Phytochemistry 70 (2009) 1696–1707 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Review Evolution of morphine biosynthesis in opium poppy Jörg Ziegler a,b, Peter J. Facchini b, René Geißler a,1, Jürgen Schmidt a, Christian Ammer a, Robert Kramell a, Susan Voigtländer a, Andreas Gesell a,2, Silke Pienkny a, Wolfgang Brandt a,* a Leibniz-Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany b Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4 article info abstract Article history: Benzylisoquinoline alkaloids (BIAs) are a group of nitrogen-containing plant secondary metabolites com- Received 7 May 2009 prised of an estimated 2500 identified structures. In BIA metabolism, (S)-reticuline is a key branch-point Received in revised form 29 June 2009 intermediate that can be directed into several alkaloid subtypes with different structural skeleton config- Available online 6 August 2009 urations. The morphinan alkaloids are one subclass of BIAs produced in only a few plant species, most notably and abundantly in the opium poppy (Papaver somniferum). Comparative transcriptome analysis Keywords: of opium poppy and several other Papaver species that do not accumulate morphinan alkaloids showed Papaver somniferum that known genes encoding BIA biosynthetic enzymes are expressed at higher levels in P. somniferum. Papaveraceae Three unknown cDNAs that are co-ordinately expressed with several BIA biosynthetic genes were iden- Morphine Benzylisoquinoline alkaloids tified as enzymes in the pathway. One of these enzymes, salutaridine reductase (SalR), which is specific Transcript profiling for the production of morphinan alkaloids, was isolated and heterologously overexpressed in its active Metabolite profiling form not only from P. somniferum, but also from Papaver species that do not produce morphinan alkaloids. Short chain dehydrogenase/reductase SalR is a member of a class of short chain dehydrogenase/reductases (SDRs) that are active as monomers and possess an extended amino acid sequence compared with classical SDRs. Homology modelling and substrate docking revealed the substrate binding site for SalR. The amino acids residues conferring salu- taridine binding were compared to several members of the SDR family from different plant species, which non-specifically reduce (À)-menthone to (+)-neomenthol. Previously, it was shown that some of these proteins are involved in plant defence. The recruitment of specific monomeric SDRs from monomeric SDRs involved in plant defence is discussed. Ó 2009 Elsevier Ltd. All rights reserved. Contents 1. Introduction . 1696 2. Integration of alkaloid and transcript profiles in Papaver species . 1699 3. Characterization of cDNAs involved in BIA metabolism based on transcriptional profiles ......................................... 1703 4. Occurrence of SalR in other Papaver species . 1703 5. Structure-function analysis of SalR . 1703 6. Relationship of SalR to other SDRs . 1705 Acknowledgments . 1706 References . 1706 1. Introduction widespread occurrence, mainly in the plant kingdom. Nine hun- dred out of 1000 plant species in which benzylisoquinoline alka- Benzylisoquinoline alkaloids (BIAs) are a structurally diverse loids (BIAs) have been detected belong to the closely related group of nitrogen-containing plant secondary metabolites with orders of the Ranunculales, Magniolales, and Laurales, but many structures have also been found in several species of the Rutaceae * Corresponding author. Tel.: +49 345 5582 1360; fax: +49 345 5582 1309. (Shulgin and Perry, 2002). Thus, the occurrence of this class of E-mail address: [email protected] (W. Brandt). plant secondary metabolites across several plant families might re- 1 Present address: Institute of Biochemistry and Biotechnology, Martin-Luther- flect both monophyletic origins and parallel evolutionary events University Halle-Wittenberg, D-06120 Halle, Germany. 2 Present address: Department of Biology, University of Victoria, Victoria, BC, (Jensen, 1995). The evolution and diversification of metabolic path- Canada V8 W 3N5. ways is believed to result from the recruitment of enzymes from 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.07.006 J. Ziegler et al. / Phytochemistry 70 (2009) 1696–1707 1697 primary metabolism. After gene duplication, random mutations in specific accumulation of distinct metabolites that are all produced new genes result in the establishment of novel catalytic functions via one pathway. (Ober and Hartmann, 2000). This mechanism was demonstrated BIA biosynthesis begins with a condensation reaction catalyzed for an enzyme in pyrrolizidine alkaloid biosynthesis. Homospermi- by norcoclaurine synthase of dopamine and para-hydroxyphenyl- dine synthase, the first enzyme in the pathway, was identified as a acetaldehyde, both of which are derived from L-tyrosine (Fig. 1). modified version of the ubiquitous enzyme deoxyhyposine syn- Interestingly, two enzymes belonging to two different protein fam- thase, which is responsible for translational regulation (Ober and ilies have been identified to catalyze the reaction. One enzyme be- Hartmann, 1999). Similarly, high sequence similarities suggest that longs to the PR10/Betv1 family of proteins and its recruitment from putrescine N-methyltransferase, which is the first committed step defence proteins is discussed (Samanani et al., 2004; Liscombe in tropane alkaloid metabolism, is derived from spermidine syn- et al., 2005). The other enzyme was classified as a member of the thase (Hashimoto et al., 1998; Teuber et al., 2007). In the same 2-oxoglutarate-dependent dioxygenase family, although it did pathway, the classification of tropinone reductases as members not possess an oxoglutarate binding site, and was active in the ab- of the short chain dehydrogenase/reductase (SDR) family suggests sence oxoglutarate (Minami et al., 2007). (S)-Norcoclaurine repre- the recruitment of these enzymes from primary metabolism (Nak- sents the first pathway intermediate with a benzylisoquinoline ajima et al., 1993). The emergence of these enzymes is characteris- skeleton from which all other BIA classes are derived. In a first ser- tic for another evolutionary process, in which new functions are ies of reactions, (S)-norcoclaurine is methylated by norcoclaurine established by small variations in existing functions. The modifica- 6-O-methyltransferase (6OMT) to (S)-coclaurine, which is N- tion of tropinone reductases led to the generation of two paralo- methylated by coclaurine N-methyltransferase (CNMT) yielding gous enzymes with opposite stereo-specificities (Nakajima et al., N-methylcoclaurine (Morishige et al., 2000; Choi et al., 2002; Oun- 1993). The two products serve as the precursors of two different aroon et al., 2003). Subsequent hydroxylation of the benzyl moiety classes of tropane alkaloids, scopolamine/hyoscyamine and the by (S)-N-methylcoclaurine 30-hydroxylase, a P450-dependent calystegines. A recently identified SDR from Cochlearia officinalis monooxygenase belonging to the CYP80B subfamily, and methyla- was suggested to represent a prototype during the development tion by (S)-30-hydroxy N-methylcoclaurine 40-O-methyltranferase of stereospecific tropinone reductase, since it forms a mixture of (4’OMT) results in the formation of (S)-reticuline (Pauli and Kut- tropine and pseudotropine from tropinone (Brock et al., 2008). chan, 1998; Huang and Kutchan, 2000; Morishige et al., 2000; Zie- The functional diversification and specialization of enzymes after gler et al., 2005). This basic benzylisoquinoline pathway is involved recruitment from common ancestors is also evident within the in the formation of most BIAs with the exception of the bisbenzyl- numerous plant secondary metabolic enzymes, which belong to isoquinolines and (most likely) the N-demethylated benzylisoquin- large protein families including chalcone synthases, terpenoid syn- olines, which are derived from precursors upstream of thases, O-orN-methyltransferases, glycosyltransferases, and P450- (S)-reticuline (Kraus and Kutchan, 1995; Pienkny et al., 2009). For dependent monooxygenases (Durbin et al., 2000; Vogt and Jones, all other BIAs, (S)-reticuline represents a major branch-point 2000; Zubieta et al., 2001, 2002; Austin and Noel, 2003; Ounaroon intermediate in the pathway. Methylations lead to the further et al., 2003; Schuler and Werck-Reichhart, 2003; Uefuji et al., 2003; elaboration of the simple benzylisoquinoline class with respect to Ziegler et al., 2005; Stenzel et al., 2006; Tholl, 2006; Inui et al., the number and positions of the methyl groups. The reactions 2007; Liscombe and Facchini, 2007). resulting in the majority of BIA structural diversity involve the for- Another strategy for the establishment of specific metabolite mation of C–C bonds between the isoquinoline ring, or the N- profiles involves molecular mechanisms that lead to the activa- methyl group, and the benzyl moiety. Depending on the positions tion or deactivation of genes. Cell- or tissue-specific accumulation of the carbon atoms, these reactions produce different classes of of plant secondary metabolites generally correlates with the BIAs, such as the pavines, isopavines, aporphines, proaporphines, expression of the corresponding biosynthetic genes, as shown and protoberberines (Shulgin and Perry, 2002). The latter group for tropinone reductases and phenylpropene O-methyltransfer- of compounds