morphinans and reticuline

Phylogenomic analysis of whole genome sequences of five benzylisoquinoline alkaloid (BIA)-producing species from the Ranunculales and Proteales orders of flowering plants revealed the sequence and timing of evolutionary events leading to the diversification of these compounds. (

Topics: Benzophenanthridines; Benzylisoquinolines; Berberine Alkaloids; Enzymes; Evolution, Molecular; Gene Duplication; Isoquinolines; Morphinans; Morphine; Multigene Family; Noscapine; Papaveraceae; Phylogeny; Plant Proteins

Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.

Topics: Base Sequence; Benzylisoquinolines; Cytochrome P-450 Enzyme System; Genetic Loci; Isoquinolines; Molecular Sequence Data; Morphinans; Mutation; Oxidation-Reduction; Papaver; Plant Proteins; Quaternary Ammonium Compounds

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.

Topics: Aldehyde Reductase; Aldo-Keto Reductases; Alkaloids; Base Sequence; Benzylisoquinolines; Bromoviridae; Carbohydrate Epimerases; Cytochrome P-450 Enzyme System; Escherichia coli; Exons; Gene Expression Regulation, Plant; Gene Fusion; Introns; Ligases; Molecular Sequence Data; Morphinans; Morphine; Open Reading Frames; Opium; Oxidation-Reduction; Papaver; Plant Proteins; Recombinant Proteins; Saccharomyces cerevisiae; Stereoisomerism

Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds.

Topics: Animals; Benzylisoquinolines; Biosynthetic Pathways; Carbohydrate Metabolism; Codeine; Genetic Engineering; Hydrocodone; Morphinans; Papaver; Saccharomyces cerevisiae; Thebaine

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis-Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.

Topics: Alcohol Oxidoreductases; Animals; Aryl Hydrocarbon Hydroxylases; Benzylisoquinolines; Codeine; Cytochrome P-450 CYP2D6; Cytochrome P450 Family 2; Humans; Kinetics; Mass Spectrometry; Microsomes, Liver; Morphinans; Morphine; Phenols; Rats; Rats, Wistar; Substrate Specificity; Thebaine

A cytochrome P450 (P450) enzyme in porcine liver that catalyzed the phenol-coupling reaction of the substrate (R)-reticuline to salutaridine was previously purified to homogeneity (Amann, T., Roos, P. H., Huh, H., and Zenk, M. H. (1995) Heterocycles 40, 425-440). This reaction was found to be catalyzed by human P450s 2D6 and 3A4 in the presence of (R)-reticuline and NADPH to yield not a single product, but rather (-)-isoboldine, (-)-corytuberine, (+)-pallidine, and salutaridine, the para-ortho coupled established precursor of morphine in the poppy plant and most likely also in mammals. (S)-Reticuline, a substrate of both P450 enzymes, yielded the phenol-coupled alkaloids (+)-isoboldine, (+)-corytuberine, (-)-pallidine, and sinoacutine; none of these serve as a morphine precursor. Catalytic efficiencies were similar for P450 2D6 and P450 3A4 in the presence of cytochrome b(5) with (R)-reticuline as substrate. The mechanism of phenol coupling is not yet established; however, we favor a single cycle of iron oxidation to yield salutaridine and the three other alkaloids from (R)-reticuline. The total yield of salutaridine formed can supply the 10 nm concentration of morphine found in human neuroblastoma cell cultures and in brain tissues of mice.

Topics: Animals; Benzylisoquinolines; Catalysis; Cell Line, Tumor; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Humans; Iron; Mice; Morphinans; Morphine; Oxidation-Reduction; Phenols; Rats

A synthase which oxidizes (S)-reticuline to 1,2-dehydroreticuline has been found to occur in seedlings of opium poppy (Papaver somniferum L.). Due to its instability, this enzyme could only be partly purified (ca. 5-fold enrichment). Partial characterization at this stage of purification showed that it does not need a redox cofactor and accepts both (S)-reticuline and (S)-norreticuline as substrates. [1-(2)H, (13)C]-(R,S)-reticuline was enzymatically converted into [1-(13)C]-dehydroreticuline, which has been identified by mass spectrometry. Release of the hydrogen atom in position C-1 of the isoquinoline alkaloid during the oxidative conversion, was exploited as a sensitive assay system for this enzyme. The enzyme has a pH optimum of 8.75, a temperature optimum of 37 degrees C and the apparent K(M) value for the substrate reticuline was shown to be 117 microM. Moreover it could be demonstrated by sucrose density gradient centrifugation that the enzyme is located in vesicles of varying size. In combination with the previously discovered strictly stereoselective and NADPH dependent 1,2-dehydroreticuline reductase the detection of this enzyme, the 1,2-dehydroreticuline synthase, provides the necessary inversion of configuration and completes the pathway from two molecules of L-tyrosine via (S)-norcoclaurine to (R)-reticuline in opium poppy involving a total number of 11 enzymes.

Topics: Alkaloids; Benzylisoquinolines; Centrifugation, Density Gradient; Enzyme Stability; Isomerism; Isoquinolines; Mass Spectrometry; Morphinans; Oxidoreductases; Papaver; Seedlings; Vacuoles