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Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy

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.

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Figure 1: Proposed two-step stereochemical inversion of (S)-reticuline to (R)-reticuline catalyzed by 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reductase (DRR) in opium poppy.
Figure 2: Maps of cDNAs and genes encoding reticuline epimerase (REPI), 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reductase (DRR).
Figure 3: Catalytic functions of PsREPI, PsDRS and PrDRS using (S)-reticuline, 1,2-dehydroreticuline and (R)-reticuline as substrates.
Figure 4: Virus-induced gene silencing in opium poppy supports the role of PsREPI in morphinan alkaloid biosynthesis.

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GenBank/EMBL/DDBJ

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Acknowledgements

We thank D. Nelson (University of Tennessee) for assigning the CYP nomenclature. This work was supported by grants from Genome Canada, Genome Alberta, the Government of Alberta, the Canada Foundation for Innovation and the Natural Sciences and Engineering Research Council of Canada to P.J.F. S.C.F. and G.A.W.B. were recipients of scholarships from the Natural Sciences and Engineering Research Council of Canada. S.C.F. also received a scholarship from Alberta Innovates Technology Futures.

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S.C.F. performed all recombinant enzyme assays, virus-induced gene silencing experiments and mass spectrometric analyses, and co-wrote the manuscript. J.M.H. constructed the yeast expression vectors, performed all qRT-PCR experiments, and co-wrote the manuscript. D.C.B. conducted and interpreted the NMR analysis. G.A.W.B. contributed to the initial gene isolations. P.J.F. directed the research, prepared the figures and tables, and edited the manuscript.

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Correspondence to Peter J Facchini.

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P.J.F., S.C.F. and G.A.W.B. have filed a patent application (Patent Cooperation Treaty Application PCT/CA2014/0511164) covering compositions and methods related to the manufacture or (R)-reticuline and precursors thereof.

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Farrow, S., Hagel, J., Beaudoin, G. et al. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat Chem Biol 11, 728–732 (2015). https://doi.org/10.1038/nchembio.1879

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