ABSTRACT
The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.
Subject(s)
Aldehyde Reductase/metabolism , Carbohydrate Epimerases/metabolism , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Plant , Morphine/biosynthesis , Papaver/metabolism , Plant Proteins/metabolism , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Alkaloids/biosynthesis , Alkaloids/chemistry , Base Sequence , Benzylisoquinolines/chemistry , Benzylisoquinolines/metabolism , Bromoviridae/genetics , Bromoviridae/metabolism , Carbohydrate Epimerases/antagonists & inhibitors , Carbohydrate Epimerases/genetics , Cytochrome P-450 Enzyme System/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Exons , Gene Fusion , Introns , Ligases/genetics , Ligases/metabolism , Molecular Sequence Data , Morphinans/chemistry , Morphinans/metabolism , Morphine/chemistry , Open Reading Frames , Opium/chemistry , Opium/metabolism , Oxidation-Reduction , Papaver/genetics , Plant Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , StereoisomerismABSTRACT
In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,O-demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O-demethylenation by CODM and protopine O-dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.
Subject(s)
Benzylisoquinolines/metabolism , Biocatalysis , Dioxygenases/metabolism , Opium/metabolism , Papaver/enzymology , Benzylisoquinolines/chemistry , Berberine Alkaloids/chemistry , Berberine Alkaloids/metabolism , Chromatography, Liquid , Formaldehyde/metabolism , Gene Silencing , Kinetics , Mass Spectrometry , Methylation , Phylogeny , Substrate Specificity , VirusesABSTRACT
Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.
Subject(s)
Alkaloids/metabolism , Benzylisoquinolines/metabolism , Gene Expression Regulation, Plant , Opium/chemistry , Papaver/metabolism , Alkaloids/chemistry , Benzylisoquinolines/chemistry , Biological Transport , Biosynthetic Pathways , Gene Expression , Genomics , Metabolic Engineering , Models, Biological , Papaver/chemistry , Papaver/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, MedicinalABSTRACT
Papaver somniferum L. (Family: Papaveraceae) is a species well known for its diverse alkaloids (100 different benzylisoquinoline alkaloids (BIAs)). L-tyrosine serves as a precursor of several specific metabolites like BIAs. It has been used as an antitussive and potent analgesic to alleviate mild to extreme pain since ancient times. The extraction of pharmaceutically important alkaloids like morphine and codeine from poppy plant reflects the need for the most suitable and standard methods. Several analytical and extraction techniques have been reported in open literature for morphine, codeine and other important alkaloids which play a vital function in drug development and drug discovery. Many studies suggest that opioids are also responsible for adverse effects or secondary complications like dependence and withdrawal. In recent years, opium consumption and addiction are the most important risk factors. Many evidence-based reviews suggest that opium consumption is directly linked or acts as a risk factor for different cancers. In this review, we highlight significant efforts related to research which have been done over the past 5 decades and the complete information on Papaver somniferum including its phytochemistry, pharmacological actions, biosynthetic pathways and analytical techniques of opium alkaloid extraction and the link between opium consumption and cancer-related updates.
Subject(s)
Alkaloids , Benzylisoquinolines , Neoplasms , Papaver , Opium/adverse effects , Opium/metabolism , Alkaloids/pharmacology , Alkaloids/metabolism , Benzylisoquinolines/pharmacology , Benzylisoquinolines/metabolism , Papaver/metabolism , Codeine/metabolism , Neoplasms/drug therapy , Neoplasms/etiology , Morphine Derivatives/metabolismABSTRACT
Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.
Subject(s)
Benzylisoquinolines/metabolism , Opium/metabolism , Papaver/enzymology , Tyrosine Transaminase/metabolism , Benzylisoquinolines/chemistry , DNA, Complementary/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant , Hydrogen-Ion Concentration , Kinetics , Papaver/genetics , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/isolation & purification , Tyrosine Transaminase/genetics , Tyrosine Transaminase/isolation & purificationABSTRACT
BACKGROUND: Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. RESULTS: A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. CONCLUSIONS: The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.
Subject(s)
Alkaloids/metabolism , Gene Expression Profiling , Plant Proteins/analysis , Proteome/analysis , Alkaloids/chemistry , Benzophenanthridines/chemistry , Benzophenanthridines/metabolism , Benzylisoquinolines/chemistry , Benzylisoquinolines/metabolism , Biological Factors/pharmacology , Biosynthetic Pathways/drug effects , Botrytis/chemistry , Cells, Cultured , Chromatography, High Pressure Liquid , Cluster Analysis , Electrophoresis, Polyacrylamide Gel , High-Throughput Nucleotide Sequencing , Isoquinolines/chemistry , Isoquinolines/metabolism , Mass Spectrometry , Molecular Sequence Data , Molecular Structure , Morphine/chemistry , Morphine/metabolism , Opium/chemistry , Opium/metabolism , Papaver/cytology , Papaver/genetics , Papaver/metabolism , Proteomics , Tyrosine/chemistry , Tyrosine/metabolismABSTRACT
The opium poppy, Papaver somniferum L., and its narcotic and analgesic alkaloids, have an ancient history of use (and abuse) by humankind. A recent article by Allen and co-workers describes the metabolic engineering of morphine biosynthesis to block morphine formation and accumulate a potentially valuable pathway intermediate, (S)-reticuline. This work highlights the potential for modifying the production of pharmaceuticals in plants, but also raises questions about the complex regulation of biosynthetic pathways.