reticuline and discretamine

Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 4'-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C6' coupling and four O-methyl groups at C6, C7, C3', and C4' as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 3' and 4' hydroxyl groups of certain alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 4'OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 4'-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 3'-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 3'- and 7'-O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O-methylation patterns of alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry.

Topics: Aporphines; Benzylisoquinolines; Berberine Alkaloids; Escherichia coli; Gene Expression Regulation, Plant; Isoquinolines; Methyltransferases; Papaveraceae; Phylogeny; Plant Proteins; Plant Roots; Plants, Medicinal; Recombinant Proteins; Substrate Specificity; Tetrahydropapaveroline

Noscapine is a benzylisoquinoline alkaloid produced in opium poppy (Papaver somniferum) and other members of the Papaveraceae. It has been used as a cough suppressant and more recently was shown to possess anticancer activity. However, the biosynthesis of noscapine in opium poppy has not been established. A proposed pathway leading from (S)-reticuline to noscapine includes (S)-scoulerine, (S)-canadine, and (S)-N-methylcanadine as intermediates. Stem cDNA libraries and latex extracts of eight opium poppy cultivars displaying different alkaloid profiles were subjected to massively parallel pyrosequencing and liquid chromatography-tandem mass spectrometry, respectively. Comparative transcript and metabolite profiling revealed the occurrence of three cDNAs encoding O-methyltransferases designated as SOMT1, SOMT2, and SOMT3 that correlated with the accumulation of noscapine in the eight cultivars. SOMT transcripts were detected in all opium poppy organs but were most abundant in aerial organs, where noscapine primarily accumulates. SOMT2 and SOMT3 showed strict substrate specificity and regiospecificity as 9-O-methyltransferases targeting (S)-scoulerine. In contrast, SOMT1 was able to sequentially 9- and 2-O-methylate (S)-scoulerine, yielding (S)-tetrahydropalmatine. SOMT1 also sequentially 3'- and 7-O-methylated both (S)-norreticuline and (S)-reticuline with relatively high substrate affinity, yielding (S)-tetrahydropapaverine and (S)-laudanosine, respectively. The metabolic functions of SOMT1, SOMT2, and SOMT3 were investigated in planta using virus-induced gene silencing. Reduction of SOMT1 or SOMT2 transcript levels resulted in a significant decrease in noscapine accumulation. Reduced SOMT1 transcript levels also caused a decrease in papaverine accumulation, confirming the selective roles for these enzymes in the biosynthesis of both alkaloids in opium poppy.

Topics: Alkaloids; Amino Acid Sequence; Benzylisoquinolines; Berberine Alkaloids; Chromatography, Liquid; Enzyme Activation; Escherichia coli; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gene Library; Gene Silencing; Isoquinolines; Metabolome; Methyltransferases; Molecular Sequence Data; Noscapine; Papaver; Phylogeny; Plant Proteins; Recombinant Proteins; RNA, Plant; Substrate Specificity; Tandem Mass Spectrometry

The flavoprotein Berberine Bridge Enzyme (BBE) catalyzes the regioselective oxidative cyclization of (S)-reticuline to (S)-scoulerine in an alkaloid biosynthetic pathway. A series of solvent and substrate deuterium kinetic isotope effect studies were conducted to discriminate between a concerted mechanism, in which deprotonation of the substrate phenol occurs before or during the transfer of a hydride from the substrate to the flavin cofactor and substrate cyclization, and a stepwise mechanism, in which hydride transfer results in the formation of a methylene iminium ion intermediate that is subsequently cyclized. The substrate deuterium isotope effect of 3.5 on k(red), the rate constant for flavin reduction, is pH-independent, indicating that C-H bond cleavage is rate-limiting during flavin reduction. Solvent isotope effects on k(red) are equal to 1 for both wild-type BBE and the E417Q mutant, indicating that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism as a possibility for catalysis by BBE. An intermediate was not detected by rapid chemical quench or continuous-flow mass spectrometry experiments, indicating that it must be short-lived.

Topics: Benzylisoquinolines; Berberine Alkaloids; Cannabis; Deuterium; Deuterium Exchange Measurement; Isotope Labeling; Kinetics; Oxidation-Reduction; Oxidoreductases, N-Demethylating; Plant Proteins

Berberine bridge enzyme catalyzes the stereospecific oxidation and carbon-carbon bond formation of (S)-reticuline to (S)-scoulerine. In addition to this type of reactivity the enzyme can further oxidize (S)-scoulerine to the deeply red protoberberine alkaloid dehydroscoulerine albeit with a much lower rate of conversion. In the course of the four electron oxidation, no dihydroprotoberberine species intermediate was detectable suggesting that the second oxidation step leading to aromatization proceeds at a much faster rate. Performing the reaction in the presence of oxygen and under anoxic conditions did not affect the kinetics of the overall reaction suggesting no strict requirement for oxygen in the oxidation of the unstable dihydroprotoberberine intermediate. In addition to the kinetic characterization of this reaction we also present a structure of the enzyme in complex with the fully oxidized product. Combined with information available for the binding modes of (S)-reticuline and (S)-scoulerine a possible mechanism for the additional oxidation is presented. This is compared to previous reports of enzymes ((S)-tetrahydroprotoberberine oxidase and canadine oxidase) showing a similar type of reactivity in different plant species.

Topics: Benzylisoquinolines; Berberine Alkaloids; Catalysis; Eschscholzia; Molecular Structure; Oxidation-Reduction; Oxidoreductases Acting on CH-CH Group Donors; Oxidoreductases, N-Demethylating; Stereoisomerism

Benzylisoquinoline alkaloids, such as the analgesic compounds morphine and codeine, and the antibacterial agents berberine, palmatine, and magnoflorine, are synthesized from tyrosine in the Papaveraceae, Berberidaceae, Ranunculaceae, Magnoliaceae, and many other plant families. It is difficult to produce alkaloids on a large scale under the strict control of secondary metabolism in plants, and they are too complex for cost-effective chemical synthesis. By using a system that combines microbial and plant enzymes to produce desired benzylisoquinoline alkaloids, we synthesized (S)-reticuline, the key intermediate in benzylisoquinoline alkaloid biosynthesis, from dopamine by crude enzymes from transgenic Escherichia coli. The final yield of (S)-reticuline was 55 mg/liter within 1 h. Furthermore, we synthesized an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, from dopamine via reticuline by using different combination cultures of transgenic E. coli and Saccharomyces cerevisiae cells. The final yields of magnoflorine and scoulerine were 7.2 and 8.3 mg/liter culture medium. These results indicate that microbial systems that incorporate plant genes cannot only enable the mass production of scarce benzylisoquinoline alkaloids but may also open up pathways for the production of novel benzylisoquinoline alkaloids.

Topics: Aporphines; Benzylisoquinolines; Berberine Alkaloids; Biotechnology; Coptis; Dopamine; Escherichia coli; Genes, Plant; Micrococcus luteus; Saccharomyces cerevisiae; Transgenes

Using post-embedding immunogold techniques the cytological localization of the two branchpoint enzymes of isoquinoline biosynthesis, berberine bridge enzyme (BBE) and (S)-tetrahydroprotoberberine oxidase (STOX), was demonstrated. Electron-microscopic examination revealed their exclusive compartmentation within vesicles. After these vesicles have fused with the central vacuole, they release their contents, resulting in a characteristic electron-dense precipitate at the tonoplast. Vesicles of similar structure could be identified in young meristematic tissues of roots or shoots from different Berberis species and Papaver somniferum L. The appearance of electron-dense osmiophilic material is strictly correlated with the alkaloid content of the tissue. Immunocytological staining of P. somniferum tissue with antibodies directed against BBE led to a characteristic labeling of electron-dense aggregates in idioblasts that are not connected to the laticifer system. This localization demonstrates the strictly cytological separation of benzophenanthridine and morphine biosyntheses within this plant.

Topics: Alkaloids; Benzylisoquinolines; Berberine; Berberine Alkaloids; Berberis; Cells, Cultured; Eschscholzia; Immunohistochemistry; Isoquinolines; Microscopy, Immunoelectron; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxidoreductases, N-Demethylating; Papaver; Plant Cells; Plants; Vacuoles

Topics: Benzyl Compounds; Benzylisoquinolines; Berberine Alkaloids; Cells, Cultured; Chromatography, Gel; Chromatography, Thin Layer; Enzymes; Hydrogen-Ion Concentration; Isoquinolines; Kinetics; Opium; Plants