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
The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.