functional characterization of a novel benzylisoquinoline o

Functional characterization of a novel benzylisoquinolineO-methyltransferase suggests its involvement in papaverinebiosynthesis in opium poppy (Papaver somniferum L)

Silke Pienkny, Wolfgang Brandt, Jurgen Schmidt, Robert Kramell and Jorg Ziegler†,*

Leibniz-Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany

Received 13 April 2009; accepted 8 May 2009; published online 13 July 2009.*For correspondence (fax +1 403 289 9311; e-mail [email protected]).†Present address: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB T2N 1N4, Canada.

SUMMARY

The benzylisoquinoline alkaloids are a highly diverse group of about 2500 compounds which accumulate in a

species-specific manner. Despite the numerous compounds which could be identified, the biosynthetic

pathways and the participating enzymes or cDNAs could be characterized only for a few selected members,

whereas the biosynthesis of the majority of the compounds is still largely unknown. In an attempt to

characterize additional biosynthetic steps at the molecular level, integration of alkaloid and transcript profiling

across Papaver species was performed. This analysis showed high expression of an expressed sequence tag

(EST) of unknown function only in Papaver somniferum varieties. After full-length cloning of the open reading

frame and sequence analysis, this EST could be classified as a member of the class II type O-methyltransferase

protein family. It was related to O-methyltransferases from benzylisoquinoline biosynthesis, and the amino

acid sequence showed 68% identical residues to norcoclaurine 6-O-methyltransferase. However, rather than

methylating norcoclaurine, the recombinant protein methylated norreticuline at position seven with a Km of

44 lM using S-adenosyl-L-methionine as a cofactor. Of all substrates tested, only norreticuline was converted.

Even minor changes in the benzylisoquinoline backbone were not tolerated by the enzyme. Accordingly,

the enzyme was named norreticuline 7–O-methyltransferase (N7OMT). This enzyme represents a novel O-

methyltransferase in benzylisoquinoline metabolism. Expression analysis showed slightly increased expres-

sion of N7OMT in P. somniferum varieties containing papaverine, suggesting its involvement in the partially

unknown biosynthesis of this pharmaceutically important compound.

Keywords: benzylisoquinoline alkaloids, O-methyltransferase, Papaver, opium poppy, papaverine biosynthe-

sis, secondary metabolism.

INTRODUCTION

Benzylisoquinoline alkaloids (BIAs) constitute of a group of

natural products with diverse structures, which are all

derived from the amino acid tyrosine. So far, about 2500

compounds have been identified, several of which exhibit

important pharmaceutical properties. Morphine is one of the

most powerful analgesics (Goodman et al., 2007), while its

precursor, codeine, is widely used as an antitussive (Chung,

2005). The benzophenanthridine sanguinarine and the pro-

toberberine alkaloid berberine exert potent antimicrobial

activities (Colombo and Bosisio, 1996). The simple benzyl-

isoquinoline papaverine is used as a vasodilator for treat-

ment of vasospasms (Brisman et al., 2006) and erectile

dysfunction (Thomas, 2002), and as a smooth muscle

relaxant (Sato et al., 2007). All these effects are attributed to

its inhibitory effect on phosphodiesterases (Boswell-Smith

et al., 2006).

The biosynthesis of all BIAs begins with the conden-

sation of the tyrosine-derived compounds dopamine and

p-hydroxyphenylacetaldehyde by norcoclaurine synthase,

yielding the basic tetrahydrobenzylisoquinoline (S)-norco-

claurine. Initial modifications include 6- and 4¢-O-methyla-

tions, N-methylation and 3¢-hydroxylation that lead to

(S)-reticuline (Figure 1). This central intermediate is exten-

sively modified in subsequent pathways leading to the

majority of benzylisoquinoline structures. Oxidative C–C

bond formation between the N-methyl group and the ortho-

carbon of the benzyl moiety results in berberine bridge

formation and initiates the biosynthesis of protoberberine

56 ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2009) 60, 56–67 doi: 10.1111/j.1365-313X.2009.03937.x

and benzophenanthridine alkaloids. The inversion of stereo-

chemistry from (S)-reticuline to its (R)-enantiomer and

subsequent carbon–carbon phenol coupling leads to pro-

morphinan and morphinan alkaloids. Both pathways have

been extensively investigated in the past years and several

cDNAs coding for biosynthetic enzymes have been obtained

(Ziegler and Facchini, 2008). This is in contrast to the

biosynthesis of papaverine, for which the pathway is not

completely understood. Tracer experiments suggested that

it might be derived from norreticuline or nororientaline

(Brochmann-Hanssen et al., 1971, 1975). Accordingly, two

additional O-methylations must occur, as well as the dehy-

drogenation of the heterocyclic ring. The results from the

feeding experiments revealed that dehydrogenation occurs

after methylation of all hydroxyl groups. However, the

participation of reticuline as an intermediate compound in

papaverine biosynthesis could not be completely ruled out.

This would require the demethylation of the nitrogen as an

additional step. Up to now, neither cDNAs nor enzyme

activities which might support one or the other metabolic

route have been reported for papaverine biosynthesis.

O-Methylation is a common theme in the biosynthesis of

BIAs and several cDNAs encoding proteins that catalyze

these reactions have been obtained (Figure 1). Norcoclau-

rine 6-O-methyltransferase (6OMT) and 3¢-hydroxy-N-meth-

ylcoclaurine 4¢-O-methyltransferase (4¢OMT) both act in the

early pathway up to reticuline (Frick and Kutchan, 1999;

Morishige et al., 2000; Ounaroon et al., 2003; Ziegler et al.,

2005; Inui et al., 2007). Scoulerine 9-O-methyltransferase

(SOMT) as well as columbamine O-methyltransferase (CoO-

MT) catalyze reactions in the protoberberine branch of

the pathway (Takeshita et al., 1995; Morishige et al., 2002).

Reticuline 7-O-methyltransferase (7OMT) leads to laudanine,

which might serve as an intermediate in papaverine biosyn-

thesis assuming N-demethylation at a later stage (Ounaroon

et al., 2003). All of these OMTs belong to the class II type

Figure 1. Benzylisoquinoline alkaloid biosynthesis.

Conversions which are catalyzed by more than one enzyme are indicated as double arrows. Question marks denote steps not unequivocally resolved yet. 4¢OMT, (S)-

3¢hydroxy N-methylcoclaurine 4¢O-methyltransferase; 6OMT, (S)-norcoclaurine 6-O-methyltransferase; 7OMT, reticuline 7-O-methyltransferase; BBE, berberine

bridge enzyme; CNMT, (S)-coclaurine N-methyltransferase; CoOMT, columbamine O-methyltransferase; COR1, codeinone reductase 1; CYP80B3, (S)-N-methyl

coclaurine 3¢hydroxylase; HPAA, p-hydroxy phenylacetaldehyde; NCS, (S)-norcoclaurine synthase; SalAT, 7(S)-salutaridinol 7-O-acetyltransferase; SalR,

salutaridine reductase; SalSyn, salutaridine synthase; SOMT, (S)-scoulerine 9-O-methyltransferase; STOX, (S)-tetrahydroprotoberberine oxidase; TYDC, tyrosine

decarboxylase. Protoberberine alkaloids are boxed in dark grey, promorphinan and morphinan alkaloids in light grey.

Benzylisoquinoline O-methyltransferase 57

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 56–67

O-methyltransferases which use S-adenosyl-L-methionine

(SAM) as the methyl donor, and do not require a metal ion

for activity. They exhibit considerable amino acid identity

of at least 30% with 6OMT and 4¢OMT showing a close

relationship of more than 50% identity. They exhibit high

substrate specificity with the highest catalytic efficiency

toward their natural substrate. Nevertheless, selected mod-

ifications in methylation patterns of the alkaloid substrate

may be tolerated by most OMTs (Takeshita et al., 1995; Frick

and Kutchan, 1999; Morishige et al., 2000, 2002; Ounaroon

et al., 2003; Ziegler et al., 2005; Inui et al., 2007). Remark-

ably, none of the OMTs accepts the in vivo substrate of

another OMT, suggesting a strict order in which O-methyl-

ation occurs in benzylisoquinoline biosynthesis. Addition-

ally, they show strong regiospecificity with their natural

substrate. However, depending on the provided substrate,

7OMT from Papaver somniferum has been shown to

perform O-methylations at the 6 and 4¢ position as well as

double methylations, and 4¢OMT from Eschscholzia califor-

nica and Coptis japonica exhibited traces of 6-O-methylating

activities (Ounaroon et al., 2003; Inui et al., 2007).

In recent publications we have reported comparative

transcript and alkaloid profiling in Papaver species showing

that most enzymes implicated in BIA metabolism show

coordinate expression and are present in one gene expres-

sion cluster exhibiting higher expression in plants which

accumulate morphinan alkaloids. Investigations of two

cDNAs from this cluster led to the characterization of

salutaridine reductase leading to morphinan alkaloids and

of 4¢OMT (Ziegler et al., 2005, 2006). In this paper, we report

the characterization of a third cDNA present in that cluster as

a novel, highly specific class II type OMT, norreticuline 7-O-

methyltransferase. The possible involvement of this enzyme

in papaverine biosynthesis is discussed.

RESULTS

Detection and sequence analysis of EST A21G11

Large-scale transcriptome and alkaloid analysis in Papaver

species yielded 69 cDNAs showing higher expression in six

P. somniferum varieties compared with 15 other Papaver

species (Ziegler et al., 2006). The comparison between

P. somniferum and the other Papaver species was chosen

because P. somniferum was bred for increased alkaloid

accumulation. Additionally, it is the only species among the

selected ones which was reported to produce morphinan

alkaloids as well as papaverine and noscapine (Shulgin and

Perry, 2002; Ziegler et al., 2005, 2006). The differentially

expressed cDNAs were distributed in six gene expression

clusters. Sequence analysis showed that no function could

be assigned to 62% of the cDNAs, based on database com-

parison. Eight coded for proteins presumably involved

in secondary metabolism, including six P450-dependent

monooxygenases. Additionally, all cDNAs already known to

encode enzymes of benzylisoquinoline biosynthesis showed

increased expression in P. somniferum varieties (Figure 2).

These included early pathway genes such as tyrosine

decarboxylase, 6OMT, and 4¢OMT, as well as genes involved

Figure 2. Gene expression analysis of Papaver species.

Two clusters consisting of expressed sequence tags exhibiting increased expression in Papaver somniferum varieties are shown. The cDNAs coding for enzymes in

benzylisoquinoline biosynthesis are indicated in blue and the cDNA characterized in this study is shown in red.

58 Silke Pienkny et al.


in the biosynthesis of specific benzylisoquinoline subclasses

such as the morphinan alkaloid-specific salutaridinol-7-O-

acetyltransferase and codeinone reductase 1, the laudanine-

specific 7OMT or the protoberberine-type specific berberine

bridge enzyme (Ziegler et al., 2006). The rationale behind

that study was the isolation and characterization of novel

cDNAs involved in benzylisoquinoline biosynthesis based

on their expression profiles. This led to the identification of

hitherto uncharacterized cDNAs, for example the morphinan

alkaloid-specific salutaridine reductase (Ziegler et al., 2006).

Another cDNA showing increased expression in P. som-

niferum varieties and which is clustered together with other

benzylisoquinoline biosynthetic genes was EST A21G11

(Figure 2). This EST with a length of 137 bp did not match to

any other sequence when submitted to the non-redundant

database on the NCBI server using either the BLASTX or

BLASTN algorithms, even when an expectation value of 10

was used as threshold. In order to obtain sequence infor-

mation for the whole cDNA, the Genome Walker kit was

applied using genomic DNA from P. somniferum with

primers designed according to the A21G11 sequence.

Sequence analysis of the resulting DNA fragment of

2287 bp revealed an open reading frame of 1071 bp distrib-

uted over two exons of 777 bp and 294 bp, respectively,

which are interrupted by an intron of 735 bp. The 137-bp

sequence of EST A21G11 is located in the 3¢ untranslated

region 85 bp downstream from the stop codon at positions

1899 to 2036. The gene encodes a protein of 357 amino acids

with a molecular mass of 39.7 kDa and an isoelectric point

of 4.98.

The translated open reading frame exhibits several

motifs, which are typical for SAM-dependent class II type

O-methyltransferase, including the catalytic histidine. Most

of the residues which are involved in SAM binding in

chalcone O-methyltransferase and isoflavone O-methyl-

transferase (Zubieta et al., 2001) are conserved in the

A21G11 sequence, as well as in other OMTs from benzyl-

isoquinoline biosynthesis (Figure 3a). Phylogenetic analysis

of several OMTs from plant secondary metabolism showed

that A21G11 forms a clade together with 6OMT from

P. somniferum and C. japonica, which is well separated

from the 4¢OMTs (Figure 3b). A21G11 exhibited the highest

similarity to 6OMT from P. somniferum, followed by 6OMT

from C. japonica (68 and 55% amino acid identity, respec-

tively). The 4¢OMTs showed between 40 and 46% amino acid

identity to A21G11. Distant from these enzymes, the recently

characterized 7OMTs from P. somniferum and E. californica

form their own group. This is reflected by the lower

percentage of identical amino acid residues (37%) between

A21G11 and both enzymes. This degree of sequence simi-

larity was also observed for other OMTs of plant secondary

metabolism, which are not active in benzylisoquinoline

biosynthesis, such as caffeic acid and flavonoid O-meth-

yltransferases.

Gene expression analysis

The transcript profiling revealed higher expression of

A21G11 in P. somniferum varieties compared to other

Papaver species, although the expression level varied in

individual plants of each variety. In only one variety (Papa;

Figure 2) was A21G11 expression elevated in all individual

plants. All P. somniferum varieties produce morphinan

alkaloids (Ziegler et al., 2006). However, electrospray Fou-

rier transform ion cyclotron resonance mass spectrometry

(ESI-FT-ICRMS) showed the most prominent peaks for

masses indicative of other BIAs in some varieties. The most

abundant masses in the varieties Papa and Fool Ori were at

m/z 340.15441 and m/z 340.15409 [M+H]+, indicating an

elemental composition of [C20H22NO4]+. A corresponding

low abundant mass signal was also present in Papaver

dubium, but was not detected in the other P. somniferum

varieties or species shown in Figure 2. Subsequent liquid

chromatography-tandem mass spectrometry (LC-MS/MS)

analyses identified the peak at m/z 340 ([M+H]+) as papav-

erine in Papa, Fool Ori and P. dubium based on a compari-

son of retention time and fragmentation pattern compared

with a papaverine standard. This alkaloid profile suggests

high A21G11 expression in plants with high papaverine

content. In order to confirm the expression data, quantita-

tive RT-PCR analysis was performed with all benzyliso-

quinoline OMTs.

This expression analysis revealed that 4 ¢OMT, 6OMT,

7OMT and A21G11 are expressed at higher levels in stems

compared with leaves, roots or seedlings (Figure 4). In

seedlings, 7OMT was expressed at a higher level than in

leaves or roots, whereas 6OMT and 4 ¢OMT expression was

higher in these tissues compared with seedlings. A21G11

was expressed at similar levels in leaves, roots and seed-

lings – levels which were very low compared with the

expression levels observed in stems. Similar expression

levels of 4 ¢OMT and 7OMT were shown (increase by 13 and

29%, respectively) in stems of the P. somniferum variety

Papa, which contained the highest levels of papaverine, and

a variety without detectable levels of papaverine. While

6OMT and A21G11 seem to exhibit minor increases in

expression, by 125 and 76%, respectively (Figure 4), there

was no statistical difference because of considerable

variation among samples.

Overexpression and functional characterization of

recombinant A21G11 protein

The open reading frame was cloned into the expression

vector pQE30 coding for a six-histidine N-terminal extension

and was over-expressed in Escherichia coli strain SG13009.

The recombinant His-tagged protein had a relative molecular

mass of 40 kDa as determined by SDS–PAGE, which com-

pared favorably with the calculated mass of 39.7 kDa de-

duced from the translation of the cDNA. A native molecular



mass of 72 kDa was determined by gel filtration on a cali-

brated Superdex HL 200 column, suggesting the dimeric

nature of the overexpressed protein (data not shown).

Based on sequence homology to class II type OMTs of

benzylisoquinoline metabolism, 16 alkaloids were tested as

possible substrates. Additionally, four phenolic compounds

(a)

(b)

Figure 3. Comparison of A21G11 to selected O-methyltransferases (OMTs) of plant secondary metabolism.

(a) Amino acid sequence alignments of selected plant OMTs. The sequence alignment was performed using the CLUSTALW application (Thompson et al., 1994) of

MegAlign (DNASTAR Inc., http://www.dnastar.com/). The protein sequence of A21G11 from Papaver somniferum (GenBank accession number FJ156103) was

aligned to P. somniferum norcoclaurine 6-O-methyltransferase (Pso-6OMT), reticuline 7-O-methyltransferase (Pso-7OMT), 3¢-hydroxy N-methylcoclaurine 4¢-O-

methyltransferase (Pso-4¢OMT) and Medicago sativa isoflavone 7-O-methyltransferase (Msa-I7OMT). Circles and box, residues involved in S-adenosyl-L-methionine

(SAM) binding; triangle, catalytic histidine according to the Msa-I7OMT structure (PDB: 1FP2).

(b) Neighbour joining phylogenetic tree of functionally characterized OMTs involved in plant secondary metabolism. The amino acid alignment was performed as

described in (a). The tree was generated and visualized with the TREECON software (Yves van de Peer, University of Konstanz, Germany). Bootstrap values in per

cent of 1000 trials are indicated. The source and accession numbers are: Eschscholzia californica Eca-4¢OMT (Inui et al., 2007); Pso-4¢OMT (AAP45314); Coptis

japonica (Cja)-4¢OMT (Q9LEL5); Cja-6OMT (Q9LEL6); Pso-6OMT (AAQ01699); A21G11 (FJ156103); Hlu-OMT2, Humulus lupulus chalcone/xanthohumol OMT

(ABZ89566); Rch-CafOMT, Rosa chinensis var spontanea caffeic acid OMT3 (BAC78828); Hlu-OMT1, H. lupulus desmethylxanthohumol OMT (ABZ89565); Eca-7OMT

(BAE79723); Pso-7OMT (AAQ01668); Pta-AEOMT, Pinus taeda hydroxycinnamic acid/hydroxycinnamoyl-CoA ester OMT (AAC49708); Cja-SOMT, C. japonica

scoulerine OMT (BAA06192); Msa-IL2¢OMT, Medicago sativa isoliquiritigenin 2¢OMT (AAB48059); TtuOMT1, Thalictrum tuberosum OMT1 (AAD29841);

Cro-Caf3OMT, Catharanthus roseus caffeic acid 3OMT (Q8W013); Ath-F3¢OMT, Arabidopsis thaliana flavonol 3¢OMT (AAB96879); Msa-CafOMT, Medicago sativa

caffeic acid OMT (P28002); Cbr-CafOMT, Clarkia breweri caffeic acid OMT (AAB71141); RchCafOMT, Rosa chinensis caffeic acid OMT (CAD29457); Cja-CoOMT,

C. japonica columbamine OMT (BAC22084); Mpi-F8OMT, Mentha piperita flavonoid 8OMT (AAR09600); Rhy-OOMT, Rosa hybrid cultivar orcinol OMT (AAM23004);

Rgr-3,5DMPOMT, Ruta graveolens 3,5-dimethoxyphenol OMT (AAX82431); Oba-CVOMT, Ocimum basilicum chavicol OMT (AAL30423); Cro-F4¢OMT, Catharanthus

roseus flavonoid 4¢OMT (AAR02420); Msa-I7OMT (AAC49928).



(caffeic acid, vanillic acid, guaiacol and catechol) which are

known to be accepted by several class II type OMTs, as well

as the flavonoid quercetin and the coumarin esculetin were

tested (Figure 5). Although homologous to 6OMT, norcocla-

urine did not serve as a substrate for A21G11, whereas

incubations with norreticuline exhibited substantial radio-

activity in the organic phase after extraction of the enzyme

assays. Reticuline, the N-methylated derivative of norreticu-

line, was not accepted as a substrate, nor were any

tetrahydrobenzylisoquinolines containing methylated nitro-

gens. Moreover, the enzyme seems to be dependent on the

presence of a methoxy group at positions 6 and/or 4¢, since

norlaudanosoline is not converted. Tetrahydrobenzyliso-

quinolines with methoxy groups at position 7 and 3¢, as in

norisoorientaline, are not tolerated as well. The BIAs of more

complex structures such as scoulerine, or the promorphin-

ans and morphinans salutaridinol, oripavine, codeine or

morphine did not serve as substrates. The same was true

for the tetrahydroisoquinoline salsolinol and the phenolic

compounds.

Structural analysis of the reaction product

The HPLC analysis of the reaction products with norreticu-

line as the substrate showed a new peak eluting 3 min after

the substrate peak, which could only be detected in incu-

bations with active enzyme (Figure 6a). The LC-MS analysis

revealed a [M+H]+ ion at m/z 330 for the product, indicating

single methylation of norreticuline ([M+H]+, m/z 316; Fig-

ure 6b). However, there are several positions which can be

O-methylated, resulting in norlaudanine or norcodamine,

respectively. Additionally, N-methylation to reticuline can-

not be ruled out. These three possibilities can be differenti-

ated by their electrospray ionization (ESI) MS-fragmentation

pattern. Tetrahydrobenzylisoquinoline alkaloids are cleaved

into their isoquinoline and benzyl moieties. Norreticuline

shows a fragment at m/z 178, which represents the

Figure 4. Gene expression analysis of benzylisoquinoline O-methyltransfe-

rases (OMTs).

The transcript levels of 3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransfer-

ase (4¢OMT), norcoclaurine 6-O-methyltransferase (6OMT), reticuline 7-O-

methyltransferase (7OMT) and norreticuline 7-O-methyltransferase (N7OMT)

in different organs of the high morphine producing variety (PaSo) and in

stems of a high papaverine producing variety (Papa) of P. somniferum were

analyzed. The relative transcript levels (2)DDCt) were calculated using elonga-

tion factor 1a as the normalizer and the DCt value of 4¢OMT in PaSo stems as

the calibrator.

Figure 5. Substrate specificity of A21G11.

The structures of compounds tested as potential

substrates of the Papaver somniferum O-meth-

yltransferase A21G11 are displayed. Grey shad-

ing denotes the converted compound.



isoquinoline part of the molecule (Figure 6; Schmidt et al.,

2005). This fragment was shifted by 14 mass units towards

higher masses in the reaction product, resulting in a corre-

sponding signal at m/z 192. This suggests that methylation

takes place at the isoquinoline moiety. In contrast, 3¢-O-

methylation would have resulted in norcodamine and the

mass of the isoquinoline fragment would have been

unchanged. However, the ESI-time-of-flight (TOF) MS data

did not show a corresponding fragment ion with a mass at

m/z 178 (Figure 6b). Furthermore, the loss of ammonia leads

to a fragment at m/z 299 for norreticuline. This fragment was

also shifted by 14 mass units in the product, yielding a signal

at m/z 313. An N-methylation would have led to a fragment

at m/z 299 because of a loss of methylamine ([M+H–

NH2CH3]+). Therefore, reticuline can be excluded as a pos-

sible product. Based on this fragmentation pattern, and

since position 6 is already methylated in norreticuline, it can

be concluded that position 7 is methylated by the enzyme,

yielding norlaudanine.

Considering the substrate and product specificity of the

A21G11 protein, it was named norreticuline 7-O-methyl-

transferase (N7OMT). The recombinant protein exhibited its

highest activity at 35�C, with half-maximum activity at 25�Cand 40�C, respectively. The pH dependence revealed a broad

optimum between pH 7.0 and pH 9.5, with strongly reduced

activity at pH 6.5 and pH 10.0. The enzyme exhibited a Km of

44 � 5 lM (n = 6) and a kcat of 0.074 sec)1 for norreticuline,

and a Km of 19 � 6 lM (n = 3) for the cofactor SAM.

DISCUSSION

Comparison of N7OMT with other OMTs in

benzylisoquinoline biosynthesis

O-Methylation is a common theme in the decoration of the

benzylisoquinoline backbone, which leads to their structural

diversity. Several benzylisoquinoline OMTs have been

characterized, some of which exhibit distinct regio- and

substrate specificities whereas some show a broader sub-

strate range. Based on substrate specificity, N7OMT showed

a very narrow substrate range with norreticuline as the only

methyl acceptor (Figure 5). The contribution of the addi-

tional N-terminal 6· histidine tag to this restrictive specificity

was not investigated. The recombinant enzyme exhibited

strong discrimination between N-methylated and N-deme-

thylated benzylisoquinolines, as it is able to accept norreti-

culine but not reticuline. Furthermore, all tested compounds

containing an N-methyl group are not converted. This is

unique among all cloned OMTs which are specific for simple

tetrahydrobenzylisoquinolines. The 4¢OMTs from E. califor-

nica, C. japonica, and P. somniferum accept laudanosoline

as well as norlaudanosoline, although there is a preference

for the N-methylated substrate (Morishige et al., 2000; Zie-

gler et al., 2005 Inui et al., 2007), whereas C. japonica 6OMT

exhibits equal activities for both compounds (Morishige

(a)

(b)

(c)

Figure 6. Product identification of norreticuline conversion by A21G11

[=norreticuline 7-O-methyltransferase (N7OMT)].

(a) The HPLC chromatograms. Upper line, norreticuline substrate; middle line,

after incubation with bacterial extracts without A21G11 protein; lower line, in

the presence of A21G11 protein.

(b) Electrospray ionization-time-of-flight (ESI-TOF) mass spectra of the

substrate norreticuline at retention time (Rt) = 17.3 min on HPLC (top panel),

and of the product at Rt = 20.0 min (lower panel).

(c) Fragmentation patterns of norreticuline, and the possible enzyme products

norlaudanine, norcodamine, and reticuline.



et al., 2000). Papaver somniferum 6OMT accepts the

N-demethylated norprotosinomenine but not protosinome-

nine (Ounaroon et al., 2003). However, this enzyme converts

the N-methylated isoorientaline, showing that modifications

at the heteroatom are tolerated for some compounds. Only

7OMT from P. somniferum also discriminates between

N-methylated and N-demethylated substrates. This enzyme

displays exclusive preference for N-methylated tetrahyd-

robenzylisoquinolines, since it is able to methylate reticuline

but not norreticuline. However, this enzyme also accepts

phenolic compounds and is not as specific for tetrahydrob-

enzylisoquinoline alkaloids as N7OMT (Ounaroon et al.,

2003).

It is found that N7OMT is not active with substrates in

which the methoxy and hydroxyl substitution pattern of the

tetrahydrobenzylisoquinoline backbone is different from

norreticuline. Even the non-methylated norlaudanosoline

is not accepted as a substrate. This compound was methy-

lated by all other tetrahydrobenzylisoquinoline OMTs when

it was tested as a substrate (Morishige et al., 2000; Ziegler

et al., 2005; Inui et al., 2007). In contrast to N7OMT, 4¢OMT,

6OMT, and 7OMT are more promiscuous with regard to the

methylation pattern of the substrates. Although we used a

wide range of compounds, we cannot exclude the possibility

that other benzylisoquinolines which have not been

included in this study are accepted by N7OMT. Nevertheless,

compared with the other benzylisoquinoline OMTs studied

so far, N7OMT exhibits stronger substrate specificity. To our

knowledge, there is only one OMT showing a similar degree

of specificity. A 3¢OMT was purified and characterized from

Argemone platyceras cell cultures and accepted only 6-O-

methylnorlaudanosoline out of a large range of substrates

(Rueffer et al., 1983b). However, molecular data for this

enzyme are not available.

None of the tetrahydrobenzylisoquinoline-specific OMTs

cloned so far accepted norreticuline as a substrate

(Morishige et al., 2000; Ounaroon et al., 2003; Ziegler et al.,

2005). Only Thalictrum tuberosum OMT2 (Frick and Kut-

chan, 1999) showed some activity with norreticuline.

However, this enzyme displayed a broad substrate range

and is 10-fold more active towards caffeic acid and

catechol. Recombinant SOMT from C. japonica also

showed little side activity with norreticuline, with 10%

conversion compared to the major substrate scoulerine

(Morishige et al., 2002). The regiospecificity has not been

determined for both enzymes. To our knowledge there is

only one example where methylation of norreticuline could

be measured in plant extracts. This activity was purified

from A. platyceras cell cultures and exhibited 1% activity

compared with the preferred substrate norlaudanosoline.

This enzyme was shown to methylate norlaudanosoline at

positions 6 and 7 in a ratio of 4:1, but the regiospecificity

for norreticuline was not determined (Rueffer et al., 1983a).

Summarizing the available data, the substrate- and regio-

specificity of N7OMT adds a new member to the range of

benzylisoquinoline OMTs.

Phylogenetic analysis revealed that N7OMT forms one

cluster together with 6OMTs (Figure 3b). Considering that

other benzylisoquinoline OMTs are grouped according to

their regiospecifities one could have expected a closer

relationship of N7OMT to 7OMTs. A similar pattern was

observed for OMTs capable of 6-O-methylation of tetrahyd-

robenzylisoquinolines. Here, the 6OMTs from C. japonica

and P. somniferum are not closely related to OMT1 from

T. tuberosum, which also possesses norcoclaurine 6-O-

methyltransferase activity. However, 7OMT from P. som-

niferum as well as OMT1 from T. tuberosum accept phenolic

compounds with a higher catalytic efficiency than their

preferred tetrahydrobenzylisoquinoline substrate (Frick and

Kutchan, 1999; Ounaroon et al., 2003). The promiscuous

nature of 7OMT as well as OMT1 from T. tuberosum

suggested that tetrahydrobenzylisoquinoline OMTs might

have evolved from OMTs of the phenylpropanoid pathway

(Frick and Kutchan, 1999; Ounaroon et al., 2003). Further

speciation to 4¢OMT and 6OMT is believed to be the result of

divergent evolution. Substrate specificity and sequence

homology suggested that 4¢OMTs have evolved further than

the 6OMTs (Ziegler et al., 2005). However, residual 6OMT

activity detected for C. japonica and E. californica 4¢OMTs

led to the assumption that 6OMT might have evolved by

speciation after duplication of a 4¢OMT gene (Inui et al.,

2007). The close relationship of N7OMT to 6OMT suggests

that in P. somniferum a duplication of 6OMT and further

speciation led to the generation of N7OMT. In contrast to

that, the evolution of N7OMT through speciation after

duplication of 7OMT seems unlikely.

Participation of N7OMT in papaverine biosynthesis

In the last decade considerable progress has been achieved

in the cloning of cDNAs implicated in benzylisoquinoline

biosynthesis. All of the basic pathway genes up to reticuline

have been elucidated, as well as a major portion of the steps

leading to berberine and morphine (Ziegler and Facchini,

2008). Considering the estimated number of 2500 benzyl-

isoquinoline structures, many biosynthetic steps remain to

be discovered, and current data on the biosynthetic sequence

of entire side pathways often reveal several options.

The biogenic origin of papaverine is one of those exam-

ples. Feeding experiments suggested reticuline and norori-

entaline as well as norreticuline to be possible pathway

intermediates (Brochmann-Hanssen et al., 1971, 1975). After

reticuline is converted by 7OMT (Ounaroon et al., 2003),

laudanine might be methylated at the 3¢ position to laud-

anosine, which is N-demethylated to tetrahydropapaverine,

the immediate precursor of papaverine (Figure 7). The need

for an N-demethylating step represents a major obstacle for

support of a reticuline-based pathway since an enzyme

catalyzing this reaction had not yet been detected in plants



with any benzylisoquinoline substrate. The incorporation of

labelled reticuline into papaverine was low compared with

nororientaline or norreticuline (Brochmann-Hanssen et al.,

1971). Additionally, feeding experiments did not suggest a

turnover of laudanine to papaverine, and laudanine accu-

mulates in P. somniferum without detectable levels of

papaverine. This indicates that methylation products of

reticuline are not likely intermediates in papaverine biosyn-

thesis (Brochmann-Hanssen et al., 1975; Schmidt et al.,

2007).

Considering N-demethylated tetrahydrobenzylisoquino-

lines as intermediates, (S)-coclaurine could be diverted

towards papaverine biosynthesis by 3¢-hydroxylation to

6-O-methylnorlaudanosoline. Further methylations might

occur, first at 3¢ followed by methylations at 4¢ and 7, when

nororientaline represents an intermediate, or in the order 4¢,7, and 3¢ in the case of a norreticuline based pathway

(Figure 7). In both pathways, a 3¢-hydroxylase is required for

conversion of coclaurine to 6-O-methylnorlaudanosoline.

The involvement of N-methylcoclaurine 3¢-hydroxylase

(CYP80B subfamily) can be ruled out, since it is strictly

dependent on the presence of an N-methyl group (Pauli and

Kutchan, 1998; Huang and Kutchan, 2000). Loeffler and Zenk

(1990) found an enzyme activity described as phenolase in

Berberis cell cultures converting coclaurine to 6-O-methyl-

norlaudanosoline. Subsequently, this compound might be

methylated at the 3¢ position in a nororientaline pathway.

Such an activity has been detected in A. platyceras cell

culture (Rueffer et al., 1983b). However, this enzyme and the

phenolase have not yet been detected in papaverine-

producing plants. It is less likely that 4¢OMT and 7OMT are

involved in that pathway: 4¢OMT exhibited no activity

towards nororientaline and 7OMT is strictly dependent on

an N-methyl group, which excludes methylation of norcod-

amine (Ounaroon et al., 2003; Ziegler et al., 2005). Norcod-

amine could not be tested as a substrate for N7OMT since

this compound was not available. Therefore, it cannot be

ruled out that N7OMT is involved in this pathway, especially

because of its preference for N-demethylated tetrahydrob-

enzylisoquinolines.

The considerable activity of 4¢OMT towards 6-O-methyl-

norlaudanosoline suggests a possible participation in a

norreticuline pathway (Morishige et al., 2000; Ziegler et al.,

2005). Together with the cloning of a norreticuline specific

N7OMT, two enzymes able to catalyze O-methylation in a

norreticuline based pathway have now been characterized

from P. somniferum. However, an enzyme catalyzing the

3¢-O-methylation of norlaudanine is still missing. The 3¢OMT

from A. platyceras has not been tested with norlaudanine

(Rueffer et al., 1983b) and the existence of a 3¢-OMT in

papaverine-producing plants has not been reported.

The feeding experiments, the properties of all available

tetrahydrobenzylisoquinoline OMTs, and the slightly higher

expression of N7OMT in a papaverine-accumulating P. som-

niferum variety, as observed from the macroarray analysis,

suggest that papaverine biosynthesis proceeds via norreti-

culine. However, since higher N7OMT expression in papav-

erine-accumulating plants cannot be clearly deduced from

the quantitative RT-PCR data, and because of the missing

links in any of the possible pathways, transgenic approaches

are required to clearly resolve the biosynthesis of papaver-

ine. Unfortunately, opium poppy transformation is a tedious

and time-consuming task, but the availability of N7OMT

cDNA provides a promising candidate for these experiments

in order to elucidate papaverine biosynthesis.

EXPERIMENTAL PROCEDURES

Plant material

The seeds of the Papaver plants were obtained either from the seedstock collection of the Department of Natural Product Biotechnol-ogy of the Leibniz Institute of Plant Biochemistry, Halle, Germany orwere the kind gift of Tasmanian Alkaloids Pty Ltd, Westbury,Tasmania, Australia. Papaver somniferum L. plants were grownoutdoors in Saxony-Anhalt, Germany.

Figure 7. Proposed scheme for papaverine

biosynthesis in Papaver somniferum.

Solid arrows denote conversions for which spe-

cific enzymes have been cloned or characterized,

or which can be performed in vitro by the

indicated enzymes, respectively. For the conver-

sions shown as broken arrows, an enzyme has

not yet been detected in papaverine-accumulat-

ing plants. 4¢OMT, (S)-3¢hydroxy N-methylcocla-

urine 4¢O-methyltransferase; 7OMT, reticuline

7-O-methyltransferase; CNMT, (S)-coclaurine

N-methyltransferase; CYP80B3, (S)-N-methyl

coclaurine 3¢hydroxylase; N7OMT, (S)-norreti-

culine 7-O-methyltransferase.



Analytical methods

The HPLC analysis was performed using an LC 1100 series Agilentsystem (http://www.agilent.com/) equipped with a Lichrospher 60RP-select B column (250 · 4 mm, 5 lm; Merck, http://www.merck.-com/) and a solvent system consisting of A, CH3CN–H2O (2:98; v/v);B, CH3CN–H2O (98:2; v/v) each with 0.1% (v/v) formic acid. Thegradient was from 0% B to 46% B in 25 min with a hold for 5 minfollowed by an increase to 100% B in 2 min and a hold for 3 min at aflow rate of 1 ml min)1. The detection wavelength was set to282 nm.

The ESI-MS measurements and LC separations were carried outon a Mariner TOF mass spectrometer (Applied Biosystems, http://www3.appliedbiosystems.com/) equipped with a Turbo Ion Spraysource (PE-Sciex) using an LC1100 series Agilent system adapted toflow rates of 0.2 ml min)1. Samples were injected on a Supersphere60 RP-Select B column (125 · 2 mm, 5 lm; Merck). The following LCconditions were used: solvent A, CH3CN–H2O (2:98; v/v) and solventB, CH3CN–H2O (98:2; v/v), 0.2% (v/v) formic acid in both solvents.The gradient increased from 0% to 46% B in 25 min, to 90% in 1 minand was held at 90% for 7 min; post time was 5 min. The TOF massspectrometer was operated in the positive ion mode, with nebulizergas (N2) flow of 0.5 L min)1, curtain gas (N2) flow of 1.5 L min)1, andheater gas flow of (N2) 7 L min)1. The spray tip potential of the ionsource was 5.5 kV, the heater and quadrupole temperature were360�C and 140�C, respectively, the nozzle potential was set to 200 V,and the detector voltage to 1.95 kV. The other settings varieddepending on tuning.

The positive ion ESI-collision-induced dissociation (CID) massspectra for the detection of papaverine in extracts from P. som-niferum var. Papa, P. somniferum var. FoolOri, and P. dubiumwere obtained from a TSQ Quantum Ultra AM system (ThermoScientific, http://www.thermo.com) equipped with a hot ESI source(HESI; electrospray voltage 3.0 kV, sheath gas nitrogen; vaporizertemperature 50�C, capillary temperature 250�C). The MS system iscoupled to a Surveyor Plus micro-HPLC (Thermo Electron, http://www.thermo.com/), equipped with a RP18 column (5 lm,150 · 1 mm, Hypersil GOLD, Thermo Scientific). A gradient systemwas used for separation, starting from H2O:CH3CN 85:15 (v/v) toH2O:CH3CN 5:95 (v/v), each containing 0.2% (v/v) acetic acid,within 15 min, followed by a hold for a further 30 min at a flowrate of 50 ll min)1. The CID mass spectra of papaverine(RT = 9.9 min) were recorded during the HPLC run with a collisionenergy of 30 eV (collision gas argon, collision gas pressure1.5 mTorr).

The ESI-CID mass spectrum of papaverine is [m/z (relativeintensity, %)]: 340 ([M+H]+, 20), 324 (95), 296 (20), 202 (100), 187(8), 171 (34). The LC-MS/MS data were in excellent agreement withthose of authentic papaverine. The fragmentation of papaverine isdescribed elsewhere (Wickens et al., 2006).

The high-resolution positive ion ESI mass spectra were obtainedas described in Ziegler et al. (2006).

Full-length cDNA cloning of N7OMT and protein expression

in E. coli

For the generation of full-length cDNA for EST A21G11, the GenomeWalker Kit (BD Biosciences, http://www.bdbiosciences.com/) wasused according to the manufacturer’s instructions. Genomic DNAisolated from P. somniferum stems was used as a template. Theendonucleases DraI, EcoRV, PvuII and StuI were used to producedifferent libraries with genomic fragments. The PCR conditions withLong PCR Enzyme Mix (Fermentas, http://www.fermentas.com/)were as follows: 29 cycles of 96�C for 10 sec, 50�C for 5 sec, 60�Cfor 4 min using the primers A21G11-GW1: 5¢-GATGCAAAGATG-

TAGTATCAGCGGGAGGG-3¢, A21G11-GW2: 5¢-GCAAGACGAAT-CACAGAATCAGATACGAAGCC-3¢, and AP1: 5¢-GTAATACGACT-CACTATAGGGC-3¢. The PCR fragments were cloned into pGEMT(Promega, http://www.promega.com/), propagated in E. coli strainXL1BlueMRF and sequenced using the ABI Prism Big Dye Termi-nator Cycle Sequencing Ready Reaction Kit (Applied Biosystems).Sequencing reactions were run after removal of excess dye on theABI Prism 3100 Sequencer (http://www.appliedbiosystems.com).Genome Walking was performed twice until the sequence of thegene’s 5¢ end could be detected.

The entire open reading frame was obtained by RT-PCR fromP. somniferum stem mRNA using MLV Reverse Transcriptase (Pro-mega) and PfuUltra� Hotstart DNA Polymerase (Stratagene, http://www.stratagene.com/) with the following primers containing BamHIand PstI restriction sites: A21G11f2: 5¢-gaatggatccATGGAAGTAGT-TAGCCAGATTG-3¢ and A21G11r2: 5¢-gctactgcagTTAATAAACCTCA-ATTATAGATTG-3¢ using the following PCR conditions: five cycles of94�C for 30 sec, 45�C for 30 sec, 72�C for 2 min followed by 25 cyclesof 94�C for 30 sec, 55�C for 30 sec, 72�C for 2 min and a finalextension at 72�C for 5 min. After A-tailing with Taq-DNA polymer-ase, the amplified PCR product was cloned into pCR2.1 (Invitrogen,http://www.invitrogen.com/) and sequenced. For heterologous over-expression the plasmid was digested with endonucleases BamHIand PstI (Fermentas), ligated into pQE32 (Qiagen, http://www.qiagen.com/), and transformed in E. coli cells (strain SG13009).

For overexpression, the cells were induced with 1 mM isopropyl-b-D-thiogalactopyranoside, incubated at 4�C overnight, then har-vested and sonicated in extraction buffer (50 mM potassiumphosphate pH 7, 300 mM NaCl, 10% (v/v) glycerol, 1 mM

b-mercaptoethanol, 1% (v/v) Tween 20, 750 lg ml)1 lysozyme).After removal of the cell debris by centrifugation, the supernatantwas loaded onto a cobalt affinity column (Talon; Clontech, http://www.clontech.com/), followed by washing with extraction bufferwithout Tween 20 and lysozyme. The His-tagged O-methyltrans-ferase was eluted in washing buffer with an imidazole concentra-tion of 200 mM. The purified protein was desalted using PD10columns (Amersham Biosciences, http://www.gelifescience.com)in storage buffer [50 mM potassium phosphate pH 7.5, 20% (v/v)glycerol]. The purity of the enzymes was checked by SDS–PAGE(12% polyacrylamide) according to Laemmli (1970). After purifica-tion the protein concentration was determined with Bradfordreagent at 595 nm (Bradford, 1976).

Enzyme assays

The standard enzyme assay reaction mixture (80 ll) consisted of50 mM potassium phosphate pH 7, 25 mM sodium ascorbate,250 lM S-adenosyl-L-methionine (SAM), [methyl-14C]SAM (20000 c.p.m., 1.58 lM), 150 lM substrate, and 4.2 lg enzyme and wasincubated for 20 min at 35�C. Substrates were obtained from thecompound collection of the department or synthesized from buy-able precursors. The enzymatic reaction was terminated by theaddition of 100 ll of 1 M NaHCO3 and products were extracted with200 ll ethyl acetate in the case of benzylisoquinoline substrates. Forother substrates, the reaction was acidified by HCl prior to extrac-tion. The organic phase was added to 4 ml of scintillation cocktailUltima GOLD MV (Perkin Elmer, http://www.perkinelmer.com/), andthe radioactivity was quantified with a Beckman Coulter LS 6500liquid scintillation counter (http://www.beckman.com/). For productidentification on LC-MS, the enzyme assays were performed with-out the addition of radioactively labelled SAM. The influence of pHon enzyme activity was monitored in sodium citrate (pH 5.5–6.5),potassium phosphate (pH 6.0–8.4), 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-HCl (pH 8.0–9.5) and glycine/NaOH (pH 9.0–10)buffered solutions.



For enzyme kinetic analysis, the assays were performed underlinear product formation conditions which were achieved byadjusting the protein concentration or the incubation time. Thesubstrate concentration ranged between 6.25 and 300 lM at aconstant SAM concentration of 250 lM. The kinetic data for SAMwere collected by varying the SAM concentration between 12.5and 250 lM at a constant norreticuline concentration of 150 lM.Although substrate inhibition was not evident up to 300 lM norret-iculine, no further increase in the velocity could be observed above aconcentration of 150 lM. Saturation curves and calculation of kineticparameters were obtained according to Michaelis–Menten kineticsin Kaleidagraph (Synergy Software, http://www.synergy.com/).

Quantitative real-time PCR analysis

Total RNA was extracted from stems, seedlings, leaves, and roots ofP. somniferum var. Paso and from stems of P. somniferum var.Papa using TRIZOL reagent (Invitrogen). The RNA was treated withRNase Inhibitor and DNaseI (Roche, http://www.roche.com/)according to the manufacturer’s instructions. Total RNA wasquantified via absorption at 260 nm and the quality was checked bygel electrophoresis. First-strand cDNA synthesis was performedusing 2 lg total RNA and Superscript II reverse transcriptase (Invi-trogen) according to the manufacturer’s protocol. The PCR ampli-fications were performed with the following oligonucleotides whichwere designed using the Primer Express software (Applied Bio-systems): 6OMT, 5¢-AACACTGGTGGAAAAGAGAGAACC-3¢ and5¢-CCTCAATTACAGATTGGACAGCAG-3¢; 4¢OMT, 5¢-AGAGAGAG-AACTGCAGAGGATTGG-3¢ and 5¢-CTTCAATGACAGACTGAATA-GCGC-3¢; 7OMT, 5¢-CTGATGATGGCACATACTACAGCTG-3¢ and5¢-GGAAATGCCGGAGTTCGAAT-3¢; N7OMT, 5¢-GATGCAGCAG-GTTTTGCTAGTTG-3¢ and 5¢-AGCTAACAAAGTCTCGCCCTCC-3¢; ef1(elongation factor 1a), 5¢-AGATGATTCCAACCAAGCCCA-3¢ and5¢-CCTTGATGACACCAACAGCAACT-3¢. Each reaction contained a20-ng RNA equivalent of cDNA, 1 pmol of gene-specific primers,and 5 ll SYBR Green PCR Master Mix (Applied Biosystems). ThePCR reaction was performed in a MX 3000P Cycler (Stratagene)using the following protocol: 95�C for 10 min, 40 cycles 95�C for30 sec, 60�C for 1 min, 72�C for 30 sec. After each PCR a meltingcurve was measured by heating the samples to 95�C for 1 min,followed by 60�C for 30 sec and a temperature increase to 95�C.Control reactions were run with untranscribed RNA. All measure-ments were performed with two technical and two biological rep-licates. For each tissue, the delta of the threshold cycle (DCt) valuesfor each gene were obtained by subtraction of the arithmetic meanCt values of the normalizing ef1 from the arithmetic mean Ct valuesof each gene. The DDCt was calculated using the DCt value of 4¢OMTin PaSo stems as the calibrator. The Ct values of the normalizer ef1were 18.45 � 0.22 for PaSo stems, 17.14 � 0.01 for leaves, 17.25 �0.01 for roots, 16.56 � 0.52 for seedlings, and 17.06 � 0.75 for Papastems.

ACKNOWLEDGEMENTS

Funding was provided by Deutsche Forschungsgemeinschaft, Bonn(SPP1152, Priority Program ‘Evolution of Metabolic Diversity’).

REFERENCES

Boswell-Smith, V., Spina, D. and Page, C.P. (2006) Phosphodiesterase inhib-

itors. Br. J. Pharmacol. 147, Suppl 1, S252–S257.

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye

binding. Anal. Biochem. 72, 248–254.

Brisman, J.L., Eskridge, J.M. and Newell, D.W. (2006) Neurointerventional

treatment of vasospasm. Neurol. Res. 28, 769–776.

Brochmann-Hanssen, E., Leung, A.Y., Fu, C.C. and Zanati, G. (1971) Opium

alkaloids X. Biosynthesis of 1-benzylisoquinolines. J. Pharm. Sci. 60, 1672–

1676.

Brochmann-Hanssen, E., Chen, C.H., Chen, C.R., Chiang, H.C., Leung, A.Y. and

McMurtrey, K. (1975) Opium alkaloids. Part XVI. The biosynthesis of

1-benzylisoquinolines in Papaver somniferum. Preferred and secondary

pathways; stereochemical aspects.J. Chem. Soc. [Perkin 1], 16, 1531–1537.

Chung, K.F. (2005) Drugs to suppress cough. Expert Opin. Investig. Drugs, 14,

19–27.

Colombo, M.L. and Bosisio, E. (1996) Pharmacological activities of Chelido-

nium majus L (Papaveraceae). Pharmacol. Res. 33, 127–134.

Frick, S. and Kutchan, T.M. (1999) Molecular cloning and functional expres-

sion of O-methyltransferases common to isoquinoline alkaloid and phe-

nylpropanoid biosynthesis. Plant J. 17, 329–339.

Goodman, A.J., Le Bourdonnec, B. and Dolle, R.E. (2007) Mu opioid receptor

antagonists: recent developments. Chem. Med. Chem. 2, 1552–1570.

Huang, F.C. and Kutchan, T.M. (2000) Distribution of morphinan and

benzo[c]phenanthridine alkaloid gene transcript accumulation in Papaver

somniferum. Phytochemistry, 53, 555–564.

Inui, T., Tamura, K., Fujii, N., Morishige, T. and Sato, F. (2007) Overexpression

of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the

rate-limiting step in benzylisoquinoline alkaloid biosynthesis in cultured

Eschscholzia californica. Plant Cell Physiol. 48, 252–262.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of

the head of bacteriophage T4. Nature, 227, 680–685.

Loeffler, S. and Zenk, M.H. (1990) The hydroxylation step in the biosynthetic

pathway leading from norcoclaurine to reticuline. Phytochemistry, 29,

3499–3503.

Morishige, T., Tsujita, T., Yamada, Y. and Sato, F. (2000) Molecular charac-

terization of the S-adenosyl-L-methionine : 3 ‘-hydroxy-N-methylcoclaurine

4 ‘-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in

Coptis japonica. J. Biol. Chem. 275, 23398–23405.

Morishige, T., Dubouzet, E., Choi, K.B., Yazaki, K. and Sato, F. (2002) Molec-

ular cloning of columbamine O-methyltransferase from cultured Coptis

japonica cells. Eur. J. Biochem. 269, 5659–5667.

Ounaroon, A., Decker, G., Schmidt, J., Lottspeich, F. and Kutchan, T.M. (2003)

(R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-meth-

yltransferase of Papaver somniferum - cDNA cloning and characterization

of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant

J. 36, 808–819.

Pauli, H.H. and Kutchan, T.M. (1998) Molecular cloning and functional

heterologous expression of two alleles encoding (S)-N-methylcoclaurine

3’-hydroxylase (Cyp80b1), a new methyl jasmonate-inducible cytochrome

P-450-dependent mono-oxygenase of benzylisoquinoline alkaloid biosyn-

thesis. Plant J. 13, 793–801.

Rueffer, M., Nagakura, N. and Zenk, M.H. (1983a) Partial purification and

properties of S-adenosylmethionine (R),(S)-norlaudanosoline-6-O-methyl-

transferase from Argemone platyceras cell cultures. Planta Med. 49, 131–

137.

Rueffer, M., Nagakura, N. and Zenk, M.H. (1983b) A highly specific O-meth-

yltransferase for nororientaline synthesis isolated from Argemone platyc-

eras cell cultures. Planta Med. 49, 196–198.

Sato, Y., He, J.X., Nagai, H., Tani, T. and Akao, T. (2007) Isoliquiritigenin, one

of the antispasmodic principles of Glycyrrhiza ularensis roots, acts in the

lower part of intestine. Biol. Pharm. Bull. 30, 145–149.

Schmidt, J., Raith, K., Boettcher, C. and Zenk, M.H. (2005) Analysis of ben-

zylisoquinoline type alkaloids by electrospray tandem mass spectrometry

and atmospheric pressure photoionization. Eur. J. Mass Spectrom. 11, 325–

334.

Schmidt, J., Boettcher, C., Kuhnt, C., Kutchan, T.M. and Zenk, M.H. (2007)

Poppy alkaloid profiling by electrospray tandem mass spectrometry and

electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding.

Phytochemistry, 68, 189–202.

Shulgin, A.T. and Perry, W.E. (2002) The Simple Plant Isoquinolines. Berkeley,

CA: Transform Press.

Takeshita, N., Fujiwara, H., Mimura, H., Fitchen, J.H., Yamada, Y. and Sato, F.

(1995) Molecular cloning and characterization of S-adenosyl-L-methio-

nine:scoulerine-9-O-methyltransferase from cultured cells of Coptis

japonica. Plant Cell Physiol. 36, 29–36.

Thomas, J.A. (2002) Pharmacological aspects of erectile dysfunction. Jpn. J.

Pharmacol. 89, 101–112.



Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improv-

ing the sensitivity of progressive multiple sequence alignment through

sequence weighting, position-specific gap penalties and weight matrix

choice. Nucleic Acids Res. 22, 4673–4680.

Wickens, J.R., Sleeman, R. and Keely, B.J. (2006) Atmospheric pressure

ionisation mass spectrometric fragmentation pathways of noscapine and

papaverine revealed by multistage mass spectrometry and in-source deu-

terium labeling. Rapid Commun. Mass Spectrom. 20, 473–480.

Ziegler, J. and Facchini, P.J. (2008) Alkaloid biosynthesis: metabolism and

trafficking. Annu. Rev. Plant. Biol. 59, 735–769.

Ziegler, J., Diaz-Chavez, M.L., Kramell, R., Ammer, C. and Kutchan, T.M.

(2005) Comparative macroarray analysis of morphine containing Papaver

somniferum and eight morphine free Papaver species identifies an O-

methyltransferase involved in benzylisoquinoline biosynthesis. Planta,

222, 458–471.

Ziegler, J., Voigtlander, S., Schmidt, J., Kramell, R., Miersch, O., Ammer,

C., Gesell, A. and Kutchan, T.M. (2006) Comparative transcript and

alkaloid profiling in Papaver species identifies a short chain dehydro-

genase/reductase involved in morphine biosynthesis. Plant J. 48, 177–

192.

Zubieta, C., He, X.Z., Dixon, R.A. and Noel, J.P. (2001) Structures of two nat-

ural product methyltransferases reveal the basis for substrate specificity in

plant O-methyltransferases. Nat. Struct. Biol. 8, 271–279.

Accession numbers for sequence data: Sequence data from this article have been deposited with the EMBL/GenBank data

libraries under the accession numbers FJ156103 for P. somniferum norreticuline 7-O-methyltransferase (N7OMT).



functional characterization of a novel benzylisoquinoline o

Documents