Papaverine 7-O-Demethylase, a Novel 2-Oxoglutarate/Fe2+-Dependent Dioxygenase from Opium Poppy ⇑ Scott C

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FEBS Letters 589 (2015) 2701–2706

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Papaverine 7-O-demethylase, a novel 2-oxoglutarate/Fe2+-dependent dioxygenase from opium poppy ⇑ Scott C. Farrow, Peter J. Facchini

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada article info abstract

Article history: Opium poppy (Papaver somniferum) produces several pharmacologically important benzylisoquino- Received 17 June 2015 line alkaloids including the vasodilator papaverine. Pacodine and palaudine are tri-O-methylated Revised 24 July 2015 analogs of papaverine, which contains four O-linked methyl groups. However, the biosynthetic ori- Accepted 27 July 2015 gin of pacodine and palaudine has not been established. Three members of the 2-oxoglutarate/Fe2+- Available online 8 August 2015 dependent dioxygenases (2ODDs) family in opium poppy display widespread O-dealkylation activity Edited by Ulf-Ingo Flügge on several benzylisoquinoline alkaloids with diverse structural scaffolds, and two are responsible for the antepenultimate and ultimate steps in morphine biosynthesis. We report a novel 2ODD from opium poppy catalyzing the efﬁcient substrate- and regio-speciﬁc 7-O-demethylation of papaverine Keywords: O 2-Oxoglutarate/Fe2+-dependent yielding pacodine. The occurrence of papaverine 7- -demethylase (P7ODM) expands the enzymatic dioxygenase scope of the 2ODD family in opium poppy and suggests an unexpected biosynthetic route to Papaverine pacodine. Pacodine Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Benzylisoquinoline alkaloid metabolism Opium poppy

1. Introduction fully established [5–11]. Two routes have been proposed; (i) the N-methyl pathway purports that papaverine is derived via the cen- Opium poppy (Papaver somniferum) remains the sole commer- tral branch-point intermediate (S)-reticuline, from which mor- cial source for pharmaceutical opiates owing to a unique ability phine and noscapine are also produced [7], whereas (ii) the N- among plants to produce morphine and codeine [1,2]. The plant desmethyl pathway proposes that the upstream pathway interme- also produces other benzylisoquinoline alkaloids (BIAs) with diate (S)-coclaurine serves as the branch point to papaverine diverse pharmacological activities including the cough suppressant [5,6,8,10]. Radioactive precursor labeling [5,6], gene-suppression and promising anticancer agent noscapine, the antimicrobial san- [8] and comparative transcriptomics studies [10] support the N- guinarine, and the vasodilator and antispasmodic papaverine [3]. desmethyl pathway as the major route to papaverine. Papaverine is tetra-O-methylated at the C6, C7, C30 and C40 posi- Opium poppy also accumulates low levels of the papaverine tions (Fig. 1). In addition of O-linked methyl groups, the analogues pacodine and palaudine, which lack O-linked methyl 1-benzylisoquinoline scaffold of papaverine contains a 30-hydroxyl, groups at C7 and C30 respectively (Fig. 1). The formation of paco- and 1,2- and 3,4-desaturations yielding three fully conjugated rings dine and palaudine was logically proposed to proceed via the dehy- compared with (S)-norcoclaurine, the initial tri-cyclic intermediate drogenation of their corresponding tri-O-methylated precursors from which all BIAs are derived. Whereas the biosynthetic pathways (S)-norcodamine and (S)-norlaudanine respectively [6]. In support leading to morphine and noscapine have been largely elucidated [4], of this proposal, suppression of the gene encoding (S)-norreticuline the relatively less complex formation of papaverine has not been 7-O-methyltransferase (N7OMT), which is responsible for the substrate and regio-specific O-methylation of (S)-norreticuline yielding (S)-norcodamine, led to a reduced accumulation of Abbreviations: P7ODM, papaverine 7-O-demethylase; 2ODD, 2-oxoglutarate/Fe2 +-dependent dioxygenase; BIA, benzylisoquinoline alkaloid; CID, collision induced papaverine and pacodine in opium poppy [8]. A flavoprotein- dissociation; F7ODM, flavone 7-O-demethylase; N7OMT, (S)-norreticuline 7-O- oxidase from opium poppy, named tetrahydropapaverine oxidase methyltransferase; TPOX, tetrahydropapaverine oxidase (TPOX) in this context, was recently shown to dehydrogenate Author contributions: SCF performed all experiments. SCF and PJF jointly designed (S)-tetrahydropapaverine yielding papaverine [9]. However, the study and drafted the manuscript. (S)-norlaudanine and (S)-norcodamine were not tested as alterna- ⇑ Corresponding author. Fax: +1 403 220 7651. tive substrates. E-mail address: [email protected] (P.J. Facchini). http://dx.doi.org/10.1016/j.febslet.2015.07.042 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2702 S.C. Farrow, P.J. Facchini / FEBS Letters 589 (2015) 2701–2706

Fig. 1. Proposed biosynthesis of papaverine, pacodine and palaudine from (S)-norcoclaurine in opium poppy showing conversions catalyzed by tetrahydropapaverine oxidase (TPOX) and papaverine 7-O-demethylase (P7ODM). Dashed arrows represent multiple enzymatic steps. The question mark indicates an unproven conversion.

The characterization of 2-oxoglutarate/Fe2+-dependent dioxy- transcriptome database using the CODM (ADD85331) amino acid genases (2ODDs) catalyzing the widespread O-dealkylation of sequence as a query. DIOX7 was amplified from Bea’s Choice stem diverse BIAs in opium poppy [12,13] prompted us to consider the cDNA using Phusion DNA polymerase and primers listed in possibility that pacodine and palaudine were formed via the Table S1. Amplicons were A-tailed using green Taq DNA poly- O-demethylation of papaverine. We report the isolation and char- merase, ligated into pGEM-T Easy (Promega), and cloned in acterization of a novel 2ODD from opium poppy that efficiently Escherichia coli XL-1 Blue (Agilent). Isolated plasmids were digested catalyzes the substrate- and regio-specific 7-O-demethylation of with SacI/KpnI restriction endonucleases and the DIOX7 fragment papaverine yielding pacodine. was ligated into the pET47b (EMD Millipore) expression vector, which was used to transform E. coli Arctic Express (Agilent). 2. Material and methods Bacteria producing DIOX7 were grown on an orbital shaker (200 rpm) at 37 °C in one liter of LB broth containing gentamicin, 2.1. Plants and chemicals streptomycin and kanamycin. During the logarithmic growth phase, recombinant gene expression was initiated by adding iso- Opium poppy chemotypes Bea’s Choice and Veronica were culti- propyl b-D-1-thiogalactopyranoside (IPTG) to a final concentration vated as described previously [14]. Papaverine was purchased from of 0.3 mM. Cultures were grown for 24 h at 10 °C at 200 rpm. Sigma. All other alkaloids were obtained as described previously Subsequently, bacterial pellets were re-suspended in protein [13]. Restriction endonucleases and Phusion DNA polymerase were extraction buffer [100 mM Tris–HCl, pH 7.4, 10% (v/v) glycerol, from New England Biolabs. Green Taq DNA polymerase was from 10 mM b-mercaptoethanol [BME], 1 mM phenylmethanesulfonyl Genscript. T4 DNA ligase and moloney murine leukemia virus fluoride], and cells were lysed at 4 °C using a French pressure cell reverse transcriptase were from Invitrogen. Talon resin was pur- (1000 psi). Recombinant protein was purified as described previ- chased from Clontech. All other reagents and materials were pur- ously [13] and desalted using a PD-10 column (GE Healthcare Life chased from Sigma, VWR, ThermoFisher, or BioShop. Sciences) with elution in 100 mM Tris–HCl, pH 7.4, 10 mM BME, and 10% (v/v) glycerol. Proteins were visualized by SDS–PAGE and 2.2. Cloning and expression of DIOX7 recombinant DIOX7 was detected by immunoblot analysis as described previously [4] (Fig. S1). Protein concentration was deter- A novel 2ODD candidate initially designated DIOX7 was identi- mined using a BCA protein assay kit (Thermo), and purified proteins fied by a tBLASTn search of an opium poppy Bea’s Choice root were used immediately or stored at 80 °C for later use. S.C. Farrow, P.J. Facchini / FEBS Letters 589 (2015) 2701–2706 2703

2.3. DIOX7 enzyme assay and 32 transients. The 1D 13C spectrum was acquired over a 41666.7 Hz spectral window with 250000 points, a 0.2 s recycle Standard DIOX7 assays were performed as described previously delay, and 512 transients. Two-dimensional (2D) 1H/13C HSQC for other 2ODDs with denatured proteins serving as negative con- spectra were acquired at 25 °C using the default VnmrJ4.0 gc2h- trols [13]. Optimal pH and temperature were determined using sqcse pulse sequence. Spectra were acquired with a 7142.9 Hz otherwise standard assay conditions. Assays were quenched with spectral window and 2048 points in the direct dimension and a six volumes of acetonitrile, reduced to dryness in a vacuum con- 35195.8 Hz spectral window and 128 increments in the indirect centrator and stored at 20 °C prior to analysis. Kinetic parameters dimension. Spectra were collected with a 0.64 s recycle delay, for DIOX7 were determined using papaverine at concentrations up and 4 transients. The 2D 1H/13C HMBC spectra were acquired using to 400 lM and a fixed concentration of 500 lM ketoglutarate the default VnmrJ4.0 gHMBCAD pulse sequence, over a 7142.9 Hz (a-KG). Briefly, assays were incubated at 25 °C in 100 mM glycine spectral window and 2048 points in the direct dimension and a buffer, pH 8.0, containing 100 ng lL1 recombinant DIOX7 protein, 42238.6 Hz spectral window and 256 increments in the indirect 10 mM sodium ascorbate, 500 lM iron sulfate, and10 mM BME. dimension. Spectra were collected with a 0.64 s recycle delay, Assays were quenched with six volumes of acetonitrile, evaporated and 8 transients. The 2D COSY spectra were acquired using the in a vacuum concentrator and stored at 20 °C until analysis. default VnmrJ4.0 gCOSY pulse sequence, over a 7142.9 Hz spectral window and 2048 points in the direct dimension and a 7142.9 Hz 2.4. LC–MS analysis of enzyme assays spectral window and 256 increments in the indirect dimension. Spectra were collected with a 0.64 s recycle delay, and 2 transients. Enzymes assays were analyzed by HPLC–MS as described previ- The 2D ROESY spectra were acquired using the default VnmrJ4.0 ously [13]. For kinetic parameter analyses, a five-point calibration ROESY pulse sequence, over a 7142.9 Hz spectral window and curve for papaverine (500 pM–5 lM) was used to determine the 2048 points in the direct dimension and a 7142.9 Hz spectral win- amount of product formed per unit of time. Kinetic constants were dow and 256 increments in the indirect dimension. Spectra were calculated based on Michaelis–Menten kinetics using Microsoft collected with a 1.5 s recycle delay, 8 transients, and a 0.2 s mixing Excel and GraphPad Prism 5. time. NMR spectra were processed using MestReNova NMR pro- cessing software (v10.0.1). All spectra were Fourier transformed, 2.5. Reaction product identification phase corrected, and baseline corrected. Window functions were applied as necessary prior to Fourier transformation and all 2D The DIOX7 reaction product of papaverine as the substrate was spectra were twofold linear predicted in the F1 dimension prior identified using mass spectrometry and NMR. Comparison of the to Fourier transformation. Chemical shifts were referenced relative 1 collision induced dissociation (CID) spectra of the reaction product to the residual solvent peaks (d4-methanol, d( H) = 3.31 ppm, d with empirical spectra available in the literature [15] provided the (13C) = 49.00 ppm). initial reaction product identification. CID data were obtained at 25 eV in MS2 whereby fragment ions were detected between 50 2.6. Gene expression analysis m/z and five atomic mass units higher than the m/z of the quasi- molecular ion. To obtain sufficient reaction product for NMR Relative transcript levels in various organs of the opium poppy analysis, DIOX7 was used to convert 15 mg of papaverine under Veronica chemotype [16] were performed on six biological repli- scaled-up standard assay conditions [500 lM a-ketoglutarate, cates by quantitative real-time PCR (qRT-PCR) as described previ- 1 mM sodium ascorbate, 500 lM iron sulfate and 44 mL glycine ously [14], and using primers listed in Table S1. Polyubiquitin 10 buffer (100 mM, pH 8.0, and 10 mM BME)] containing 5 mL of puri- and elongation factor-1a were used as reference genes and yielded fied recombinant enzyme (200 ng lL1). After 16 h, the reaction consistent results for all target genes. Efficiencies for all primer sets was filtered through a 0.22 lM PVDF filter (Millipore), spiked with were approximately equal and always >90%. 5% (v/v) HCl, and the solution was subjected to solid phase extraction using Strata-XC cartridges (6 mL, 300 mg, Phenomenex). 2.7. LC/MS analysis of papaverine and pacodine Cartridges were equilibrated with methanol followed by 5% (v/v) HCl. Samples were loaded onto the cartridges, washed with 5% Papaverine and pacodine levels in different organs of the opium (v/v) HCl and 100% methanol, and the reaction product was eluted poppy chemotype Veronica were determined by HPLC–MS/MS. with 50:50 acetonitrile:methanol containing 5% (v/v) ammonium Opium poppy tissues were flash frozen and ground to a powder hydroxide, dried in a vacuum concentrator, and stored at 20 °C. under liquid nitrogen using a TissueLyser II (Qiagen) at 30 Hz for Sample residue was re-suspended in 0.08% (v/v) acetic acid con- 1 min with a 30 mL grinding jar and a 10-mm stainless steel ball. taining 5% (v/v) acetonitrile and the reaction product was purified Ground plant material was lyophilized and extracted with 30 mL on an Agilent 1200 equipped with a Luna C18(2) semi-preparative acetonitrile per gram dry weight. Extracts were incubated for column (5 lm, 25 10 mm, Phenomenex). Sample was injected 16 h at 20 °C and subsequently centrifuged at 4 °C and onto the column in 100 lL aliquots and pacodine was separated 14000g for 10 min to remove insoluble debris. The supernatant with a gradient of 0.08% acetic acid/acetonitrile (95/5, v/v, solvent was transferred to a new tube and evaporated to dryness under A) and acetonitrile (solvent B). The elution profile was monitored vacuum. Samples were reconstituted in 10 mM ammonium acetate by absorbance at 210 and 284 nm. Fractions containing the reac- (pH 5.5) containing 5% (v/v) acetonitrile, and 10 lL were injected tion product were pooled and lyophilized. onto a Hypersil Gold C18 HPLC column (1.8 lm, 2.1 50 mm; For NMR analysis, the dried powder was dissolved in 220 lLof Thermo), and compounds were separated using a gradient elution d4-methanol (Cambridge Isotope Laboratories) and placed in an of 10 mM ammonium acetate (pH 5.5) containing 5% (v/v) acetoni- 8 inch 3 mm NMR tube. NMR spectra were acquired immedi- trile [solvent A] and acetonitrile [solvent B]. The flow rate was ately at 25 °C using an Agilent DD2 spectrometer (t(1H) 500 lL min1 and the gradient was started at 0% solvent B, which = 699.809 MHz, t(13C) = 175.983 MHz) equipped with a 5 mm vari- was increased linearly to 7.5% (v/v) by 1.5 min and then immedi- able temperature 1H-19F{13C/15N} Triple Resonance Cold Probe and ately to 25% by 1.6 min. Solvent B was increased linearly to 33% VnmrJ4.0 acquisition software using the standard sequences. The by 5 min, 92% by 7 min, and 98% by 7.6 min. At 8.6 min solvent B one-dimensional (1D) 1H NMR spectrum was acquired over a was reduced to initial conditions by 8.7 min for 4 min of re- 7142.9 Hz spectral window with 71154 points, a 5 s recycle delay, equilibration. Analytes were subjected to multiple reaction 2704 S.C. Farrow, P.J. Facchini / FEBS Letters 589 (2015) 2701–2706 monitoring using transitions established for papaverine (m/z 340.2 > 202.1 and 340.2 > 324.1) and pacodine (326.1 > 188.1 and 326.2 > 310.1) and quantifications were performed using a calibration curve of authentic papaverine.

3. Results

3.1. Isolation of DIOX7

The DIOX7 cDNA contained an open reading frame of 1110 nucleotides encoding a polypeptide of 364 amino acids with a predicted molecular weight of 41.3kDa (Fig. S1). DIOX7 contained the catalytic triad HX(D/E)XnH motif conserved in all 2ODDs [17] and the a-KG binding domain predicted from the crystal structure from Arabidopsis thaliana leucoanthocyanidin dioxygenase [18]. DIOX7 shares considerable sequence identity with previously characterized O-demethylases and O,O-demethylenases from opium poppy: codeine O-demethylase (CODM; 75%), protopine O-dealkylase (PODA; 73%) and thebaine 6-O-demethylase (T6ODM; 71%). The only other characterized 2ODD O-demethylase is ﬂavone 7-O-demethylase (F7ODM) from sweet basil (Ocimum basilicum) [19], which shares 49% sequence identity with DIOX7.

3.2. Expression and characterization of DIOX7

Affinity purified, epitope-tagged DIOX7 was detected by SDS– PAGE and immunoblot analysis, and displayed an empirical molecular weight of approximately 41kDa (Fig. S1). In the presence of a-KG and other reaction components required by 2ODDs, DIOX7 converted papaverine (m/z 340) to a reaction product at m/z 326 (Fig. 2A). The loss of 14 atomic mass units from papaverine (m/z 340) suggested that O-demethylation had occurred, which was supported by CID analysis of the reaction product (m/z 326) indicating the loss of an O-linked methyl group from the A-ring of papaverine (Fig. 2B). Although the CID spectrum could not be used to determine whether O-demethylation occurred at C6 or C7, NMR analysis on the purified reaction product showed that DIOX7 was specific for the C7 O-linked methyl group, and the product was unequivocally identified as pacodine (Fig. 2B and Table S2). Since Fig. 2. (A) HPLC–MS-extracted ion chromatograms of enzyme assays using native no other alkaloids were accepted as substrates (Fig. S2), DIOX7 or denatured P7ODM with papaverine (m/z 340) as the substrate showing O- was renamed papaverine 7-O-demethylase (P7ODM). demethylation and the formation of pacodine (m/z 326). (B) Identification of reaction product using papaverine as the substrate by collision-induced dissociation mass spectrometry showing the loss of a methyl group from the A-ring of 3.3. Biochemical properties of P7ODM papaverine.

P7ODM activity was stable across a broad temperature range from 0 to 25 °C, but was steadily inactivated along a linear temper- 3.5. Pacodine and papaverine levels in different opium poppy organs ature gradient from between approximately 30 and 55 °C(Fig. 3A). P7ODM also showed optimal activity between pH 8 and 9 in gly- Papaverine levels were more than three hundred-fold higher in cine buffer (Fig. 3B). Kinetic analysis of P7ODM was in agreement all organs compared with pacodine (Fig. 4C). Interestingly, both with the Michaelis–Menten model (Fig. 3C) and exhibited a Km of alkaloids showed a similar distribution profile with higher levels 1 83 ± 13, a Vmax of 5.53 ± 0.38 picokatal, a Kcat of 0.09 s , and an in flower buds, followed by stems, leaves and roots (Fig. 4C). The 1 1 enzymatic efficiency (Kcat/Km) of 1099 s M . Substrate or pro- accumulation of papaverine and pacodine were significantly duct inhibition was also detected (Fig. 3C). The kinetic values for (P < 0.05) lower in root compared with aerial organs. P7ODM fall within the range of those calculated for T6ODM, CODM and PODA, and the presence of product or substrate inhibi- 4. Discussion tion appears to be a common feature among the characterized opium poppy O-demethylases [12,13]. The limited substrate acceptance of P7ODM with papaverine (Fig. S2) is surprising considering the broad substrate range 3.4. P7ODM transcript levels in different opium poppy organs observed for T6ODM, CODM and PODA [13], suggesting that P7ODM has more stringent requirements for substrate docking P7ODM transcript levels were low in all organs, although stem and coordination in the active site. In a previous study [13], showed lower abundance than roots and leaves (Fig. 4A and T6ODM and PODA showed low and trace O-dealkylation activity Fig. S3A). Compared with CODM and T6ODM, transcript levels with (R,S)-tetrahydropapaverine and papaverine, respectively, of P7ODM were substantially lower in all organs (Fig. 4B and indicating that minor amino acid substitutions between P7ODM Fig. S3B). and T6ODM/PODA facilitate the efficient O-dealkylation of S.C. Farrow, P.J. Facchini / FEBS Letters 589 (2015) 2701–2706 2705

is not known whether TPOX (Fig. 1) catalyzes the dehydrogenation of (S)-norcodamine to form pacodine; thus, dehydrogenation of (S)-norcodamine must also be considered as a possible route to pacodine. The low P7ODM transcript levels in opium poppy plants (Fig. 4) suggest a potential requirement of conditional cues for gene expression. Biosynthetic genes involved in plant specialized metabolism are often inducible [21,22]. Signiﬁcantly, a massive induction in gene expression, including those encoding several BIA biosynthetic enzymes, is triggered by treatment of opium poppy cell cultures with a fungal elicitor [23]. Although cDNAs encoding P7ODM were not detected in the elicitor-induced opium poppy cell culture EST database [23], alternative sources of induction might occur in the plant. The broad temperature optimum of P7ODM suggests that abiotic stressors could be required for P7ODM expression. In A. thaliana, cold stress up-regulates several specialized

Fig. 3. In vitro characterization of afﬁnity-puriﬁed P7ODM. (A) Temperature optimum. (B) pH optimum. (C) Steady-state enzyme kinetics. Values represent the mean ± S.D. of three replicates.

papaverine by P7ODM. Insight into residues conferring stringency could be obtained through mutagenizing the regions of T6ODM and PODA thought to confer substrate specificity. A redesign of CODM has already been achieved by similar techniques [20], and when referencing the CODM sequence [17], substitutions to amino acids in non-conserved regions of P7ODM provide rational targets for understanding the O-dealkylation of papaverine by P7ODM. The biosynthesis of pacodine and palaudine has been attributed to the aromatization of (S)-norcodamine and (S)-norlaudanine, respectively [5,6,8]. However, TPOX did not accept (S)-norreticuline [9] suggesting that tetra-O-methylation of the papaveroline scaffold is required for dehydrogenation (Fig. 1). The discovery of P7ODM is the first direct biochemical evidence for the formation of pacodine via the direct 7-O-demethylation of papaverine (Fig. 1). Corroboration of the biochemical data (Fig. 3) using virus- induced gene silencing (VIGS) was not possible owing to the low P7ODM transcript levels in opium poppy plants (Fig. 4). However, the calculated Km of P7ODM is within the physiological concentration of papaverine [13,16] and the calculated efficiency for the reaction supports an activity for native P7ODM in papaverine Fig. 4. Relative abundance of P7ODM transcripts in opium poppy determined using O-demethylation. Furthermore, the low concentration of pacodine qRT-PCR with elongation factor-1a as the reference gene. Values represent the (Fig. 4C) is in agreement with the low P7ODM transcript abundance mean ± S.D. of six biological replicates. (A) Relative levels of P7ODM transcripts in in all organs (Fig. 4A and Fig. S3A) supporting a role for P7ODM in different plant organs. Values with the same letter were not significantly different (P > 0.05) as determined using a two-tailed, unpaired t-test. (B) Relative levels of pacodine synthesis. Interestingly, papaverine and pacodine accu- P7ODM, T6ODM and CODM transcripts in different organs. (C) Papaverine and mulation show the same general profile in different organs pacodine levels in different opium poppy organs. Values represent the mean ± S.D. (Fig. 4C), which might correlate with the occurrence of P7ODM. It of six biological replicates. 2706 S.C. Farrow, P.J. Facchini / FEBS Letters 589 (2015) 2701–2706 metabolism genes including 2ODDs that operate in flavonoid and 1-benzylisoquinolines in Papaver somniferum. J. Chem. Soc. Perkin Trans. 1, glucosinolate biosynthesis, and a 2ODD potentially involved in 1531–1537. [7] Han, X., Lamshoft, M., Grobe, N., Ren, X., Fist, A.J., Kutchan, T.M., Spiteller, M. alkaloid biosynthesis [24]. and Zenk, M.H. (2010) The biosynthesis of papaverine proceeds via In general, the O-demethylation activity of P7ODM and related (S)-reticuline. Phytochemistry 71, 1305–1312. 2ODDs from opium poppy could be part of a transmethylation sys- [8] Desgagné-Penix, I. and Facchini, P.J. (2012) Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to tem involved in the maintenance of steady-state alkaloid levels papaverine in opium poppy. Plant J. 72, 331–344. [25]. As suggested for the biosynthesis of sanguinarine [13], [9] Hagel, J.M., Beaudoin, G.A.W., Fossati, E., Ekins, A., Martin, V.J.J. and Facchini, P. O-dealkylation might also alleviate the accumulation of pathway J. (2012) Characterization of a flavoprotein oxidase from opium poppy catalyzing the final steps in sanguinarine and papaverine biosynthesis. J. intermediates that cause feedback regulation in BIA metabolism. Biol. Chem. 287, 42872–42983. Papaverine has widespread uses in medicine including for the [10] Pathak, S., Lakhwani, D., Gupta, P., Mishra, B.K., Shukla, S., Asif, M.H. and treatment of erectile dysfunction [26], smooth muscle spasm Trivedi, P.K. (2013) Comparative transcriptomics analysis using high papaverine mutant of Papaver somniferum reveals pathway and [27], is a cryo-preservative for blood vessels [28], is used in uncharacterized steps of papaverine biosynthesis. PLoS ONE 8, e65622. angioplasty and coronary artery surgery [29], and is a second line [11] Winterstein, E. and Trier, G. (1910) Die Alkaloide: Eine Monographie der prophylaxis for migraine headaches [30]. Alternatively, the Naturlichen Basen, Borntrager, Berlin, DE, p. 307, Borntrager, Berlin, DE. pharmacological properties of pacodine have not been reported. [12] Hagel, J.M. and Facchini, P.J. (2010) Dioxygenases catalyze the O-demethylation steps of morphine biosynthesis in opium poppy. Nat. However, given the structural similarity to papaverine, pacodine Chem. Biol. 6, 273–275. or analogs thereof might exhibit similar or novel biological [13] Farrow, S.C. and Facchini, P.J. (2013) Dioxygenases catalyze O-demethylation activities. and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism. J. Biol. Chem. 288, 28997–29012. Previous phylogenetic analysis of 2ODDs from opium poppy [14] Dang, T.-T.T. and Facchini, P.J. (2012) Characterization of three and other species [19] showed a close relationship between O-methyltransferases involved in noscapine biosynthesis in opium poppy. F7ODM from sweet basil and opium poppy 2ODDs, which suggests Plant Physiol. 159, 618–631. [15] Wickens, J.R., Sleeman, R. and Keely, B.J. (2006) Atmospheric pressure a conservation of features required for O-dealkylation. With chemical ionization mass spectrometric fragmentation pathways of several available 2ODDs to compare, elucidation of the structural noscapine and papaverine revealed by multistage mass spectrometry and basis for O-dealkylation is possible. In a broader context, the in-source deuterium labeling. Rapid Commun. Mass Spectrom. 20, 473–480. [16] Desgagné-Penix, I., Farrow, S.C., Cram, D., Nowak, J. and Facchini, P.J. (2012) O-dealkylation of small molecules is considered rare in plant Integration of deep transcript and targeted metabolite profiles for eight metabolism [19], yet all plants contain several uncharacterized cultivars of opium poppy. Plant Mol. Biol. 79, 295–313. 2ODDs [17]. A fundamental understanding of protein features [17] Farrow, S.C. and Facchini, P.J. (2014) Functional diversity of 2-oxoglutarate/Fe (II)-dependent dioxygenases in plant metabolism. Front. Plant Met. Chemodiv. conferring O-dealkylation reactions will also allow prediction of 524, 1–15. O-dealkylation functions among uncharacterized 2ODDs. [18] Wilmouth, R.C., Turnbull, J.J., Welford, R.W.D., Clifton, I.J., Prescott, A.G. and Schofield, C.J. (2002) Structure and mechanism of anthocyanidin synthase Acknowledgements from Arabidopsis thaliana. Structure 10, 93–103. [19] Berim, A., Kim, M-.J. and Gang, D.R. (2015) Identification of a unique 2-oxoglutarate-dependent flavone 7-O-demethylase completes the elucidation We thank Dr. Darcy Burns (University of Toronto) for assistance of the lipophilic flavone network in basil. Plant Cell Physiol. 56, 126–136. with the interpretation of NMR spectra. This work was supported [20] Runguphan, W., Glenn, W.S. and O’Connor, S.E. (2012) Redesign of a dioxygenase in morphine biosynthesis. Chem. Biol. 19, 674–678. through funding from the Natural Sciences and Engineering [21] Van der Fits, L. and Memelink, J. (2000) ORCA3, a jasmonate-responsive Research Council of Canada. P.J.F. holds the Canada Research transcriptional regulator of plant primary and secondary metabolism. Science Chair in Plant Metabolic Processes Biotechnology. S.C.F. is the 5477, 295–297. [22] Cheong, Y.H., Chang, H.-S., Gupta, R., Wang, X., Zhu, T. and Luan, S. (2002) recipient of a Natural Sciences and Engineering Research Council Transcriptional profiling reveals novel interactions between wounding, of Canada Alexander Graham Bell Graduate Scholarship and an pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Alberta Innovates Technology Futures Graduate Scholarship. Physiol. 129, 661–677. [23] Zulak, K.G., Cornish, A., Daskalchuk, T.E., Deyholos, M.K., Goodenowe, D.B., Gordon, P.M.K., Klassen, D., Pelcher, L.E., Sensen, C.W. and Facchini, P.J. (2007) Appendix A. Supplementary data Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism. Planta 225, 1085–1106. 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