previously reported that overexpression of hyoscyamine 6β-hydroxylase in Atropa belladonna efficiently converts this species' main alkaloid, hyoscyamine, to scopolamine (12). This successful metabolic engineering of a medicinal plant has raised prospects for biotechnological applications of secondary metabolite production, but fundamental difficulties remain in transforming the host plants (e.g., Catharanthus; ref. 13). Our recent attempts to improve the production of putrescine-derived alkaloids and isoquinoline alkaloids by molecular engineering are reported.
Nicholas J Walton - One of the best experts on this subject based on the ideXlab platform.
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Metabolism of N-alkyldiamines and N-alkylnortropinones by transformed root cultures of Nicotiana and Brugmansia
Phytochemistry, 1999Co-Authors: Henry D Boswell, Nicholas J Walton, David J. Robins, Richard J Robins, Birgit Dräger, Adrian J. Parr, Andreas Portsteffen, John Eagles, Carol A Mcclintock, Chi WongAbstract:Abstract A range of analogues of N -methylputrescine and tropinone were fed to transformed root cultures of Nicotiana rustica and/or a Brugmansia candida × aurea hybrid. These cultures were made by the transformation of the relevant plant species with Agrobacterium rhizogenes . A number of the metabolites, notably those showing a relatively modest alteration in the N -alkyl substituent, were metabolized in vivo to form homologues of the normal alkaloids biosynthesized by these roots. These products were identified by GC/MS and comparison with some synthetic reference materials. Analogues with major alterations in the size of the N -alkyl substituent were not metabolized at all. In the N. rustica cultures, the analogues fed at 1 mM significantly affected the profile of normal alkaloids, with up to a 4-fold diminution in nicotine being found in the presence of N - n -propylputrescine. The ratio between alkaloids of the pyrrolidine series and the piperideine series was also affected. In contrast, the presence of the analogues in the B. candida × aurea hybrid culture at 1 mM did not inhibit or substantially interfere with the accumulation of the normal spectrum of alkaloids. The potential for using these cultures to make complex novel products from simple precursors is discussed.
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Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.
Planta, 1994Co-Authors: Nicholas J Walton, Henry D Boswell, Richard J Robins, Abigael C J Peerless, Michael J C Rhodes, David J. RobinsAbstract:Putrescine- N -methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes . The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K _m values for putrescine and S -adenosyl- l -methionine of 0.31 mM and 0.10 mM, respectively, and K _i values for S -adenosyl- l -homocysteine and N -methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino- trans -but-2-ene. Little activity was observed with 1,4-diamino- cis -but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K _i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.
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formation of n ethyl s nornicotine by transformed root cultures of nicotiana rustica
Phytochemistry, 1993Co-Authors: Henry D Boswell, Allan B Watson, Nicholas J Walton, David J. RobinsAbstract:Abstract N-Ethylputrescine dihydrochloride has been synthesized by an improved procedure and it is converted by transformed root cultures of Nicotiana rustica into the nicotine analogue, N′-ethyl-S-nornicotine, preferentially in the optically active S-form, with an efficiency similar to that of the corresponding natural process.
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Formation of N′-ethyl-S-nornicotine by transformed root cultures of Nicotiana rustica
Phytochemistry, 1993Co-Authors: Henry D Boswell, Allan B Watson, Nicholas J Walton, David J. RobinsAbstract:Abstract N-Ethylputrescine dihydrochloride has been synthesized by an improved procedure and it is converted by transformed root cultures of Nicotiana rustica into the nicotine analogue, N′-ethyl-S-nornicotine, preferentially in the optically active S-form, with an efficiency similar to that of the corresponding natural process.
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enzymes of n methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of datura stramonium and atropa belladonna
Planta, 1990Co-Authors: Nicholas J Walton, Richard J Robins, Abigael C J PeerlessAbstract:The activities of enzymes related to the biosynthesis of N-Methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine δ-Nmethyltransferase and δ-N-methylornithine decafboxylase were undetectable, indicating that δ-N-methylornithine is an unlikely intermediate in the formation of N-Methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-14C]ornithine, l-[U-14C]arginine, [U-14C]agmaine and [1,4-14C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl-α-difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl-α-difluoromethylornithine. Together with the demonstration that label was incorporated from [U-14C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-14C] ornithine and l-[U-14C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.
David J. Robins - One of the best experts on this subject based on the ideXlab platform.
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Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura.
Phytochemistry, 1999Co-Authors: Henry D Boswell, David J. Robins, Richard J Robins, Birgit Dräger, W. R. Mclauchlan, Andreas Portsteffen, N. J. WaltonAbstract:The enzymes N-Methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.
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Metabolism of N-alkyldiamines and N-alkylnortropinones by transformed root cultures of Nicotiana and Brugmansia
Phytochemistry, 1999Co-Authors: Henry D Boswell, Nicholas J Walton, David J. Robins, Richard J Robins, Birgit Dräger, Adrian J. Parr, Andreas Portsteffen, John Eagles, Carol A Mcclintock, Chi WongAbstract:Abstract A range of analogues of N -methylputrescine and tropinone were fed to transformed root cultures of Nicotiana rustica and/or a Brugmansia candida × aurea hybrid. These cultures were made by the transformation of the relevant plant species with Agrobacterium rhizogenes . A number of the metabolites, notably those showing a relatively modest alteration in the N -alkyl substituent, were metabolized in vivo to form homologues of the normal alkaloids biosynthesized by these roots. These products were identified by GC/MS and comparison with some synthetic reference materials. Analogues with major alterations in the size of the N -alkyl substituent were not metabolized at all. In the N. rustica cultures, the analogues fed at 1 mM significantly affected the profile of normal alkaloids, with up to a 4-fold diminution in nicotine being found in the presence of N - n -propylputrescine. The ratio between alkaloids of the pyrrolidine series and the piperideine series was also affected. In contrast, the presence of the analogues in the B. candida × aurea hybrid culture at 1 mM did not inhibit or substantially interfere with the accumulation of the normal spectrum of alkaloids. The potential for using these cultures to make complex novel products from simple precursors is discussed.
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Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.
Planta, 1994Co-Authors: Nicholas J Walton, Henry D Boswell, Richard J Robins, Abigael C J Peerless, Michael J C Rhodes, David J. RobinsAbstract:Putrescine- N -methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes . The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K _m values for putrescine and S -adenosyl- l -methionine of 0.31 mM and 0.10 mM, respectively, and K _i values for S -adenosyl- l -homocysteine and N -methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino- trans -but-2-ene. Little activity was observed with 1,4-diamino- cis -but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K _i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.
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formation of n ethyl s nornicotine by transformed root cultures of nicotiana rustica
Phytochemistry, 1993Co-Authors: Henry D Boswell, Allan B Watson, Nicholas J Walton, David J. RobinsAbstract:Abstract N-Ethylputrescine dihydrochloride has been synthesized by an improved procedure and it is converted by transformed root cultures of Nicotiana rustica into the nicotine analogue, N′-ethyl-S-nornicotine, preferentially in the optically active S-form, with an efficiency similar to that of the corresponding natural process.
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Formation of N′-ethyl-S-nornicotine by transformed root cultures of Nicotiana rustica
Phytochemistry, 1993Co-Authors: Henry D Boswell, Allan B Watson, Nicholas J Walton, David J. RobinsAbstract:Abstract N-Ethylputrescine dihydrochloride has been synthesized by an improved procedure and it is converted by transformed root cultures of Nicotiana rustica into the nicotine analogue, N′-ethyl-S-nornicotine, preferentially in the optically active S-form, with an efficiency similar to that of the corresponding natural process.
Yasuyuki Yamada - One of the best experts on this subject based on the ideXlab platform.
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INAUGURAL ARTICLE by a Recently Elected Academy Member:Metabolic engineering of plant alkaloid biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Fumihiko Sato, Takashi Hashimoto, Akira Hachiya, Ken-ichi Tamura, Kum-boo Choi, Takashi Morishige, Hideki Fujimoto, Yasuyuki YamadaAbstract:Higher plants constitute one of our most important natural resources. They provide not only foodstuffs, fibers, and woods, but many chemicals, such as oils, flavorings, dyes, and pharmaceuticals. Although plants are renewable resources, some species are becoming more difficult to obtain in sufficient amounts to meet increasing demands. Destruction of natural habitats and technical difficulties in cultivation also are driving the drastic reductions in plant availability. For example, it is claimed that the demand for paclitaxel, a potent anticancer compound, could endanger forests of Taxus brevifolia (Pacific yew) because of the low paclitaxel content (40–100 mg/kg of bark) in and slow growth of the trees (1). For many natural chemicals it is possible to synthesize alternatives from petroleum, coal, or both. The economic limitations of chemical synthesis and the pollution that accompanies this type of chemical synthesis, however, have led to the development of cell culture and molecular engineering of plants for the production of important and commodity chemicals. Plant cell and organ culture offer promising alternatives for the production of chemicals because totipotency enables plant cells and organs to produce useful secondary metabolites in vitro (2). Cell culture is also advantageous in that useful metabolites are obtained under a controlled environment, independent of climatic changes and soil conditions. In addition, the products are free of microbe and insect contamination. Fermentation technology also can be used to produce desired metabolites and can be optimized to maintain high and stable yields of known quality by cellular and molecular breeding techniques to further improve productivity and quality. After extensive empirical trials, some metabolites are now being produced by large-scale cell culture (e.g., shikonin and berberine; ref. 2), but the numbers of compounds that are producible commercially by cell culture technology are still very few. The main limitations are low productivity and the necessity of the down-stream processing of the desired compounds. Molecular engineering of secondary metabolites has the potential to increase productivity and improve product composition. The Solanaceae produce a range of biologically active alkaloids that include nicotine and the tropane alkaloids (3). Tropane alkaloids, such as hyoscyamine (atropine) and scopolamine (hyoscine), which are found mainly in Hyoscyamus, Duboisia, Atropa, and Scopolia species, together with their semisynthetic derivatives, are used as parasympatholytics that competitively antagonize acetylcholine. Both the tropane ring moiety of the tropane alkaloids and the pyrrolidine ring of nicotine are derived from putrescine by way of N-Methylputrescine (MP) (Fig. (Fig.1).1). Because putrescine is metabolized to polyamines such as spermidine and spermine, the N-methylation of putrescine catalyzed by putrescine N-methyltransferase (PMT) is the first committed step in the biosynthesis of these alkaloids. Figure 1 Biosynthetic pathways of tropane alkaloids and nicotine. Tropane alkaloids and nicotine are derived from diamine putrescine produced from ornithine by ornithine decarboxylase (ODC), arginine, or both (28, 29). Putrescine is N-methylated by PMT then ... Isoquinoline alkaloids are some of the major metabolites successfully produced by plant cell culture. So far, about 60 have been isolated from plant cell cultures (ref. 4 and references therein). One example is berberine, a benzylisoquinoline alkaloid obtained from Coptis (Ranunculaceae) that is used as an antibacterial agent. Berberine biosynthesis in plant cells has been well investigated at the enzyme level (5–7). The biosynthetic pathway leading from l-tyrosine to berberine has 13 different enzymatic reactions that involve a norcoclaurine synthase, an N-methyltransferase, three O-methyltransferases (OMTs), a hydroxylase, a berberine bridge enzyme, a methylenedioxy ring-forming enzyme, and a tetrahydro protoberberine oxidase (Fig. (Fig.2).2). cDNAs of several enzymes in this pathway have been isolated and characterized: norcoclaurine 6OMT (8), a hydroxylase (9), 3′-hydroxy-N-methyl-coclaurine 4′OMT (8), a berberine bridge enzyme (10), and (S)-scoulerine 9-O-methyltransferase (SMT) (11). Figure 2 Schematic biosynthetic pathway for a variety of isoquinoline alkaloids. 1, l-tyrosine decarboxylase; 2, phenolase; 3, l-tyrosine transaminase; 4, p-hydroxyphenylpyruvate decarboxylase; 5, (S)-norcoclaurine synthase; 6, (S)-adenosyl-l-methionine:norcoclaurine ... We previously reported that overexpression of hyoscyamine 6β-hydroxylase in Atropa belladonna efficiently converts this species' main alkaloid, hyoscyamine, to scopolamine (12). This successful metabolic engineering of a medicinal plant has raised prospects for biotechnological applications of secondary metabolite production, but fundamental difficulties remain in transforming the host plants (e.g., Catharanthus; ref. 13). Our recent attempts to improve the production of putrescine-derived alkaloids and isoquinoline alkaloids by molecular engineering are reported.
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differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures
Plant Molecular Biology, 1998Co-Authors: Shunsuke Imanishi, Yasuyuki Yamada, Takashi Hashimoto, Katsuhito Hashizume, Makiko Nakakita, Hisae Kojima, Yoshikatsu Matsubayashi, Youji Sakagami, Kenzo NakamuraAbstract:A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-Methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.p>
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diamine oxidase from cultured roots of hyoscyamus niger its function in tropane alkaloid biosynthesis
Plant Physiology, 1990Co-Authors: Takashi Hashimoto, Akira Mitani, Yasuyuki YamadaAbstract:Diamine oxidase was partially purified from cultured roots of Hyoscyamus niger L. that produce considerable amounts of tropane alkaloids, and then characterized. N-Methylated amines inhibited the activity of the enzyme more strongly than the corresponding primary amines. N-Methylputrescine was the best substrate of those studied, the respective Km values for it and for putrescine and cadaverine being 0.33, 2.85, and 6.25 millimolar. The specificity constants Vmax/Km for putrescine and cadaverine were 11 and 1% of the constant for N-Methylputrescine. Marked specificity for the N-methylated diamine would enable the Hyoscyamus enzyme to function specifically in tropane alkaloid biosynthesis.
Henry D Boswell - One of the best experts on this subject based on the ideXlab platform.
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Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura.
Phytochemistry, 1999Co-Authors: Henry D Boswell, David J. Robins, Richard J Robins, Birgit Dräger, W. R. Mclauchlan, Andreas Portsteffen, N. J. WaltonAbstract:The enzymes N-Methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.
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Metabolism of N-alkyldiamines and N-alkylnortropinones by transformed root cultures of Nicotiana and Brugmansia
Phytochemistry, 1999Co-Authors: Henry D Boswell, Nicholas J Walton, David J. Robins, Richard J Robins, Birgit Dräger, Adrian J. Parr, Andreas Portsteffen, John Eagles, Carol A Mcclintock, Chi WongAbstract:Abstract A range of analogues of N -methylputrescine and tropinone were fed to transformed root cultures of Nicotiana rustica and/or a Brugmansia candida × aurea hybrid. These cultures were made by the transformation of the relevant plant species with Agrobacterium rhizogenes . A number of the metabolites, notably those showing a relatively modest alteration in the N -alkyl substituent, were metabolized in vivo to form homologues of the normal alkaloids biosynthesized by these roots. These products were identified by GC/MS and comparison with some synthetic reference materials. Analogues with major alterations in the size of the N -alkyl substituent were not metabolized at all. In the N. rustica cultures, the analogues fed at 1 mM significantly affected the profile of normal alkaloids, with up to a 4-fold diminution in nicotine being found in the presence of N - n -propylputrescine. The ratio between alkaloids of the pyrrolidine series and the piperideine series was also affected. In contrast, the presence of the analogues in the B. candida × aurea hybrid culture at 1 mM did not inhibit or substantially interfere with the accumulation of the normal spectrum of alkaloids. The potential for using these cultures to make complex novel products from simple precursors is discussed.
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Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.
Planta, 1994Co-Authors: Nicholas J Walton, Henry D Boswell, Richard J Robins, Abigael C J Peerless, Michael J C Rhodes, David J. RobinsAbstract:Putrescine- N -methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes . The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K _m values for putrescine and S -adenosyl- l -methionine of 0.31 mM and 0.10 mM, respectively, and K _i values for S -adenosyl- l -homocysteine and N -methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino- trans -but-2-ene. Little activity was observed with 1,4-diamino- cis -but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K _i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.
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formation of n ethyl s nornicotine by transformed root cultures of nicotiana rustica
Phytochemistry, 1993Co-Authors: Henry D Boswell, Allan B Watson, Nicholas J Walton, David J. RobinsAbstract:Abstract N-Ethylputrescine dihydrochloride has been synthesized by an improved procedure and it is converted by transformed root cultures of Nicotiana rustica into the nicotine analogue, N′-ethyl-S-nornicotine, preferentially in the optically active S-form, with an efficiency similar to that of the corresponding natural process.
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Formation of N′-ethyl-S-nornicotine by transformed root cultures of Nicotiana rustica
Phytochemistry, 1993Co-Authors: Henry D Boswell, Allan B Watson, Nicholas J Walton, David J. RobinsAbstract:Abstract N-Ethylputrescine dihydrochloride has been synthesized by an improved procedure and it is converted by transformed root cultures of Nicotiana rustica into the nicotine analogue, N′-ethyl-S-nornicotine, preferentially in the optically active S-form, with an efficiency similar to that of the corresponding natural process.
