ARTICLE Published online: 1 JULY 2015 | doi: 10.1038/nchembio.1879

Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy Scott C Farrow1, Jillian M Hagel1, Guillaume A W Beaudoin1,3, Darcy C Burns2 & Peter J Facchini1* 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.

T

he pentacyclic morphinan alkaloids in opium poppy (P. somniferum) feature five chiral carbons that limit the efficiency of chemical synthesis1. As a result, agricultural production of opium poppy remains the sole commercial source for pharmaceutical opiate production. Since the isolation of morphine in 1806, the intricate links between opium poppy and the human condition have motivated extensive research on the biosynthesis of morphinan alkaloids2. Substantial progress toward pathway elucidation was achieved during the 1960s, and this supported a key hypothesis3 that morphine was derived via 1-benzylisoquinoline alkaloid metabolism4. (S)-Reticuline emerged as the central 1-benzylisoquinoline intermediate, with its stereochemical inversion to (R)-reticuline purported to be a pivotal gateway reaction given that only the (R)-conformer could undergo subsequent phenol coupling to the morphinan scaffold5. With the exception of the S-to-R epimerization of reticuline, all genes responsible for perceived conversions from dopamine and 4-hydroxyphenylacetaldehyde to morphine have been isolated2 (Supplementary Results, Supplementary Fig. 1). The stereochemical inversion of (S)-reticuline has been proposed to proceed via a 1,2-dehydroreticulinium cation intermediate, and two distinct enzymes implicated in successive oxidation and reduction reactions have been at least partially purified from opium poppy (Fig. 1). 1,2-Dehydroreticuline synthase (DRS) was partially enriched and reported to convert (S)-reticuline to 1,2-dehydroreticuline in the absence of a cofactor6, whereas 1,2-dehydroreticuline reductase (DRR) was purified to apparent homogeneity and showed a strict requirement for NADPH7. Although the genes encoding DRS and DRR have not been isolated, a potential clue to their identity is apparent from the results of RNA interference (RNAi)-mediated silencing of the codeinone reductase (COR) gene family in opium poppy. COR is an aldo-keto reductase (AKR) operating several steps downstream of (S)-reticuline (Supplementary Fig. 1), yet silencing COR resulted in the accumulation of (S)-reticuline rather than codeinone8. Possible explanations for this unexpected phenotype include off-target gene silencing9. In this paper, we report the isolation and characterization of genes encoding reticuline epimerase (REPI), DRS and DRR from opium poppy and the common field poppy (P. rhoeas).

RESULTS COR paralog search identifies CYP82Y2 in opium poppy

The possible off-target co-suppression of DRR transcripts in CORsilenced opium poppy plants prompted a search for COR paralogs8,9. We isolated two COR paralogs, one of which showed an in-frame extension of the coding region upstream of the predicted AKR (PsDRR1) domain (Fig. 2a). The upstream (PsDRS) domain was annotated as a member of the CYP82 family, and we designated it CYP82Y2. We amplified the entire coding region from opium poppy by RT-PCR, ruling out an assembly artifact as the source of the fusion. An independent AKR (PsAKR2) displaying 84% identity to the PsDRR1 region of CYP82Y2 showed no evidence of fusion (Fig. 2a). We also identified full-length orthologs encoding fusion proteins sharing 100% and 96% amino acid identity with opium poppy CYP82Y2 in Papaver setigerum and P. bracteatum, respectively. In contrast, the most similar orthologs in P. rhoeas encoded independent CYP82 (PrDRS) and AKR (PrDRR) proteins sharing 76% and 86% amino acid identity with the respective domains of the opium poppy fusion (Fig. 2a). Phylogenetic analysis indicated a close relationship between DRS and other CYP82 family members involved in the formation of benzylisoquinoline alkaloids (Supplementary Fig. 2a), whereas DRR is related to AKR enzymes involved in cocaine biosynthesis10 and chalcone metabolism11, in addition to several COR variants (Supplementary Fig. 2b). Isolation of the corresponding genes from opium poppy and P. rhoeas showed conservation in the approximate location of introns (Fig. 2b).

CYP82Y2 is reticuline epimerase

As defined below, we named the opium poppy CYP82Y2 fusion reticuline epimerase (PsREPI), and we designated corresponding CYP polypeptides from opium poppy and P. rhoeas (Fig. 2) as 1,2-dehydroreticuline synthase (PsDRS and PrDRS, respectively). Similarly, we named active AKR polypeptides from opium poppy and P. rhoeas (Fig. 2) 1,2-dehydroreticuline reductase (PsDRR1 and PrDRR, respectively). We then coexpressed constructs containing a CYP in Saccharomyces cerevisiae with a plant cytochrome P450 reductase (CPR). We tested the following complete or partial polypeptides for catalytic activity: (i) the CYP82Y2 fusion (pCPR/ PsREPI), (ii) the isolated CYP domain (pCPR/PsDRS), (iii) the

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. 2Department of Chemistry, University of Toronto, Toronto, Ontario, Canada. 3Current address: Horticultural Sciences Department, University of Florida, Gainesville, Florida, USA. e-mail: [email protected]

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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. CYP719B1 converts (R)-reticuline to salutaridine, which undergoes a multistep transformation to morphine.

isolated AKR domain (pPsDRR1) and (iv) the independent AKR (pPsAKR2) from opium poppy, and (v) the non-fused CYP82Y2 (pCPR/PrDRS) and (vi) AKR (pPrDRR) from P. rhoeas. The recombinant CYP and CPR proteins in S. cerevisiae microsomes were validated by immunoblot analysis (Supplementary Fig. 3a). His-tagged AKR domains (pPsDRR1, pPsAKR2 and pPrDRR) were expressed in E. coli and purified by affinity chromatography (Supplementary Fig. 3b,c). Microsomal preparations or purified proteins were assayed using (S)-reticuline, 1,2-dehydroreticuline or (R)-reticuline as substrates. Under standard reaction conditions, PsREPI converted (S)-reticuline to ~5% 1,2-dehydroreticuline and ~10% (R)-reticuline (Fig. 3). When supplied with 1,2-dehydroreticuline, PsREPI produced ~80% (R)-reticuline, but (R)-reticuline was not accepted as a substrate. PsDRS and PrDRS each converted (S)-reticuline to 1,2-dehydroreticuline, although the opium poppy CYP domain showed a higher turnover (>50%) and neither enzyme yielded (R)-reticuline (Fig. 3). Using assay conditions favoring alkaloid substrate reduction, PsDRR1 and PrDRR both converted 1,2-dehydroreticuline to (R)-reticuline, although the opium poppy AKR domain was substantially more efficient (Supplementary Figs. 4 and 5; Supplementary Table 1). Under conditions favoring oxidation, PsDRR1 and PrDRR converted (R)-reticuline, but not (S)-reticuline, to 1,2-dehydroreticuline (Supplementary Fig. 4b). PsAKR2 (Fig. 2) showed no activity with any tested substrate (Supplementary Fig. 6).

Requirement for N-methylated substrates

Physiological characterization of reticuline epimerase

We used virus-induced gene silencing (VIGS) to determine the physiological role of PsREPI in opium poppy (Fig. 4). The pTRV2REPI-5ʹ construct targeted the 5ʹ-untranslated region and a short portion of the PsREPI coding region (Supplementary Fig. 12). The pTRV2-COR1.1 construct contained (Supplementary Fig. 13) the fragment used previously for the RNAi-mediated silencing of the COR gene family in opium poppy8, and the pTRV2-REPI-a construct targeted a corresponding region in the AKR domain of PsREPI (Supplementary Fig. 14). The pTRV2-REPI-5ʹ construct was designed to test the physiological role of REPI, whereas the pTRV2-REPI-a and pTRV2-COR1.1 constructs were included to a

Assaying PsDRS and PrDRS with various 1-benzylisoquinoline alkaloids as potential substrates (Supplementary Fig. 7) revealed that only three were efficiently accepted: (S)-reticuline, (S)-3ʹ-hydroxyN-methylcoclaurine and (S)-N-methylcoclaurine. PsDRS showed the highest turnover with (S)-reticuline, whereas PrDRS was most active with (S)-N-methylcoclaurine and exhibited lower activity toward (S)-reticuline. Corresponding alkaloids lacking an N-methyl group were not accepted as substrates, and 1-benzylisoquinolines with C7 or C3ʹ O-linked methyl groups were not converted, perhaps because of greater steric hindrance. PsREPI also catalyzed the stereochemical inversion of (S)-Nmethylcoclaurine (Supplementary Fig. 8). PsDRR1 and PrDRR reduced 1,2-dehydro-N-methylcoclaurine to N-methylcoclaurine, although the low product yield precluded determination of the enantiomer (Supplementary Fig. 8). PsREPI, PsDRS and PrDRS all showed a pH optimum of ~7.8 using (S)-reticuline as substrate. In contrast, reduction of 1,2-dehydroreticuline by PsDRR1 and PrDRR exhibited a broad pH optimum between 6 and 9 (Supplementary Fig. 9), and oxidation of (R)-reticuline by PsDRR1 and PrDRR was optimal at pH ~8.8. Using (S)-reticuline as substrate, PsREPI, PsDRS and PrDRS displayed a relatively low Km of 4 mM, whereas PsDRR1 and PrDRR showed Km values of 13 mM for the reduction of 1,2-dehydroreticuline (Supplementary Table 1; Supplementary Fig. 10). PsDRR1 and PrDRR showed higher catalytic efficiency for the reduction of 1,2-dehydroreticuline than for the oxidation 2

of (R)-reticuline, with PsDRR1 displaying considerably more efficiency than PrDRR in the conversion of 1,2-dehydroreticuline to (R)-reticuline. Two contaminants with m/z 344 in the 1,2-dehydroreticuline standard were also transformed to compounds with m/z 346 in PsDRR1 and PrDRR assays (Supplementary Fig. 11a,b). Structural elucidation of the m/z 346 reaction products by NMR (Supplementary Table 2) identified them as diastereomers of a-hydroxyreticuline, indicating that the two contaminants were enantiomers of a-hydroxy-1,2-dehydroreticuline (Supplementary Fig. 11c,d). Unfortunately, assignment of the enantiomers could not be confirmed.

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Figure 2 | Maps of cDNAs and genes encoding reticuline epimerase (REPI), 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reductase (DRR). Abbreviations: Ps, P. somniferum; Pr, P. rhoeas. (a) Fulllength cDNAs showing the location of DRS and DRR domains in the REPI fusion and the alignment of independent DRS and DRR orthologs. Black and white boxes represent open reading frames and linker sequences between DRS and DRR domains, respectively. (b) Structure of genes encoding PsREPI, PrDRS, PrDRR and PsAKR2. Boxes and solid lines represent exons and introns, respectively. bp, base pairs.

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ARTICLE

Nature chemical biology doi: 10.1038/nchembio.1879 help assess the possibility that the previously reported pathway block at (S)-reticuline8 resulted from off-target suppression of REPI transcript levels. Relative REPI transcript abundance was significantly reduced in plants subjected to VIGS using all three constructs as compared to those transformed with empty vector (Fig. 4a), and the resulting alkaloid profiles were remarkably similar in both the latex and roots (Fig. 4b–d; Supplementary Figs. 12–14). A substantial increase in reticuline levels was accompanied by accumulation of the related O-methylated alkaloids codamine, laudanine and laudanosine (Fig. 4b). Notably, the ratio of (S)- to (R)-reticuline increased from approximately 3:1 in empty-vector controls to almost 50:1 in REPI and COR co-silenced plants (Fig. 4c; Supplementary Figs. 12–14). Levels of morphine, codeine and thebaine were substantially reduced and were consistently associated with a significant increase in the accumulation of noscapine, which is derived from (S)-reticuline. To ensure that the observed phenotypes did not result from off-target silencing, we performed qRT-PCR on all known morphine biosynthetic genes (Supplementary Fig. 15). No off-target silencing effects were detected in plants subjected to VIGS using the pTRV2-REPI-5ʹ construct. In contrast, reciprocal suppression of REPI and COR transcripts was observed using the pTRV2-REPI-a and pTRV2COR1.1 constructs owing to substantial (77%) nucleotide sequence identity between PsDRR1 and COR1.1 (Supplementary Table 3). Off-target silencing of SalR was observed using pTRV2-REPI-a and pTRV2-COR1.1 despite low nucleotide sequence identity with either REPI or COR1.1. Consistent with those of all known alkaloid biosynthetic genes, REPI transcripts were most abundant in stems (Supplementary Fig. 16).

DISCUSSION

A fusion protein consisting of CYP and AKR domains catalyzes the S-to-R stereochemical inversion of reticuline in opium poppy. The independent DRR purified from opium poppy seedlings7 could represent a member of the DRR family absent in our stem transcriptome12. Similarly, the previously detected oxidase also might occur only in seedlings6. In addition to the lack of a cofactor requirement, further evidence that the previously isolated oxidase was not a CYP82Y2 is provided by the reported substrate norreticuline, which is not accepted by PsDRS or PrDRS (Supplementary Fig. 7). Of 1,2-Dehydroreticuline

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potential relevance, (S)-tetrahydroprotoberberine oxidase has been reported to yield dehydrobenzoisoquinolines from corresponding substrates including norreticuline13,14. Dehydrogenation of (S)-reticuline by DRS represents a new reaction type in the CYP82 family. Other CYP82 members catalyze ring hydroxylations in benzylisoquinoline alkaloid biosynthesis15–18. Desaturation is not a common CYP function in plants, although sterol desaturases19,20 and desaturating flavone synthases21 have been reported. CYP-mediated desaturation of sterols is not thought to involve a hydroxylated intermediate22, although hydroxylation and subsequent dehydration are relevant in flavone formation23. Similarly, DRS could catalyze the stereospecific hydroxylation of (S)-reticuline at C1, followed by loss of water from the hemiaminal yielding the 1,2-dehydroreticulinium cation intermediate with no apparent conversion to Z- and/or E-enamines. In aqueous solution at pH >8, equilibrium with enamine isomers has been reported for 1,2-dehydroreticuline24. Although the acidic conditions used for LC-MS analyses would mask enamines formed during REPI and DRS assays (Fig. 3), labeling studies have ruled out enamine formation during the S-to-R transition of reticuline25, implying tight coordination or shielding of the intermediate. The stereoselective hydrogenation of a carbon-nitrogen double bond by DRR also represents a previously unknown AKR reaction type. Most AKRs catalyze the stereoselective reduction of aldehyde or carbonyl functional groups to primary or secondary alcohols, respectively, although members of the AKR1D family (i.e., 5b-reductases) catalyze irreversible steroid double-bond reductions26. Selective pressure on binding affinity was arguably greater for the CYP domain of PsREPI, which is reflected in the lower Km of PsDRS as compared with PsDRR1 (Supplementary Table 1). However, the potential impact of a gene fusion on biosynthetic performance is not apparent solely from kinetic data. Enzyme fusions can contribute to increased product yield without substantial alterations in Km or catalytic efficiency owing to the extreme proximity of active sites27,28. Efficient substrate channeling and coordination of the reaction intermediate through electrostatic interactions would further reduce selective pressure on the binding affinity of PsDRR1. Substrate channeling and domain-domain interactions have been reported for enzyme fusions involved in microbial folate metabolism29. In plants, domain-domain

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Figure 3 | Catalytic functions of PsREPI, PsDRS and PrDRS using (S)-reticuline, 1,2-dehydroreticuline and (R)-reticuline as substrates. (a) Extracted ion chromatograms generated by LC-MS showing the conversion of (S)-reticuline (m/z 330) to 1,2-dehydroreticuline (m/z 328) by PsREPI, PsDRS and PrDRS, and the formation of reticuline from 1,2-dehydroreticuline by PsREPI. (b) Chiral separation showing the formation of (R)-reticuline by PsREPI from either (S)-reticuline or 1,2-dehydroreticuline. All LC-MS product peaks displayed identical quasimolecular ions and collision-induced dissociation spectra compared with authentic standards (Supplementary Table 4). No conversions were detected in negative control assays (pCPR). Data are representative of at least four independent replicates. For clarity, reaction substrates and products are highlighted in blue and orange, respectively. nature chemical biology | ADVANCE ONLINE PUBLICATION | www.nature.com/naturechemicalbiology

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Nature chemical biology doi: 10.1038/nchembio.1879

Figure 4 | Virus-induced gene silencing in opium poppy supports the role of PsREPI in morphinan alkaloid biosynthesis. (a) Relative REPI and COR transcript abundance in REPI-silenced (REPI-5ʹ and REPI-a) and COR-silenced (COR1.1) plants compared with controls transformed with empty vector (pTRV2). (b) Accumulation of reticuline and O-methylated derivatives in the latex of REPI- and COR-silenced plants as compared with controls. (c) Relative abundance of S and R enantiomers of reticuline in REPI- and COR-silenced plants as compared with controls. (d) Relative abundance of major alkaloids in the latex of REPI- and COR-silenced plants as compared with controls. Values throughout represent the mean ± s.d. of 16 biological replicates. Asterisks represent significant differences determined using an unpaired, two-tailed Student’s t test (P < 0.05).

interactions are well-established features of bifunctional diterpene synthases, such as abietadiene synthase, which catalyzes two mechanistically distinct sequential reactions in separate active sites30. Substrate channeling in PsREPI, combined with possible membrane-anchored proximity to salutaridine synthase (CYP719B1), could confer functional superiority over independent DRS and DRR enzymes. Moreover, coordination of the 1,2-dehydroreticuline cation intermediate would prevent tautomerization to an enamine24. Metabolic engineering strategies using artificial protein fusions further underscore the value of integrating separate pathway enzymes into a single catalytic unit31,32. Performing VIGS using highly conserved regions of COR1.1 and REPI caused a reciprocal reduction in transcript abundance and precisely mimicked the alkaloid phenotype (Fig. 4; Supplementary Figs. 13 and 14) resulting from RNAibased silencing of COR8. In contrast, VIGS performed using a REPI-specific region produced the same alkaloid phenotype without co-suppression of COR transcript levels (Fig. 4; Supplementary Fig. 12), confirming the physiological role of REPI. The increased accumulation of O-methylated 1-benzylisoquinolines was consistent with metabolic redirection of (S)-reticuline away from morphine biosynthesis. Reduced flux to morphine was also reflected in the elevated accumulation of noscapine, which is derived from (S)-reticuline. Co-silencing of SalR (Supplementary Fig. 15) was unexpected based on its low nucleotide sequence identity with REPI and COR (Supplementary Table 3), although off-target transcript suppression is sometimes associated with post-­transcriptional gene silencing18. The stereochemical inversion of small molecules has been reported in amino acid metabolism, sugar degradation, cofactor repair and natural-product biosynthesis in plants and microbes, and it is generally catalyzed by single enzymes33–36. The evolution of a gene fusion combining CYP and AKR catalytic functions has established a single-enzyme solution to a potential metabolic bottleneck in morphine biosynthesis. The existence of REPI also 4

supports the suggestion that gene fusions provide evidence of functional interaction within metabolic pathways37. Gene clusters are regarded as mechanisms of functional association within metabolic pathways38. Recently, a ten-gene cluster consisting of most of the noscapine biosynthetic genes was identified in the opium poppy39, but the role of genetic linkage in the morphine branch pathway is not yet known. The REPI fusion might have arisen from a deletion in between two tightly linked genes, combining the CYP and AKR domains into a single translation product. The exclusive detection of REPI in morphinan alkaloid–producing Papaver species suggests a relatively recent fusion event. Despite the isolation of independent orthologs encoding DRS and DRR in P. rhoeas, reports are rare on the accumulation of 1,2-dehydroreticuline or (R)-reticuline in plants. However, the relatively common occurrence of dimeric (R,S)-, (S,R)- and (R,R)-bisbenzylisoquinolines40 suggests a potential physiological role for independent DRS and DRR enzymes. The correlation between morphinan alkaloid biosynthesis and the REPI fusion suggests that an efficient mechanism for (R)-reticuline production was required to sustain metabolic flux into the branch pathway. Interestingly, a search of the 1000 Plants database41 showed that P. rhoeas contains orthologs encoding proteins with 75–80% amino acid similarity to salutaridine synthase, salutaridine reductase and salutaridinol 7-O-acetyltransferase, which collectively convert (R)-reticuline to thebaine in opium poppy. Functional characterization of these homologs could reveal additional bottlenecks in morphinan alkaloid metabolism beyond those potentially overcome by the REPI fusion. Received 20 April 2015; accepted 25 June 2015; published online 1 July 2015

METHODS

Methods and any associated references are available in the online version of the paper.

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Nature chemical biology doi: 10.1038/nchembio.1879 Accession codes. GenBank/EMBL: sequence data in this article have been deposited under accession numbers KP985715–KP985724.

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Acknowledgments

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.

Author contributions

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.

Competing financial interests

The authors declare competing financial interests: details accompany the online version of the paper.

Additional information

Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/reprints/index. html. Correspondence and requests for materials should be addressed to P.J.F.

nature chemical biology | ADVANCE ONLINE PUBLICATION | www.nature.com/naturechemicalbiology

5

ONLINE METHODS

Alkaloids. (S)-Reticuline oxalate was a gift from Tasmanian Alkaloids. 1,2-Dehydroreticuline iodide, (S)-coclaurine and (R,S)-3ʹ-hydroxycoclaurine were purchased from Toronto Research Chemicals. (R)-Reticuline was purchased from Santa Cruz Biotechnology. Other alkaloids were enzymatically synthesized. Briefly, (R,S)-norreticuline was prepared with ~80% yield from (R,S)-3ʹ-hydroxycoclaurine and S-adenosyl-L-methionine (SAM) using purified, recombinant Coptis japonica 3ʹ-hydroxy-N-methylcoclaurine 4ʹ-O-methyltransferase (Cj4ʹOMT)42. (S)-NMethylcoclaurine was prepared with >95% yield from (S)-coclaurine and SAM using purified, recombinant C. japonica coclaurine N-methyltranseferase (CjCNMT)43. The reaction was extracted with ethyl acetate to remove a byproduct, (S)-N,Ndimethylcoclaurine. (S)-N-Methylcoclaurine was purified using a Strata-XC cartridge (Phenomenex) equilibrated with methanol and 50 mM sodium phosphate buffer, pH 7. Cartridges were rinsed with water and methanol, and the alkaloid was eluted using 5% (v/v) ammonia in 70% (v/v) 1:1 methanol:acetonitrile. N-Methyl1,2-dehydrococlaurine was prepared with ~50% yield from (S)-N-methylcoclaurine incubated with NADPH and a microsomal preparation containing PrDRS and Artemesia annua cytochrome P450 reductase. (S)-3ʹ-Hydroxy-N-methylcoclaurine was prepared with ~95% yield from (S)-3ʹ-hydroxycoclaurine and SAM using purified CjCNMT in the presence of sodium ascorbate. Owing to the small quantity of starting material available for biotransformation by PrDRS, 3ʹ-hydroxy-Nmethyl-1,2-dehydrococlaurine could not be prepared as a substrate for DRR assays. a-Hydroxyreticulines were produced from 1,2-dehydroreticuline and NADPH using purified PsDRR1, and purified using a Strata-X cartridge equilibrated with methanol and 5% (v/v) ammonia. The reaction was basified to pH 10 with ammonium hydroxide, loaded onto the cartridge, which was rinsed with 5% (v/v) ammonia and 5% (v/v) methanol, and eluted in 1:1 methanol:acetonitrile. a-Hydroxyreticulines were isolated on a Luna C18(2) semi-preparative HPLC column (250 mm × 10 mm; Phenomenex). Norcodamine, norlaudanine and norlaudanosine were prepared from (R,S)-norreticuline, and (S)-codamine, (S)-laudanine and (S)-laudanosine were prepared from (S)-reticuline, using SAM and O-methyltransferases targeting 7-O-methyl and/or 3ʹ-O-methyl groups (NCBI Nucleotide accession numbers KP176693 and KP176698). Identities of prepared alkaloids were confirmed by NMR or by using high-performance liquid chromatography-mass spectrometry (HPLC-MS) in comparison with published data (Supplementary Table 4). Gene isolation and expression vector construction. The codeinone reductase gene (COR1.1; NCBI Nucleotide accession number AF108432) was used to query P. somniferum stem transcriptome databases12 by tBLASTn, and two aldo-keto reductase candidate contigs (AKR1 and AKR2) were identified showing considerable sequence identity to COR1.1. The AKR1 contig displayed an extension that annotated as a cytochrome P450 monoxygenase (CYP), upstream and in-frame with the AKR coding region. Transcriptome databases in the PhytoMetaSyn (http://www.phytometasyn.com) and the 1000 Plants41 (https://www.bioinfodata.org/Blast4OneKP/home) projects were queried using the predicted CYP-AKR translation product, and fulllength orthologs were identified in P. setigerum and P. bracteatum. In contrast, independent orthologs of the CYP and AKR domains were found in P. rhoeas with no evidence of fusion. Coding regions for the P. somniferum AKR1 domain (renamed PsDRR1) and PsAKR2 and for the P. rhoeas AKR (renamed PrDRR) were amplified from corresponding stem cDNA libraries using Phusion DNA polymerase (New England BioLabs) and primers listed in Supplementary Table 5. Amplicons were A-tailed using green Taq DNA polymerase (Genscript) and cloned into pET47b (EMD Millipore). Constructs were used to transform the E. coli expression strains Arctic Express (Agilent) or Rosetta (Novagen). The coding region for P. somniferum CYPAKR1 (renamed PsREPI) and the isolated CYP domain (renamed PsDRS) and for the P. rhoeas CYP (PrDRS) were synthesized for insertion into the NotI and SpeI restriction sites of the dual expression vector pESC-leu2d (Agilent) along with Artemisia annua cytochrome P450 reductase (CPR; NCBI Nucleotide accession number DQ318192)44. The expression constructs were used to transform the protease-deficient S. cerevisiae YPL154c strain PEP4. Recombinant protein production in Escherichia coli. Cultures of E. coli harboring pET47b were grown on an orbital shaker (200 rpm) to log phase in 1 L of LB medium containing gentamicin, streptomycin and kanamycin (Arctic Express) or chloramphenicol and kanamycin (Rosetta). Recombinant His-tagged protein production was initiated by the addition of 1 mM isopropyl b-d-1-thiogalactopyranoside (IPTG). To produce PsDRR1 and PsAKR2, cultures were grown for 24 h at 15 °C, whereas cultures were grown for 4 h at 28 °C to produce PrDRR. For purification, bacterial pellets were resuspended in 10 mL of protein extraction buffer [100 mM sodium phosphate buffer, pH 7.4, 10% (v/v) glycerol, 300 mM sodium chloride, 1 mM PMSF], and cells were lysed at 4 °C using a French pressure cell (1,000 psi). After centrifugation (10,000g) to remove insoluble debris, the supernatant was incubated on ice with buffer-equilibrated Talon resin (Clontech) on an orbital shaker (60 rpm) for 60 min. The protein-charged resin was washed twice with 10 mL of cold extraction buffer containing 2.5 mM imidazole, and proteins were eluted stepwise with increasing concentrations (10 to 200 mM) of imidazole in extraction buffer. Total proteins from a 40 mM imidazole elution were desalted using a PD-10 column (EMD Millipore) and 100 mM sodium phosphate buffer [pH 7.4, doi:10.1038/nchembio.1879

10% (v/v) glycerol]. Recombinant proteins were analyzed by SDS-PAGE to assess yield and purity, and immunoblot analysis was performed using a-His primary antibodies (Genscript, catalog number A00186-100; 1:10,000 dilution) and goat-antimouse, horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, catalog number 170-5047; 1:10,000 dilution). Total protein concentration was determined using a BCA Protein Assay kit (Thermo Scientific). Calculated protein concentrations were adjusted based on gel densitometry. Recombinant protein production in Saccharomyces cerevisiae. Freshly transformed yeast cultures harboring pESC-leu2d constructs were grown on an orbital shaker (250 rpm) to log phase in 500 mL of Synthetic Complete dropout medium lacking leucine (SC-leu) and 2% (w/v) glucose. Cultures were subsequently centrifuged (4,000g) and resuspended in 1 L of protein induction medium containing SC-leu supplemented with 0.2% (w/v) glucose, 1% (w/v) raffinose and 1.8% (w/v) galactose. Cultures were grown for an additional 6 h at 28 °C and cells were harvested by centrifugation. Microsomes were prepared based on a standard protocol45. Briefly, after initial treatment with TEK buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 100 mM NaCl, 20 mM KCl, 10 mM MgCl2), cells were re-suspended in 5 mL of cold TESB (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) in a 50 mL tube. Ice-cold 0.5 mm glass beads (Thermo Scientific) were added up to the meniscus and cells were lysed using 1 min rounds of vigorous shaking with intermittent incubations on ice for 1 min. The process was performed for 4 to 7 rounds until at least 70% of the cells were lysed. Glass beads were rinsed 4 times with 5 mL of TESB, insoluble debris was removed by centrifugation at 15,000g and the supernatant was subjected to sucrose-gradient separation of microsomes by ultracentrifugation for 1 h at 45,000g and 4 °C. Microsomal pellets were re-suspended in 1 mL of 50 mM HEPES, pH 7.5, and recombinant proteins were detected by immunoblot analysis46 and assayed immediately. Reductase assays. Standard reduction (forward) reaction assays included 50 ng/ mL purified recombinant protein, 20 mM alkaloid and 500 mM NADPH in 100 mM sodium phosphate buffer, pH 7, in a total volume of 50 mL. Incubations were for 45 s (PsDRR1) or 4 min (PrDRR) at 37 °C. For oxidative (reverse) reaction assays, 20 mM alkaloid and 500 mM NADP+ were used as substrates in 100 mM citratesodium hydroxide buffer, pH 8.8. Denatured recombinant proteins served as negative controls. Reactions were quenched with 6 volumes of acetonitrile and reduced to dryness. For forward reactions, kinetic parameters were determined using 1,2-dehydroreticuline at concentrations up to 300 µM and at a fixed concentration of 500 mM NADPH. For reverse reactions, kinetic parameters were measured using (R)-reticuline at concentrations up to 300 µM and at a fixed concentration of 500 mM NADP+. Substrate range experiments were conducted using the same assay conditions as above. Cytochrome P450 assays. Standard cytochrome P450 assays were performed using 25 µL of prepared microsomes, 2 mM alkaloid and 250 mM NADPH in 50 mM HEPES, pH 7.5, in a total volume of 50 mL. Incubations were for 1 h at 37 °C. Microsomes prepared from S. cerevisiae YPL154c harboring pESC-leu2d containing only the A. annua CPR served as the negative control. Assays were quenched with 6 volumes of acetonitrile and reduced to dryness. Kinetic parameters were determined using the same assay conditions with up to 64 µM (S)-reticuline and at a fixed concentration of 250 µM NADPH. Enzyme assay analyses. Enzyme assays were analyzed using an Agilent 1200 HPLC coupled to an Agilent 6410B triple-quadrupole MS. Samples were re-suspended to 1 µM alkaloid in solvent A [0.08% (v/v) acetic acid:acetonitrile (95:5)], and 2 µL was injected onto a Hypersil gold SB C18 column (2.1 mm × 50 mm, Thermo Scientific). Analytes were eluted using a gradient of solvent A and solvent B (100% acetonitrile) and at a flow rate of 600 µL/min. Gradient conditions were as follows: solvent B ramped linearly from 0 to 50% (v/v) over 6 min; solvent B increased linearly to 99% by 7 min and remained constant until 8.1 min; solvent B then returned immediately to 0% followed by a 3 min re-equilibration period. Analytes were subjected to positive ion electrospray ionization using optimized source conditions (gas temperature, 350 °C; gas flow rate, 10 L/min; nebulizer gas pressure, 50 psi; capillary voltage 4,000 V, fragmentor voltage 110 V) and were subsequently detected by full scan MS operating in positive polarity. Quadrupole 1 and 2 were set to RF-only with quadrupole 3 scanning from 200 to 700 mass-to-charge (m/z) ratio. Relative enzyme activity was calculated as the percent turnover of each substrate using the formula: product peak area / (substrate peak area + product peak area) × 100. Subsequently, the compound with the highest turnover for each protein was set to 100% and the activity of other substrates was expressed as a percentage of the maximum. For kinetic analyses, a five-point calibration curve of 1,2-dehydroreticuline or (R)-reticuline (500 pM to 5 µM) was established. For chiral separations, assays were analyzed by HPLC-UV using an Agilent 1200 HPLC coupled to a diode array detector. Samples were re-suspended using solvent A containing 0.1% (v/v) diethylamine and 20 mM ammonium bicarbonate to 10 µM, and 20 µL was injected onto a Cellulose-1 chiral column (2.1 mm × 150 mm, Phenomenex). Analytes were eluted at a flow rate of 250 µL/min using isocratic conditions [80% solvent A containing 0.1% (v/v) diethylamine and 20% solvent B (v/v)] and monitored at 284 nm. For kinetic analyses, a five-point calibration curve of (R)-reticuline (10 nM to 100 µM) nature chemical biology

was established. Constants were calculated based on Michaelis-Menten kinetics using GraphPad Prism 5 software. Reaction product identification. The 25 eV collision-induced dissociation (CID) spectra of reaction products were compared with empirical spectra from authentic standards (Supplementary Table 4). Fragment ions were detected between 50 m/z and 5 atomic mass units higher than the m/z of quasi-molecular ions. In some cases, reaction product annotation was based on reported CID spectra, or was inferred when yields were low and neither authentic standards or published spectra were available. As an example of the inference process, the reduced m/z of (S)-­ N-methylcoclaurine by 2 atomic mass units in some assays implied a two-electron oxidation yielding N-methyl-1,2-deydrococlaurine. The empirical CID spectra of the inferred N-methyl-1,2-deydrococlaurine reaction product also displayed similar diagnostic features compared with the 1,2-dehydroreticuline authentic standard. NMR of a-hydroxyreticuline. Two compounds corresponding to m/z 346 were produced in sufficient quantity for isolation by semi-preparative HPLC, although the peaks could not be separated. The dried compounds were dissolved in 220 mL d4-methanol and placed in an 8-inch × 3-mm tube. NMR spectra were acquired at 25 °C using an Agilent DD2 spectrometer (u(1H) = 699.758 MHz, u(13C) = 175.973 MHz) equipped with a 5 mm variable temperature 1H 19F(13C/15N) Triple Resonance Cold Probe and VnmrJ4.0 software. One-dimensional 1H spectra were acquired using the standard VnmrJ4.0 s2pul pulse sequence. The spectrum was acquired over a 7,142.9 Hz window with 71,154 points, a 5 s recycle delay, and 32 transients. Two-dimensional 1H/13C HSQC spectra were acquired at 25 °C using the default VnmrJ4.0 gc2hsqcse pulse sequence. Spectra were acquired with a 7,142.9 Hz window and 2048 points in the direct dimension and a 35,195.8 Hz spectral window and 256 increments in the indirect dimension. Spectra were collected with a 0.64 s recycle delay and 4 transients. Two-dimensional 1H/13C HMBC spectra were acquired using the default VnmrJ4.0 gHMBCAD pulse sequence. Spectra were acquired over a 5,733.9 Hz window and 2,048 points in the direct dimension, and a 42,238.6 Hz window and 256 increments in the indirect dimension. Spectra were collected with a 0.64 s recycle delay and 32 transients. Two-dimensional COSY spectra were acquired using the default VnmrJ4.0 gCOSY pulse sequence. Spectra were acquired over a 7,142.9 Hz window and 2,048 points in the direct dimension, and a 7,142.9 Hz window and 512 increments in the indirect dimension. Spectra were collected with a 0.64 s recycle delay and 2 transients. Two-dimensional ROESY spectra were acquired using the default VnmrJ4.0 ROESY pulse sequence. Spectra were acquired over a 7,142.9 Hz window and 2,048 points in the direct dimension, and a 7,142.9 Hz window and 256 increments in the indirect dimension. Spectra were collected with a 1.5 s recycle delay and 16 transients, and processed using MestReNova software (v9.0.1). All spectra were Fourier transformed, phase corrected, and baseline corrected. Window functions were applied as necessary before Fourier transformation, and all two-dimensional spectra were twofold linear predicted in the F1 dimension before Fourier transformation. Chemical shifts were referenced relative to the residual solvent peaks (d4-MeOD, d(1H) = 3.31 ppm, d(13C) = 49.00 ppm). Virus-induce gene silencing. Virus-induced gene silencing (VIGS) was performed using the tobacco rattle virus (TRV) vector system47. TRV is a bipartite positive sense RNA virus consisting of TRV RNA1 (pTRV1) and TRV RNA2 (pTRV2). pTRV1 encodes an RNA-dependent RNA polymerase, which upon systemic infection of the plant permits propagation of target sequence cloned into the multiple cloning site of pTRV2 (ref. 48). A unique sequence encompassing parts of the 5ʹ-untranslated region and open reading frame was used for the construction of a pTRV2 vector to suppress PsREPI transcript levels (pTRV2-REPI-5ʹ). Alternatively, homologous regions of the PsREPI and PsCOR1.1 were used to construct two additional pTRV2 vectors (pTRV2-REPI-a and pTRV2-COR1.1). Fragments of COR1.1 and PsREPI were amplified using primers listed in Supplementary Table 5. Amplicons were

individually cloned into pTRV2, and assembled vectors were transformed into Agrobacterium tumefaciens49. Apical meristems and young leaves of 2- to 3-week-old seedlings were infiltrated50. Empty pTRV2 was used as a negative control, and the pTRV2-PDS construct encoding p ​ hytoene desaturase was used as a positive infiltration control49. Infiltrated plants were cultivated in the greenhouse for 8 to 12 weeks. Latex, root and stem samples were harvested for alkaloid and transcript analyses49,50. Typically, 20 to 30 plants were infiltrated with A. tumefaciens harboring pTRV1 and one pTRV2 construct. In approximately 70% to 80% of the infiltrated plants, a mobilized fragment of the pTRV2 construct was detected by RT-PCR using the primers listed in Supplementary Table 5. Relative transcript abundance was determined by qRT-PCR. Chromatography and mass spectrometry data used for alkaloid analysis of plants subjected to VIGS are provided in Supplementary Table 6. Sixteen biological replicates and three technical replicates were analyzed for each construct using three reference genes: polyubiquitin 10, ELF-1a, and actin. Efficiencies for all primer sets were approximately equal and always >90%. The entire VIGS experiment was performed in duplicate with essentially identical results. Gene expression analysis. Opium poppy organs from chemotype Veronica12 were flash frozen in liquid N2 before excision. Relative transcript abundance was determined by qRT-PCR using cDNA synthesized from isolated total RNA and the primers listed in Supplementary Table 5. Six biological replicates and three technical replicates were analyzed for each gene using three reference genes: polyubiquitin 10, ELF-1a, and actin. Efficiencies for all primer sets were approximately equal and always >90%. Phylogenetic analysis. Alignments were performed using the ClustalX algorithm51 and phylogenetic relationships were analyzed using the Geneious 6 software package (Biomatters). 42. Morishige, T., Tsujita, T., Yamada, Y. & Sato, F. Molecular characterization of the S-adenosyl-L-methionine:3ʹ-hydroxy-N-methylcoclaurine 4ʹ-Omethyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica. J. Biol. Chem. 275, 23398–23405 (2000). 43. Choi, K.B., Morishige, T., Shitan, N., Yazaki, K. & Sato, F. Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica. J. Biol. Chem. 277, 830–835 (2002). 44. Ro, D.K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006). 45. Pompon, D., Louerat, B., Bronine, A. & Urban, P. Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 272, 51–64 (1996). 46. Dang, T.T.T. & Facchini, P.J. Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy. FEBS Lett. 588, 198–204 (2014). 47. Liu, Y., Schiff, M. & Dinesh-Kumar, S.P. Virus-induced gene silencing in tomato. Plant J. 31, 777–786 (2002). 48. Dinesh-Kumar, S.P., Anandalakshmi, R., Marathe, R., Schiff, M. & Liu, Y. Virus-induced gene silencing. Methods Mol. Biol. 236, 287–294 (2003). 49. Desgagné-Penix, I. & Facchini, P.J. Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy. Plant J. 72, 331–344 (2012). 50. Farrow, S.C. & Facchini, P.J. Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy. J. Biol. Chem. 288, 28997–29012 (2013). 51. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882 (1997).

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