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Originally published In Press as doi:10.1074/jbc.M611908200 on March 27, 2007

J. Biol. Chem., Vol. 282, Issue 20, 14741-14751, May 18, 2007
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Molecular Cloning and Characterization of Tetrahydroprotoberberine cis-N-Methyltransferase, an Enzyme Involved in Alkaloid Biosynthesis in Opium Poppy*Formula

David K. Liscombe1 and Peter J. Facchini2

From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

Received for publication, December 29, 2006 , and in revised form, March 23, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
S-Adenosyl-L-methionine:tetrahydroprotoberberine cis-N-methyltransferase (EC 2.1.1.122 [EC] ) catalyzes the conversion of (S)-stylopine to the quaternary ammonium alkaloid, (S)-cis-N-methylstylopine, as a key step in the biosynthesis of protopine and benzophenanthridine alkaloids in plants. A full-length cDNA encoding a protein exhibiting 45 and 48% amino acid identity with coclaurine N-methyltransferase from Papaver somniferum (opium poppy) and Coptis japonica, respectively, was identified in an elicitor-treated opium poppy cell culture expressed sequence tag data base. Phylogenetic analysis showed that the protein belongs to a unique clade of enzymes that includes coclaurine N-methyltransferase, the predicated translation products of the Arabidopsis thaliana genes, At4g33110 and At4g33120, and bacterial S-adenosyl-L-methionine-dependent cyclopropane fatty acid synthases. Expression of the cDNA in Escherichia coli produced a recombinant enzyme able to convert the protoberberine alkaloids stylopine, canadine, and tetrahydropalmatine to their corresponding N-methylated derivatives. However, the protoberberine alkaloids tetrahydroxyberbine and scoulerine, and simple isoquinoline, benzylisoquinoline, and pavine alkaloids were not accepted as substrates, demonstrating the strict specificity of the enzyme. The apparent Km values for (R,S)-stylopine and S-adenosyl-L-methionine were 0.6 and 11.5 µM, respectively. TNMT gene transcripts and enzyme activity were detected in opium poppy seedlings and all mature plant organs and were induced in cultured opium poppy cells after treatment with a fungal elicitor. The enzyme was detected in cell cultures of other members of the Papaveraceae but not in species of related plant families that do not accumulate protopine and benzophenanthridine alkaloids.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The benzylisoquinoline alkaloids are a large and diverse group of plant secondary metabolites, many of which possess potent pharmacological and other bioactive properties. Opium poppy (Papaver somniferum) is an extensively cultivated medicinal plant that produces more than 80 benzylisoquinoline alkaloids, including the narcotic analgesics morphine and codeine and the antimicrobial sanguinarine. The effectiveness of sanguinarine as an inhibitor of fungal and bacterial growth suggests that it protects the plant against pathogens (1) and has prompted its use in oral hygiene products as an anti-plaque agent (2).

Sanguinarine biosynthesis begins with the condensation of dopamine and 4-hydroxyphenylacetaldehyde by norcoclaurine synthase (NCS)3 (3, 4) to yield (S)-norcoclaurine (Fig. 1). Dopamine is formed by the decarboxylation of tyrosine and/or dihydroxyphenylalanine by tyrosine/dopa decarboxylase (5). Norcoclaurine 6-O-methyltransferase (6OMT) and coclaurine N-methyltransferase (CNMT) convert (S)-norcoclaurine to (S)-N-methylcoclaurine (6–8). The cytochrome P450-dependent monooxygenase (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B3) catalyzes the 3'-hydroxylation of (S)-N-methylcoclaurine (9, 10) prior to the formation of (S)-reticuline by 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT) (8, 11, 12). (S)-Reticuline represents the last common intermediate in the biosynthesis of both sanguinarine and morphine (13). BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in the branch pathway leading to protopine and benzophenanthridine alkaloids, such as sanguinarine (Fig. 1). Alternatively, (S)-reticuline is epimerized to (R)-reticuline in the first steps of the morphinan alkaloid pathway in opium poppy. (S)-Scoulerine is converted to (S)-stylopine by the formation of two methylenedioxy bridges by the cytochrome P450-dependent monooxygenases cheilanthifoline synthase and stylopine synthase (14, 15). (S)-Tetrahydroprotoberberine cis-N-methyltransferase (TNMT) converts (S)-stylopine to (S)-cis-N-methylstylopine, which is subsequently hydroxylated by the cytochrome P450-dependent monooxygenase, N-methylstylopine 14-hydroxylase (16). The initial reaction product tautomerizes to protopine, which is hydroxylated to yield dihydrosanguinarine by another cytochrome P450-dependent monooxygenase, protopine 6-hydroxylase (17). Subsequent oxidation by dihydrobenzophenanthridine oxidase yields sanguinarine (18).


Figure 1
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  		FIGURE 1.
Protopine and benzophenanthridine alkaloid biosynthesis in opium poppy. Biosynthetic enzymes for which corresponding genes have been isolated are labeled. The abbreviations used are: CYP80B3,(S)-N-methylcoclaurine 3'-hydroxylase; TYDC, tyrosine/dopa decarboxylase.

 
Several S-adenosyl-L-methionine (AdoMet)-dependent O-methyltransferases involved in the biosynthesis of a variety of plant secondary metabolites have been characterized at the molecular level (7, 11, 12, 19, 20) and structural levels (21–23). However, few studies have focused on the molecular biochemical characterization of AdoMet-dependent N-methyltransferases (24–27). At least two N-methyltransferases are involved in benzylisoquinoline alkaloid biosynthesis (Fig. 1). CNMT has been purified (28), and corresponding cDNAs have been isolated from Coptis japonica (6), Thalictrum flavum (29), and P. somniferum (8). TNMT protein has been isolated from Eschscholzia californica and Corydalis vaginans cell cultures (30) and purified from Sanguinaria canadensis cell cultures (31). However, a cDNA encoding TNMT has not been reported. TNMT catalyzes the AdoMet-dependent methylation of the tertiary nitrogen of (S)-stylopine to produce the quaternary ammonium compound (S)-cis-N-methylstylopine, which is the ultimate precursor to protopine and the benzophenanthridine alkaloids (32). The transfer of a methyl group to a tertiary nitrogen atom is a relatively rare reaction in plant metabolism. Only two cDNAs encoding plant N-methyltransferases that catalyze the formation of low molecular weight quaternary ammonium compounds have been reported (33, 34). Both of these enzymes are involved in the biosynthesis of the osmoprotective metabolite betaine.

We report the identification and functional characterization of a cDNA encoding TNMT from opium poppy. TNMT is the only AdoMet-dependent N-methyltransferase in plant alkaloid metabolism known to produce a quaternary ammonium compound, and the first enzyme specific to protopine and benzophenanthridine alkaloid biosynthesis to be characterized at the molecular biochemical level.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material—Opium poppy (P. somniferum cv. Marianne) plants were cultivated in growth chambers as described previously (8). Cell suspension cultures were maintained at 23 °C under continuous fluorescent light with a flux rate of 35 µmol s–1 m–2 on 1B5C medium (35) consisting of B5 salts and vitamins, 1 g liter–1 hydrolyzed casein, 20 g liter–1 of sucrose, and 1 mg liter–1 of 2,4-dichlorophenoxyacetic acid (8). P. somniferum and Papaver bracteatum cell cultures were treated with an elicitor prepared from Botrytis cinerea mycelia as described previously (8).

Chemicals—(R,S)-Tetrahydropalmatine (80% (w/w)) from Corydalis yahsusuo was purchased from Ethnogarden Botanical (Barrie, Ontario, Canada). (R,S)-Tetrahydroberberine ((R,S)-canadine), DL-pavine hydrochloride, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, and 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydrobromide were purchased from Sigma-Aldrich. (S)-Scoulerine was purchased from Indofine Chemical (Hillsborough, NJ). S-Adenosyl-L-[methyl-14C]methionine (1850 GBq mol–1) was purchased from American Radiolabeled Chemicals (St. Louis, MO). All other chemicals were purchased from Sigma-Aldrich.

Chemical Synthesis of 2,3,9,10-Tetrahydroxyberbine—(R,S)-Tetrahydropalmatine was demethylated using 6 molar equivalents of BBr3 according to the procedure of McOmie et al. (36). The product was identified by low resolution EI-MS (m/z: 299 (26), 178 (47), 164 (40), 162 (47), 136 (13)), and the loss of O-methyl groups was confirmed by 1H-NMR.

Chemical Synthesis of Stylopine—(R,S)-Stylopine was synthesized from 2,3,9,10-tetrahydroxyberbine based on the method of Castillo et al. (37). Briefly, a solution of 1 g of 2,3,9,10-tetrahydroxyberbine in 10 ml of hexamethylphosphoramide was added to a stirred solution of 0.4 g of NaH in 16 ml of hexamethylphosphoramide over 10 min. Subsequently, CH2I2 (0.9 g) was added, and the solution was stirred for 20 min. The reaction was quenched with ice water (50 ml) and extracted with Et2O (3 x 15 ml). The extract was dried over MgSO4 and concentrated under reduced pressure. Stylopine was precipitated with the addition of CHCl3/MeOH and collected by vacuum filtration through a 0.5-µm FH membrane (Millipore, Bedford, MA). Product identity was confirmed by low resolution EI-MS (m/z: 323 (30), 179 (15), 174 (11), 148 (100)) and was in agreement with previous reports (38–40).

Nucleic Acid Isolation and Analysis—The synthesis and sequencing of an elicitor-treated opium poppy cell culture cDNA library and the assembly of the corresponding expressed sequence tag (EST) data base were described previously (41). A full-length cDNA encoding TNMT was identified by screening the EST data base in silico using the tBLASTn algorithm (42) in DNATools 6.0. CNMT cDNAs from P. somniferum (8), T. flavum (29), and C japonica (6) were used as query sequences. Total RNA was isolated using the method of Cheng and Seemann (43). Gel blot hybridization analysis was performed as described previously (3) using a random-primer 32P-labeled probe derived from the full-length TNMT cDNA.

Heterologous Expression of TNMT and At4g33120 cDNAs—The TNMT open reading frame (ORF) was amplified from the full-length cDNA using Pfx polymerase (Invitrogen). The sense primer contained a BamHI site and was specific for the 5'-end of the ORF (4E9-BamHI; 5'-TAGGATCCAATGGGTTCAATAGATG-3'), whereas the antisense primer contained an XhoI site and was complimentary to the 3'-end (4E9Term; 5'-TACTCGAGCTTCTTCTTGAAAAGCAGC-3'). A full-length cDNA of the Arabidopsis thaliana gene At4g33120 (stock number U22568 [GenBank] ) was obtained from the Arabidopsis Biological Resource Center (Columbus, OH) and used as a template to amplify the corresponding ORF. The sense primer contained a BamHI site and was specific for the 5'-end of the ORF (AtNMT-F; 5'-ATGCGGATCCAATGGAGAAGATTATTGACG-3'), whereas the antisense primer contained an EcoRI site and was complimentary to the 3'-end (AtNMT-R; 5'-TACAGAATTCTCATTTCTTCTTGAAGAGG-3'). Amplicons were ligated into pRSETB (Invitrogen) using the engineered restriction sites to produce pTNMT and pDLAt120, respectively. Escherichia coli strain ER2566 pLysS (New England Biolabs, Beverly, MA) was transformed with pTNMT or pDLAt120. Cultures were shaken at 30 °C in LB medium containing ampicillin (50 µg–1 ml) and chloramphenicol (34 µg–1 ml) to an A600 of 0.6 and subsequently induced with 0.3 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for 4.5 h at 30 °C. Cells were harvested by centrifugation at 5000 x g for 5 min. E. coli strain Rosetta (DE3) pLysS (Novagen, Madison, WI) was transformed with pTNMT, and cultures were shaken at 30 °C in LB medium containing ampicillin (50 µg–1 ml) and chloramphenicol (34 µg–1 ml) to an A600 of 0.6. Cultures were induced with 0.2 mM IPTG for 17 h at 22 °C and harvested at 5000 x g for 5 min. Bacterial pellets were resuspended in buffer A (100 mM Tris-HCl, pH 7.8, 12 mM beta-mercaptoethanol) containing pepstatin A(5 µM) and leupeptin (5 µM) and lysed using a French press at 15,000 p.s.i. Lysates were cleared by centrifugation at 10,000 x g for 20 min and desalted on PD-10 columns (Amersham Biosciences).

Purification of Recombinant TNMT—E. coli ER2566 pLysS cells harboring the pTNMT plasmid were used to produce recombinant TNMT, which was precipitated from the crude bacterial lysate using 20–40% (w/v) ammonium sulfate and resuspended in binding buffer (20 mM NaPO4, pH 7.4; 500 mM NaCl). After desalting, recombinant polyhistidine-tagged TNMT was purified by nickel affinity chromatography using a HiTrap chelating HP column (Amersham Biosciences) according to the manufacturer's instructions and concentrated by ultrafiltration.

Immunoblot Analysis—Soluble proteins (25 µg) were fractionated by SDS-PAGE and transferred to BioTrace NT nitrocellulose membranes (Pall Life Sciences, Pensacola, FL). Protein blots were incubated with mouse monoclonal anti-polyhistidine clone HIS-1 (Sigma) diluted 1:3000 in Blotto (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5% (w/v) milk protein) for 12 h, washed in TBST (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% (v/v) Tween 20), and incubated for 1 h with alkaline phosphatase-conjugated anti-mouse secondary antibodies (Bio-Rad). The membranes were washed in TBST and developed in alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.5; 100 mM NaCl; 5 mM MgCl2) containing 20 µM nitro blue tetrazolium and 20 µM 5-bromo-4-chloro-3-indolyl phosphate as substrates (44).

Enzyme Assays and Characterization Using Plant or Recombinant Proteins—Plant tissues were ground to a fine powder under liquid nitrogen using a mortar and pestle. Powdered tissues were extracted in buffer A. Debris were removed by centrifugation, and the supernatant was desalted using a PD-10 column (Amersham Biosciences). E. coli Rosetta (DE3) pLysS harboring the pTNMT plasmid was used to produce recombinant TNMT. A 20–40% (w/v) ammonium sulfate-precipitated protein fraction containing most of the soluble recombinant TNMT protein was used for enzyme characterization. TNMT activity was determined by incubating 10–15 µg of extracted soluble protein in buffer A containing 100 pmol (185 kBq) of S-adenosyl-L-[methyl-14C]methionine, 25 or 50 nmol of (R,S)-stylopine, and 12 mM beta-mercaptoethanol in a total volume of 50 µl. The buffers used to determine optimum pH were 100 mM sodium phosphate (pH 5–7) and 100 mM Tris-HCl (pH 7–9), both of which contained 12 mM beta-mercaptoethanol. After an incubation period of 30 min at 37 °C, the reactions were stopped with the addition of 1 N NaOH (5 µl), and the entire volume of each reaction was applied to a silica gel 60 F254 TLC plate (EM Science, Gibbstown, NJ). TLC plates were developed in CHCl3/MeOH/NH3 (300:150:3 (v/v/v)) (30) and analyzed using a bio-imaging analyzer (FUJIX BAS 1000, Fujifilm, Tokyo, Japan). Radiolabeled spots were quantified using MacBAS software (Fujifilm). Controls were performed using an aliquot of each protein extract that had been denatured by incubation in boiling water for 15 min. Protein concentration was determined according to the method of Bradford (45). Kinetic data were analyzed using Kaleidagraph 3.6 (Synergy Software, Reading, PA).


Figure 2
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  		FIGURE 2.
Neighbor-joining tree derived from selected plant O- and N-methyltransferases. Amino acid sequences were aligned using ClustalW (48). The tree was constructed and bootstrap analysis was performed using TREECON (49). Internal labels give bootstrap frequencies for each clade as percentages of 1,000 iterations. GenBank accession numbers are listed under "Experimental Procedures."

 
Product Identification—Due to substrate availability, (R,S)-tetrahydropalmatine was used for product identification. Approximately 1 mg of crude bacterial lysate from E. coli producing recombinant TNMT was combined in buffer A with 2.5 µmol of (R,S)-tetrahydropalmatine and 2.5 µmol of S-adenosyl-L-methionine in a total volume of 2 ml and was incubated overnight at 37 °C. The assay mixture was extracted three times with ethyl acetate, and the organic fraction was subsequently concentrated under reduced pressure and extracted twice with 1 M HCl. The aqueous phase was evaporated to dryness and analyzed by low resolution EI-MS. The resulting mass spectrum (m/z: 369 (3), 355 (71), 192 (9), 190 (26), 164 (100), 149 (69)) was in agreement with that of N-methyltetrahydropalmatine (39, 46, 47), indicating that N-methyl transfer had occurred.

Phylogenetic Analysis—Amino acid alignments were performed using the CLUSTALW (48) algorithm in MegAlign (DNAStar, Inc.). The neighbor-joining phylogeny was generated and bootstrap analysis was performed with TREECON (49). GenBankTM accession numbers for the sequences used are: T. flavum CNMT (AAU20766 [GenBank] ); Mycobacterium tuberculosis PcaA (ZP_00880145); C. japonica CNMT (BAB71802 [GenBank] ); P. somniferum CNMT (AAP45316 [GenBank] ); Mesorhizobium loti CFAPS (BAB53730 [GenBank] ); Burkholderia vietnamiensis CPFAS (ZP_00420926); A. thaliana At4g33110 ORF (AAM65762 [GenBank] ); A. thaliana At4g33120 ORF (NP_195038 [GenBank] ); T. flavum SOMT (AAU20770 [GenBank] ); C. japonica SOMT (BAA06192 [GenBank] ); Lycopersicon esculentum PEANMT (AAG59894 [GenBank] ); Coffea arabica DMXNMT1 (BAC75663 [GenBank] ); C. arabica MXNMT (BAB39216 [GenBank] ); C. arabica XNMT (BAB39215 [GenBank] ); Camellia sinensis DMXNMT (BAB12278 [GenBank] ); Clarkia breweri SAMT (AAF00108 [GenBank] ); Atropa belladonna PNMT (BAA82264 [GenBank] ); Datura stramonium PNMT (CAE47481 [GenBank] ); C. japonica CoOMT (BAC22084 [GenBank] ); P. somniferum 6OMT (AAQ01669 [GenBank] ); T. flavum 6OMT (AAU20765 [GenBank] ); C. japonica 6OMT (BAB08004 [GenBank] ); P. somniferum 4'OMT (AAP45313 [GenBank] ); T. flavum 4'OMT (AAU20768 [GenBank] ); C. japonica 4'OMT (BAB08005 [GenBank] ); Ocimum basilicum EOMT (AAL30424 [GenBank] ); O. basilicum ChOMT (AAL30423 [GenBank] ); Mentha x piperita FOMT (AY337459 [GenBank] ); Limonium latifolium BANMT (AAP03058 [GenBank] ); P. somniferum CaOMT (AAQ01670 [GenBank] ); Thalictrum tuberosum CaOMT (AAD29841 [GenBank] ); Antirrhinum majus BOMT (AAF98284 [GenBank] ); O. basilicum COMT1 (AAD38189 [GenBank] ); O. basilicum COMT2 (AF154918 [GenBank] ); C. breweri EOMT (AAC01533 [GenBank] ); C. breweri COMT (O23760 [GenBank] ); P. somniferum 7OMT (AAQ01668 [GenBank] ); Ammi majus COMT (AAR24095 [GenBank] ).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a TNMT cDNA and Phylogenetic Analysis—The recently reported molecular cloning of cDNAs encoding CNMT from P. somniferum (8), T. flavum (29), and C. japonica (6) provided query sequences to identify a full-length cDNA encoding opium poppy TNMT in an EST data base by in silico mining. We previously used this approach to identify cDNAs encoding opium poppy NCS isoforms with T. flavum NCS as the query sequence (4). TNMT cDNA contained a 1,074-bp ORF flanked by an 86-bp 5'-untranslated region and a 266-bp 3'-untranslated region followed by a polyadenylate tract (supplemental Fig. S1). The TNMT ORF encoded a predicted translation product of 358 amino acids with a molecular mass of 40.8 kDa and an isoelectric point (pI) of 5.3. Analysis of the TNMT amino acid sequence using the TargetP server did not reveal the presence of any subcellular targeting sequences, suggesting that TNMT is located in the cytosol (50).


Figure 3
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  		FIGURE 3.
Alignment of the deduced amino acid sequence of P. somniferum TNMT with P. somniferum CNMT, C. japonica CNMT, T. flavum CNMT, the A. thaliana At4g33120 gene product, and M. tuberculosis PcaA, an AdoMet-dependent cyclopropane synthase. Residues identical to TNMT have black backgrounds; residues that are similar to TNMT are shaded in gray. Asterisks denote conserved residues known to be involved in S-adenosyl-L-methionine binding (51). The conserved AdoMet-dependent methyltransferase region, motif I (53), is underlined. Sequence alignment was performed using ClustalW (48). GenBank accession numbers are listed under "Experimental Procedures."

 
Several plant-derived and AdoMet-dependent O- and N-methyltransferases were subjected to phylogenetic analysis to examine the possible evolutionary relationships among these enzymes (Fig. 2). High bootstrap support was indicated for a monophyletic clade that included PsTNMT, coclaurine N-methyltransferases (PsCNMT, CjCNMT, TfCNMT), bacterial AdoMet-dependent cyclopropane fatty acid synthases (MtPcaA, MlCFAPS, BhCPFAS), and the predicated translation products of the A. thaliana genes At4g33110 and At4g33120. The At4g33120 gene product was the less divergent of the two A. thaliana proteins. Strong support was also observed for a separate monophyletic clade including purine N-methyltransferases from C. arabica and C. sinensis (CaDMXNMT, CaMXNMT, CaXNMT, CsDMXNMT) and salicylic acid carboxyl methyltransferase from C. breweri (CbSAMT). Putrescine N-methyltransferases from D. stramonium and A. belladonna (DsPNMT, AbPNMT) formed a distinct monophyletic clade with strong support. Notably, opium poppy tetrahydroprotoberberine cis-N-methyltransferase (PsTNMT), L. esculentum phosphoethanolamine N-methyltransferase (LePEANMT), and L. latifolium beta-alanine N-methyltransferase (LlBANMT) all catalyze the AdoMet-dependent N-methylation of a tertiary amine in the formation of a quaternary ammonium compound, yet these enzymes are unrelated in this phylogeny, indicating that they evolved independently.

The amino acid sequence alignment shown in Fig. 3 compares PsTNMT with other members of its phylogenetic clade (Fig. 2). As expected, opium poppy TNMT shares considerable sequence identity with CNMT proteins from P. somniferum (48%), T. flavum (46%), and C. japonica (45%). The predicted translation products of At4g33110 and At4g33120 (Fig. 3) also exhibit considerable (44%) sequence identity to opium poppy TNMT. Surprisingly, a representative AdoMet-dependent mycolic acid cyclopropane synthase from M. tuberculosis, MtPcaA, shows 21–24% identity to plant TNMT and CNMTs. The crystal structure of MtPcaA in complex with S-adenosylhomocysteine, the byproduct of AdoMet-dependent methylation, has revealed the amino acid residues responsible for interaction with the AdoMet cofactor (51). Several of these critical AdoMet-binding residues, marked in Fig. 3 by asterisks, are conserved in plant N-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis.

Heterologous Expression of TNMT—The identity of the isolated cDNA as TNMT was confirmed by the production of recombinant protein in E. coli ER2566 pLysS harboring the TNMT expression construct pTNMT. The recombinant enzyme displayed an apparent molecular mass of ~48 kDa, which was expected due to N- and C-terminal peptide fusions derived from the expression vector (Fig. 4, A and B). The recombinant polyhistidine-tagged TNMT was detected only in total protein extracts from E. coli harboring the pTNMT plasmid (Fig. 4B). Using (R,S)-stylopine as the alkaloid substrate, TNMT activity was detected only in soluble protein extracts of E. coli expressing the full-length TNMT cDNA (Fig. 4C).

Due to substrate availability, the product of the enzyme-catalyzed reaction was investigated using (R,S)-tetrahydropalmatine. A large scale enzyme assay containing ~1 mg of total soluble protein from E. coli harboring pTNMT, 2.5 µmol of (R,S)-tetrahydropalmatine, and 2.5 µmol of unlabeled AdoMet was incubated overnight at 37 °C. The reaction mixture was extracted with ethyl acetate and acidified, and the resulting aqueous phase was evaporated to dryness. EI-MS analysis (supplemental Fig. S2) revealed the expected mass spectrum for N-methyltetrahydropalmatine (39, 46, 47), confirming that the isolated cDNA encodes TNMT.


Figure 4
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  		FIGURE 4.
Heterologous expression of the TNMT cDNA in E. coli. A, SDS-PAGE analysis of total protein extracts (25 µg) from non-induced (–IPTG) and induced (+IPTG) E. coli cells harboring the pRSETB empty vector control or the pTNMT expression vector. Purified, recombinant TNMT is also shown. B, Western blot analysis performed on protein samples from A using a polyhistidine tag monoclonal antibody shows the occurrence of recombinant proteins. Numbers on the left in A and B show the migration and molecular weight of standard protein markers. C, TNMT activity was assayed in total soluble protein extracts (10 µg).

 
Although TNMT polypeptide accumulation was most abundant in IPTG-induced cultures of E. coli harboring pTNMT (Fig. 4, A and B), the recombinant enzyme was also detected in total protein extracts of control cultures harboring pTNMT (Fig. 4, A and B). However, markedly higher levels of TNMT activity were detected in soluble protein fractions from E. coli cultures not treated with IPTG (Fig. 4C). Neither polyhistidine-tagged protein accumulation nor TNMT activity was detected in extracts of cells containing the empty pRSETB vector (Fig. 4, B and C).

Polyhistidine-tagged, recombinant TNMT was purified by nickel-affinity chromatography, as shown in Fig. 4A. Although the purified protein was active, TNMT activity was difficult to stabilize after nickel-affinity chromatography due to the sensitivity of the recombinant opium poppy enzyme to divalent cations. The addition of 1 mM chloride salts of Ni2+, Co2+, Ca2+, or Mn2+ to enzyme assays using 2 µg of 20–40% (w/v) ammonium sulfate-precipitated protein from E. coli cultures harboring pTNMT inhibited N-methyltransferase activity by 72, 71, 62, and 41%, respectively. This inhibition could be mostly prevented by the inclusion of 10 mM EDTA in the assay mixture. In contrast, the addition of 1 mM MgCl2 to the reaction mixture had no effect on TNMT activity. Our data are in agreement with a previous report that TNMT from S. canadensis was also inhibited by Co2+, Ca2+, and Mn2+ ions (31).

The A. thaliana protein encoded by the At4g33120 gene was also produced in E. coli and tested for TNMT activity. No activity was detected when (R,S)-stylopine, (R,S)-tetrahydropalmatine, or dimethoxytetrahydroisoquinoline was used as a potential substrate (data not shown).

Enzymatic Properties of Recombinant TNMT—The pH optimum for TNMT activity was 7.5, and half-maximal activity was estimated to occur at 6.8 and 8.2 (Fig. 5A). Recombinant TNMT showed maximum activity at 37 °C, and half-maximal activity would be expected at 12 and 47 °C (Fig. 6B). Varying the concentration of (R,S)-stylopine from 0.5 to 32 µM at a saturating concentration (20 µM) of AdoMet or the concentration of AdoMet from 0.625 to 40 µM, at a saturating concentration (50 µM) of (R,S)-stylopine, produced typical Michaelis-Menten substrate saturation kinetics. Apparent Km values for (R,S)-stylopine and AdoMet of 0.6 and 11.5 µM, respectively were calculated from Lineweaver-Burk plots (Fig. 5, C and D). The incorporation of radioactivity from [methyl-14C]AdoMet was used to demonstrate the strict substrate specificity of recombinant TNMT (Fig. 6). Only the protoberberine alkaloids (R,S)-stylopine, (R,S)-canadine, and (R,S)-tetrahydropalmatine were accepted as substrates. No radiolabeled products were detected when the simple isoquinoline alkaloids dimethoxytetrahydroisoquinoline and methylisoquinolinediol, the benzylisoquinoline alkaloid norlaudanosoline, the protoberberine alkaloids (R,S)-tetrahydroxyberbine and (S)-scoulerine, or the pavine alkaloid (R,S)-pavine were tested as substrates (Fig. 6).

Developmental and Inducible Gene Expression—RNA gel blot hybridization analysis showed that TNMT gene transcripts accumulate most abundantly in roots, to a lesser extent in leaves, and at lower levels in stems and flower buds (Fig. 7A). In contrast, the highest levels of TNMT activity were measured in stem and leaf tissues, with lower levels detected in roots and flower buds (Fig. 7B). Neither TNMT transcripts (Fig. 7C) nor enzyme activity (Fig. 7D) were detected in opium poppy seedlings 1 day after seed imbibition. However, both TNMT transcript levels and enzyme activity were detected 4 days after imbibition and remained relatively constant during seedling growth. TNMT transcripts were not detected in control opium poppy cell cultures but were rapidly induced within 2 h after elicitor treatment (Fig. 7E). Subsequently, TNMT transcript levels gradually decreased between 10 and 100 h after elicitor treatment (Fig. 7E). In contrast, TNMT activity increased from basal levels to a maximum 50 h after elicitation followed by a gradual decrease between 50 and 100 h (Fig. 7F).


Figure 5
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  		FIGURE 5.
General enzymatic properties of recombinant TNMT. A, the effect of pH on TNMT activity. B, the effect of temperature on TNMT activity. C, double-reciprocal plots showing the effect of substrate concentration on the reaction velocity for (R,S)-stylopine. D, double-reciprocal plots showing the effect of substrate concentration on the reaction velocity for S-adenosyl-L-methionine. The apparent Km values for (R,S)-stylopine and S-adenosyl-L-methionine were calculated as the negative reciprocals of the x-intercepts in C and D, respectively.

 
Occurrence of TNMT Activity in Other Benzylisoquinoline Alkaloid-producing Species—Cell cultures of several benzylisoquinoline-producing plants representing four different plant families were assayed for TNMT activity by measuring the incorporation of radioactivity from [methyl-14C]AdoMet when (R,S)-stylopine was supplied as a substrate (Fig. 8). (R,S)-Stylopine can only be methylated at the lone tertiary nitrogen. Alkaloid production was induced in cell cultures of P. somniferum and P. bracteatum, which do not normally produce alkaloids under these conditions, by treatment with elicitor prepared from mycelia of B. cinerea (8). Cell cultures of the other plant species tested produce alkaloids constitutively. Protoberberine and/or benzophenanthridine alkaloid accumulation was confirmed in these cell cultures by TLC analysis using authentic alkaloids as standards (data not shown). TNMT activity was detected in elicitor-treated cell cultures of P. somniferum and P. bracteatum and untreated cultures of E. californica, S. canadensis, Chelidonium majus, and Argemone mexicana (Fig. 8), which are all members of the Papaveraceae. Little or no TNMT activity was detected in cell cultures of Nandina domestica, Tinospora cordifolia, T. flavum, which represent the Berberidaceae, Menispermaceae, and Ranunculaceae, respectively (Fig. 8). TNMT activity was also not detected in Nicotiana tabacum, which was used as a negative control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We report the molecular cloning and characterization of a full-length cDNA encoding TNMT from opium poppy. This novel AdoMet-dependent N-methyltransferase was identified by tBLASTn analysis of an elicitor-treated opium poppy cell culture EST data base using CNMTs as query sequences. Functional expression of the TNMT cDNA in E. coli showed that the polyhistidine-tagged enzyme catalyzed the conversion of certain tetrahydroprotoberberine alkaloids to their corresponding N-methylated quaternary ammonium derivatives. Catalytic function was confirmed through purification to homogeneity of the polyhistidine-tagged protein (Fig. 4). Substantially more soluble and active recombinant enzyme was produced in E. coli strain Rosetta (DE3) pLysS when compared with E. coli strain ER2566 pLysS due to the occurrence of 29 rare bacterial codons in the TNMT cDNA. Moreover, bacterial cultures grown in the absence of IPTG showed a reduced rate of TNMT synthesis, which increased the proper assembly of the active enzyme (Fig. 4).

The catalytic properties of recombinant opium poppy TNMT are generally in agreement with those reported for the purified or partially purified enzyme from related plants. The predicted molecular mass of opium poppy TNMT (40.8 kDa) is comparable with that of the purified, denatured enzyme from S. canadensis (39 kDa) determined by SDS-PAGE (31). However, gel filtration chromatography of TNMT in purified partially purified protein extracts from S. canadensis, E. californica, and C. vaginans revealed native molecular masses of 70, 78, and 72 kDa, respectively (30, 31), suggesting that native TNMT is a homodimer. The temperature and pH optima for recombinant opium poppy TNMT (Fig. 5) are similar to those reported for native TNMT from S. canadensis, E. californica, and C. vaginans (30, 31). TNMT has been reported to specifically utilize (S)-enantiomers of tetrahydroprotoberberine substrates. Although we were unable to address the enantiomeric selectivity of opium poppy TNMT, the Km value determined for (R,S)-stylopine (0.6 µM) would reflect a Km for (S)-stylopine of 0.3 µM if (R)-stylopine was not an accepted substrate. This is lower than the Km values for (S)-stylopine of 3.1 and 4.0 µM reported for TNMT from E. californica and C. vaginans (30), respectively. Opium poppy TNMT exhibited an apparent Km for AdoMet of 11.5 µM, which is comparable with that reported for TNMT from E. californica (30). In contrast, opium poppy TNMT displayed a Km value for AdoMet ~10-fold higher than those determined for TNMT from S. canadensis and C. vaginans (30, 31). Opium poppy TNMT displayed a narrow substrate range with only tetrahydroprotoberberine substrates possessing dimethoxy (i.e. tetrahydropalmatine and canadine) or methylenedioxy (i.e. stylopine and canadine) functionalities at C-2/C-3 and C-9/C-10 as acceptable substrates (Fig. 6). The same substrate specificity profile was reported for native TNMT from E. californica and C. vaginans (30).


Figure 6
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  		FIGURE 6.
Substrate specificity of recombinant TNMT. The assay mixtures contained 100 mM Tris-HCl (pH 7.8), 12 mM beta-mercaptoethanol, 1 mM EDTA, 2 µM [methyl-14C]S-adenosyl-L-methionine, 500 µM alkaloid substrate, and 5 µg of protein. Assays were incubated for 30 min at 37 °C. Abbreviation: nd, not detected.

 
Phylogenetic analysis supports a monophyletic origin for the two known N-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis (Fig. 2). The downstream metabolic role suggests that TNMT resulted from gene duplication after the more ancient recruitment of CNMT. This is supported by the widespread occurrence of TNMT activity in the Papaveraceae but not in members of plant families that accumulate benzylisoquinoline but not specifically protopine or benzophenanthridine alkaloids (Fig. 8). The predicted translation product of the A. thaliana gene At4g33120, a close homolog of CNMT (6) and TNMT, did not catalyze the N-methylation the simple isoquinoline dimethoxytetrahydroisoquinoline, which was accepted by C. japonica CNMT (28), or the protoberberine alkaloids (R,S)-stylopine and (R,S)-tetrahydropalmatine, which are substrates of TNMT (Fig. 6). The similarity of the A. thaliana At4g33110 and At4g33120 gene products to CNMT and TNMT potentially reveals a latent genetic fingerprint for alkaloid biosynthesis in a plant species no longer able to produce these secondary metabolites (4). TNMT is a unique plant enzyme that catalyzes the formation of quaternary ammonium compounds. Aside from specialized protein lysine N-methyltransferases (52), only two cDNAs encoding plant N-methyltransferases that catalyze the formation of low molecular weight quaternary ammonium compounds have been reported. However, both phosphoethanolamine N-methyltransferase (24) and beta-alanine N-methyltransferase (26) are involved in the biosynthesis of betaines, which are important osmoprotectants in high salinity environments (33, 34). These enzymes, along with other N-methyltransferases involved in the biosynthesis of caffeine and other alkaloids (24, 26, 27), do not share a close phylogenetic relationship with TNMT (Fig. 2).

Although TNMT and CNMTs share limited sequence similarity with cyclopropane synthases, several residues known to be involved in AdoMet binding in MtPcaA are strictly conserved (Fig. 3). The crystal structure of MtPcaA in complex with S-adenosylhomocysteine demonstrates how Ser-34 shares a hydrogen bond with a carboxylate oxygen, and Gly-72 and Ile-136 interact with the nitrogen of the amino acid portion of AdoMet. Moreover, Gly-74, Thr-94, and Gln-99 are known to share hydrogen bonds with ribosyl hydroxyl moieties (51). The glycine-rich sequence containing Gly-72 and Gly-74 ((E/D)XGXGXG), often referred to as motif I (Fig. 3), is highly conserved in many AdoMet-dependent methyltransferases and is also present in TNMT, CNMTs, and MtPcaA (6, 51, 53). Structure and sequence conservation shows that MtPcaA exhibits a structural fold most similar to the small molecule subclass of methyltransferases (53). The conservation of critical residues involved in AdoMet binding suggests that TNMT and CNMTs also maintain this core small molecule methyltransferase fold. A methyltransferase fold model also predicts that AdoMet-binding motifs reside in the N-terminal domains of TNMT and CNMTs. As such, amino acid residues that confer substrate specificity are likely located in the C-terminal regions of these proteins (53). In the absence of crystal structures for CNMTs and TNMT, residues that confer substrate and reaction specificity with respect to secondary or tertiary nitrogen methylation might be identified among C-terminal residues that are conserved in CNMTs but unique to TNMT.


Figure 7
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  		FIGURE 7.
Relative abundance of TNMT gene transcripts (A, C, and E) and TNMT enzyme activity (B, D, and F) in organs (A and B), developing seedlings (C and D), and elicitor-treated cell cultures (E and F) of opium poppy. RNA gel blot hybridization analysis was performed using total RNA (15 µg), which was fractionated, transferred to a nylon membrane, and hybridized at high stringency to the 32P-labeled TNMT cDNA. Gels were stained with ethidium bromide prior to blotting to ensure equal loading. Error bars represent the standard error of the mean for three independent measurements.

 


Figure 8
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  		FIGURE 8.
TNMT activity in cell cultures of various benzylisoquinoline alkaloid-producing plant species. Assays contained 2 µg of total soluble protein extract, 500 µM (R,S)-stylopine, and 2 µM [methyl-14C]S-adenosyl-L-methionine and were incubated for 20 min at 37 °C.

 
The incongruity between TNMT gene transcript abundance and enzyme activity in opium poppy organs suggests that this step in alkaloid metabolism might be subject to translational and/or post-translational regulation. The detection of TNMT gene transcripts and enzyme activity in roots (Fig. 5, A and B) supports the biosynthesis of the protopine and benzophenanthridine alkaloids, which accumulate predominantly in this organ (54). However, the relatively high level of TNMT gene transcripts and enzyme activity in other plant organs suggests an involvement in additional alkaloid biosynthetic pathways. Noscapine and narcotoline are major benzylisoquinoline alkaloids found in all opium poppy organs (54), and similar phthalideisoquinolines are found in other members of the Papaveraceae and Menispermaceae (4). Noscapine has been used as an antitussive agent and has been recently purported to possess anticancer properties (55). The biosynthesis of phthalideisoquinoline alkaloids remains poorly understood, although (S)-scoulerine and (S)-canadine have been proposed as noscapine pathway intermediates (56, 57). The conversion of (S)-scoulerine to (S)-canadine involves an O-methyltransferase (58) and a methylenedioxy bridge-forming cytochrome P450 (59). The preference of (R,S)-canadine as a substrate (Fig. 6) and the occurrence of the enzyme in organs that accumulate abundant noscapine, but not sanguinarine, suggest that TNMT also participates in the biosynthesis of noscapine since both (S)-N-methylcanadine and noscapine possess N-methyl, 2,3-methylendioxy, and 9,10-dimethoxy substitutions (57). BBE gene transcripts and enzyme activity, required for (S)-scoulerine formation, have also been reported in the aerial organs of opium poppy (60).

In opium poppy seedlings, TNMT transcripts and enzyme activity were first detected 4 days after seed imbibition, which correlates the accumulation of sanguinarine ~5 days after germination (9). The rapid activation of TNMT supports a key defensive role for various benzylisoquinoline alkaloids early in plant development. The inducible accumulation of TNMT transcripts in elicitor-treated cell cultures (Fig. 7) reflects the coordinated temporal induction of all other known genes involved in sanguinarine biosynthesis in opium poppy (8). Moreover, the accumulation profiles of N-methylstylopine (41), other benzophenenthridine alkaloid pathway intermediates (41), and sanguinarine (41, 60) are in agreement with the temporal induction of TNMT activity in elicitor-treated opium poppy cell cultures.

The molecular cloning and characterization of opium poppy TNMT adds an important catalytic functionality to the repertoire of enzymes involved in benzylisoquinoline alkaloid biosynthesis. The phylogenetic relationship between CNMT and TNMT provides new insights into the evolutionary recruitment of enzymes into plant alkaloid pathways. The availability of the TNMT cDNA also provides an opportunity to elucidate the catalytic mechanism of tertiary versus secondary nitrogen methylation via the comparative structural biology of two unique, yet related, AdoMet-dependent N-methyltransferases.


   FOOTNOTES
 
* This work was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Strategic and Discovery grants (to P. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) DQ028579 [GenBank] . Back

Formula The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures and supplemental references. Back

1 The recipient of an NSERC postgraduate scholarship. Back

2 Holds the Canada Research Chair in Plant Metabolic Processes Biotechnology. To whom correspondence should be addressed. Tel.: 403-220-7651; Fax: 403-289-9311; E-mail: pfacchin{at}ucalgary.ca.

3 The abbreviations used are: NCS, (S)-norcoclaurine synthase; AdoMet, S-adenosyl-L-methionine; TNMT, (S)-tetrahydroprotoberberine cis-N-methyltransferase; CNMT, (S)-coclaurine N-methyltransferase; 4'OMT, (S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase; 6OMT, (S)-norcoclaurine 6-O-methyltransferase; 7OMT, (R,S)-reticuline 7-O-methyltransferase; BANMT, beta-alanine N-methyltransferase; BOMT, benzoic acid carboxyl methyltransferase; CaOMT, catechol O-methyltransferase; CFAPS, cyclopropane-fatty-acyl-phospholipid synthase; ChOMT, chavicol O-methyltransferase; COMT, caffeic acid O-methyltransferase; CoOMT, columbamine O-methyltransferase; CPFAS, cyclopropane-fatty-acyl-phospholipid synthase; CYP80B3, (S)-N-methylcoclaurine 3'-hydroxylase; DMXNMT, dimethylxanthine N-methyltransferase; EOMT, (iso)eugenol O-methyltransferase; FOMT, flavonoid 8-O-methyltransferase; MXNMT, 7-methylxanthine N-methyltransferase; PcaA, cyclopropane fatty acid synthase; PEANMT, phosphoethanolamine N-methyltransferase; PNMT, putrescine N-methyltransferase; SAMT, salicylic acid carboxyl methyltransferase; SOMT, scoulerine 9-O-methyltransferase; XNMT, xanthosine methyltransferase; EI-MS, electron impact-mass spectrometry; EST, expressed sequence tag; ORF, open reading frame; IPTG, isopropyl-1-thio-beta-D-galactopyranoside. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Natalia Loukanina and Dr. Jörg Ziegler for helpful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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J. M. Hagel, A. M. Weljie, H. J. Vogel, and P. J. Facchini
Quantitative 1H Nuclear Magnetic Resonance Metabolite Profiling as a Functional Genomics Platform to Investigate Alkaloid Biosynthesis in Opium Poppy
Plant Physiology, August 1, 2008; 147(4): 1805 - 1821.
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N. Ikezawa, K. Iwasa, and F. Sato
Molecular Cloning and Characterization of CYP80G2, a Cytochrome P450 That Catalyzes an Intramolecular C-C Phenol Coupling of (S)-Reticuline in Magnoflorine Biosynthesis, from Cultured Coptis japonica Cells
J. Biol. Chem., April 4, 2008; 283(14): 8810 - 8821.
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