Title:

Molecular cloning and characterization of coclaurine N-

methyltransferase from cultured cells of Coptis japonica*

Authors:

Kum-Boo Choi‡, Takashi Morishige‡, Nobukazu Shitan‡, Kazufumi

Yazaki‡, §, and Fumihiko Sato‡, §,**

Addresses:

‡, Division of Applied Life Sciences, Graduate School of Agriculture,

Kyoto University, Kyoto 606-8502, Japan

§, Division of Integrated Life Science, Graduate School of Biostudies,

Kyoto University, Kyoto 606-8502, Japan

**, To whom all correspondence should be addressed:

Dr. Fumihiko Sato

Tel:+81-(75)753-6380,

Fax:+81-(75)753-6398.

E-mail: fumihiko@kais.kyoto-u.ac.jp

Running title:

Coptis coclaurine N-methyltransferase

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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Summary

S-Adenosyl-L-methionine: coclaurine N-methyltransferase (CNMT)

converts coclaurine to N-methylcoclaurine in isoquinoline alkaloid

biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was

used to amplify the corresponding cDNA fragment, and later to isolate

full-length cDNA using 5’- and 3’-RACE. The nucleotide sequence and

predicted amino acid sequence showed that the cDNA encoded 358

amino acids which contained a putative S-adenosyl-L-methionine

binding domain and showed relatively high homology to tomato

phosphoethanolamine-N-methyltransferase. Recombinant protein was

expressed in E. coli and its CNMT activity was confirmed. Recombinant

CNMT was purified to homogeneity and enzymological characterization

confirmed that Coptis CNMT has quite broad substrate specificity; i.e.

not only for 6-O-methylnorlaudanosoline and norreticuline but also for

6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution of N-

methyltransferases in secondary metabolism is discussed based on

sequence similarity.

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Introduction:

S-Adenosyl-L-methionine: coclaurine N-methyltransferase

(CNMT) (1-3) catalyzes the transfer of a methyl group from S-adenosyl-

L-methionine to the amino group of the tetrahydrobenzylisoquinoline

alkaloid coclaurine. This is a unique N-methyltransferase in the

biosynthesis of benzylisoquinoline alkaloids (Fig. 1). This enzyme is

thought to be important because N-methylation of coclaurine strongly

enhances the 4’-O-methylation activity of 3’-hydroxy-N-

methylcoclaurine 4’-O-methyltransferase and enables the sequential

metabolic conversion of substrates (4, 5). Furthermore, the enzymatic

activity at this important step is rather low relative to the entire

biosynthetic pathway (1, 6-8). Thus, we purified this CNMT and

characterized its properties. Previous studies have clearly indicated that

CNMT is non-stereospecific and has broad substrate specificity; Coptis

enzyme methylated even simple dihydroxyisoquinoline alkaloids.

Whereas several O-methyltransferases have been characterized at the

molecular level (4, 9-12), there have been very few molecular studies of

N-methyltransferases in secondary metabolite biosynthesis in plants (13,

14). The differences in the primary structures, including the S-adenosyl

methionine binding site, of NMTs and OMTs reported so far suggest

that molecular isolation of CNMT based on the structural similarity of

methyltransferases would be very difficult. Thus, we adapted the

conventional strategy to isolate cDNA based on the amino acid sequence

of purified enzyme. Whereas our purified CNMT fraction still contained

two protein bands of about 45 kD, careful inspection of the

chromatographic behavior of the proteins and enzyme activity suggested

that the slow-moving 45 kD polypeptide would encode CNMT (1).

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Based on this observation, we determined the N-terminal amino acids,

isolated the corresponding cDNA fragment, and finally the full-length

cDNA. The nucleotide sequence of cDNA suggested that the deduced

amino acid sequence showed some similarity to known NMTs such as

phosphoethanolamine NMT. Further characterization of recombinant

protein heterologously expressed in Escherichia coli confirmed that this

isolated cDNA encoded CNMT. Recombinant CNMT was purified to

homogeneity and its enzymological properties were characterized. The

evolution of CNMT involved in secondary metabolism is discussed

based on the sequence similarity of this novel NMT.

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Experimental Procedures:

Plant material- Cultured Coptis japonica cells with high berberine

productivity were maintained as described previously (15). Ten-day-old

cultured cells were harvested and used for the extraction of mRNA.

Chemicals- Anabasine, (R,S)-1-[(3,4-dihydroxyphenyl)methyl]-

1,2,3,4-tetrahydro-6,7-isoquinolinediol (norlaudanosoline), 6,7-

dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, 1,2,3,4-

tetrahydroisoquinoline, 1-methyl-6,7-dihydroxy-1,2,3,4-

tetrahydroisoquinolinehydrobromide, 1,2,3,4-tetrahydro-3-isoquinoline

carboxylic acid hydrochloride and (+)-emetine dihydrochroride hydrate

were purchased from Aldrich. (R)-Coclaurine and (S)-coclaurine were

gifts from Dr. N. Nagakura, Kobe Women’s College of Pharmacy. Other

alkaloids were gifts from Mitsui Petrochemical Industries Ltd. All other

chemicals used were of the highest purity available.

Amino acid sequence analysis- To determine N-terminal amino acid

sequences, 100 µg of a preparation of purified CNMT (1) was

electrotransferred on a polyvinylidene difluoride (PVDF) membrane

(Nihon Milipore, Yonezawa, Japan) from a gel after sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described

elsewhere (9). A protein band of 45 kDa with slower mobility than an

accompanying band was cut from the PVDF membrane and the amino

acid sequence of the peptide was determined with a protein sequencer

(model 477A/120A; Applied Biosystems).

cDNA library construction of Coptis japonica- Total RNA was

prepared from 10 g of 10-day-old cultured Coptis cells and poly(A)+

RNA was isolated from 880 µg of total RNA using oligotex (dT)30-

Super (TaKaRa) according to the conventional protocol (16). A cDNA

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library was constructed according to the method of Gubler and Hoffman

(17). In brief, the first strand of cDNA was synthesized using 3 µg of

poly(A)+ RNA, oligo (dT) linker-primer, RAV-2 reverse transcriptase

(Stratagene), and SuperScript II reverse transcriptase (Stratagene). After

the second strand of cDNA was synthesized, blunt-end adaptor

containing the EcoR I-Not I-BamH I restriction site was ligated into

synthesized cDNA, and the cDNA fragments were then ligated into

pDR196 vector (18).

Amplification of the cDNA fragment for the N-terminal sequence-

Degenerated primers were designed from the N-terminal amino acid

sequence determined and the codon usage estimated from known Coptis

sequences (4, 9). The sense primer USP was 5’-

GCIGTIGARGCIAARCARAC-3’ to AVEAKQT and the antisense

primer UAP was 5’-TCRACRAHTAYAGYAGYAT-3’ to YDDIKQL;

I= inosine, R=A or G, H=A or T or C, Y=C or T. The N-terminal cDNA

fragment was amplified for 30 cycles: 30 sec at 94 ºC, 30 sec at 50 ºC,

and 45 sec at 72 ºC. The PCR product was subcloned into pGEM-T

vector (Promega) and its nucleotide sequence was determined. To isolate

the 3’ fragment of CNMT cDNA, primers were designed based on the

nucleotide sequence of the amplified cDNA fragment and vector

sequence. The sense primer UFSP and the antisense primer UFAP were

5’-GCTGTGGAAGCAAAGCAAACAAAGAAGGCAGC-3’ and 5’-

ACGACTCACTATAGGGCGAATTGG-3’, respectively. Rapid

amplification of the 5’ end of cDNA (5’RACE) was carried out by the

procedure of Frohman (19) with a modification to obtain full-length

cDNA. The sense primer 5RSP for 5’ RACE was designed for a PMA1

promoter in the pDR196 vector (18). The antisense primers, 5RAP1 and

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5RAP2, were designed for 5’RACE based on the cDNA sequence as

follows; 5’-CAATTGTGAATCCAAGGTCTCAACCTCTGTTGC-3’

and 5’-CTCTCGCAGTATAAATCCAGCATCGC-3’, respectively.

PCR was performed for 30 cycles using a step program; 30 sec at 94 ºC,

at 30 sec 52 ºC, and 30 sec at 72 ºC. PCR fragments were isolated and

subcloned into pGEM-T, and their nucleotide sequences were

determined.

Heterologous expression of CNMT- To express CNMT in E. coli,

full-length and partially truncated cDNAs were amplified by PCR using

30 cycles of 30 sec at 94 ºC, 90 sec at 60 ºC, and 30 sec at 72 ºC. Sense

primers (UESP: 5’-

GTTGCCATGGCTGTGGAAGCAAAGCAAACAAAGAAGGC-3’;

UESP-45N: 5’-

GTTGCCATGGACTTGTTAAAACAGTTGGAGCTGGGC-3’) were

designed to construct full-length and truncated cDNA and to introduce

an Nco I site (underlined). An antisense primer (UEAP; 5’-

GCGGAATTCACGACTCACTATAGGGCGAATTGG-3’) was used to

introduce an EcoR I site (underlined). The full-length cDNA was cloned

into the pET-21d vector (Novagen, WI) and its nucleotide sequence was

confirmed by DNA sequencing. Both expression vectors were

introduced in E. coli BL21 (DE3). Recombinant protein production was

induced in E. coli grown at 37 ºC with shaking in LB medium by adding

1 mM isopropylthiogalactoside, and E. coli was then incubated at 30 ºC

for an additional 3 h. Recombinant protein was extracted from E. coli in

100 mM Tris-HCl buffer (pH 7.5) containing 10 mM ascorbate and 20

mM 2-mercaptoethanol and used for the enzyme assay after desalting

through an NAP-5 column (Amersham Pharmacia Biotech).

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DNA sequence analysis- DNA sequencing was performed in both

directions in a DSQ-2000L automated sequencer (Shimadzu, Kyoto,

Japan) with a Thermosequence fluorescent-labeled primer cycle

sequencing kit (Amersham Pharmacia Biotech).

CNMT assay- CNMT activity was measured in 100 mM potassium

phosphate (pH 7.0), 25 mM sodium ascorbate, 0.1 mM norreticuline, 1

mM S-adenosyl-L-methionine (AdoMet), and the enzyme preparation at

30 ºC for 30 min. Although (S)-coclaurine is the true intermediate in

berberine biosynthesis, norreticuline was used as the substrate for

routine assays of CNMT due to its ready availability and similar product

formation in the reaction, as described previously (1). After the enzyme

reaction, proteins were denatured with methanol and the reaction

product was analyzed by HPLC (mobile phase, 35 % acetonitrile and

1 % acetic acid; column, LiChrospher 100 RP-18 (4 x 250 mm; Cica-

Merck); flow rate, 0.5 ml/min; detection, absorbance measurement at

280 nm). Product formation was confirmed by liquid chromatography-

mass spectroscopy (LCMS-2010, Shimadzu, Kyoto, Japan). To

determine the substrate specificity of CNMT, transfer of the 3H-labeled

methyl group of S-adenosyl-L-[methyl 3H]-methionine (NEN Life

Science Products, radioactivity; 1.5 MBq/µmol) to the product was

measured with 1 mM substrate as described previously. The activity was

shown as a relative incorporation value with 1 mM norreticuline as a

reference substance (1).

Purification of recombinant CNMT from E. coli lysate- Large

amounts of recombinant CNMT were produced in 600 ml culture of E.

coli. Unless otherwise noted, CNMT was purified as described

previously (1). In brief, CNMT was purified by Phenyl Sepharose, Q-

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Sepharose, and Mono P chromatography. All of the buffers used

contained 20 mM 2-mercaptoethanol and 10 % glycerol. The purified

enzyme was kept at -20 ºC in the presence of ca 40 % glycerol until use.

Other methods- The subunit molecular mass of the enzyme was

estimated by SDS-PAGE (10 % polyacrylamide), and the molecular

mass of the native enzyme was determined by gel filtration

chromatography on a Superose 12 column (Amersham Pharmacia

Biotech) in fast protein liquid chromatography. Protein concentration

was determined according to Bradford (20) with bovine serum albumin

as the standard.

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Results:

Isolation of cDNA for CNMT- In our previous report (1), we purified

CNMT 340-fold from cultured Coptis cells, but this highly purified

fraction still contained two protein bands. Based on a detailed analysis

of the chromatographic elution profile and enzyme activity, we

estimated that the slow-moving polypeptide should correspond to

CNMT. To obtain molecular information on CNMT, purified CNMT

was blotted on PVDF membranes and its N-terminal amino acid

sequence was determined. These 33 amino acids were determined to be

as shown in Fig. 2.

Since several attempts to isolate full-length cDNA with degenerated

primers designed for this N-terminal sequence failed, we tried to amplify

the corresponding cDNA for this N-terminal amino acid sequence.

Primers were designed based on the codon usage predicted from known

Coptis genes (4, 9) and cDNA was amplified under conditions of rather

low stringency. cDNA fragments of a corresponding length (ca 100 bp)

were amplified, subcloned and sequenced. The nucleotide sequences of

these amplified fragments indicated that a predicted amino acid

sequence of a clone corresponded to the determined N-terminal

sequence of CNMT (data not shown).

Based on the nucleotide sequence determined in a cloned PCR

fragment, strict complementary primers were designed to obtain almost

a full-length 3’-fragment of CNMT. The amplified fragment (1.3 kb)

was subcloned into pGEM-T vector and sequenced. The amino acid

sequence predicted from the determined nucleotide sequence showed the

presence of a putative AdoMet binding domain, which indicated that the

correct sequence was amplified. A 5’-fragment of CNMT was obtained

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by 5’-RACE using an internal nucleotide sequence in the 3’-fragment of

CNMT. After the sequence of this 5’-fragment was determined, full-

length cDNA was re-cloned from the cDNA library using UESP and

UEAP primers, and the nucleotide sequence was confirmed.

Nucleotide sequence and predicted amino acid sequences- Isolated

cDNA clone pCJCNMT1 contained 1,274 nucleotides with an open

reading frame that encoded 358 amino acids with a deduced Mr of

41,733 (DDBJ/GenBank/EMBL accession number AB061863) (Fig. 2).

Using the deduced amino acid sequence, we searched for homologous

sequences in the DDBJ/GenBank database using the BLAST search

program (http://blast.genome-ad.jp). BLAST search showed that the

deduced amino acid sequence had a relatively high similarity to

cyclopropane-fatty-acyl-phospholipid synthase of Mesorhizobium loti

(36 % identity; 108 aa/293 aa) and phosphoethanolamine N-

methyltransferase of Lycopersicon esculentum (28 % identity; 35 aa/124

aa), whereas the highest similarity (about 50 % identity; 152 aa/299 aa)

was found for a hypothetical protein of Arabidopsis thaliana (T05192).

O-Methyltransferases identified in berberine biosynthesis showed much

lower homology (about 10 % identity; 42 aa/358 aa). Phylogenic

analysis clearly indicated that OMTs from Coptis belong to a different

branch than CNMT (Fig. 3).

Expression of the recombinant polypeptide in E. coli and its CNMT

activity- Its amino acid sequence suggested that isolated cDNA would

encode CNMT of Coptis. To confirm the identity of this clone,

recombinant protein was produced in E. coli and enzyme activity was

determined. Enzyme assay with the crude E. coli lysate clearly showed

that the enzyme extract prepared from E. coli expressing pCJCNMT1

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had methylation activity for norreticuline (data not shown), whereas the

extract of E. coli carrying the control pET-21d vector showed no

enzymatic activity. Further LC-MS analysis confirmed that recombinant

CNMT formed reticuline from norreticuline in the presence of AdoMet

(Fig. 4). These results indicated that the isolated clone correctly encodes

Coptis CNMT. Partially truncated CNMT was also produced to examine

the importance of the N-terminal sequence. This 15-amino acid deletion

did not change the enzyme activity, whereas the protein was not purified

and the specific activity was not determined.

Purification of recombinant CNMT and its characterization- Since

CNMT purified from cultured Coptis cells was not homogeneous, we

purified recombinant CNMT to homogeneity and characterized its

enzymological properties. Recombinant CNMT was successfully

purified to homogeneity from E. coli lysate only after 3 column

chromatographies but at a 5-fold higher yield than from cultured Coptis

cells (Table 1, Fig. 5). Finally, purified recombinant CNMT showed

much higher specific activity than native CNMT purified from Coptis

cells, probably due to the high expression in transformed E. coli and the

following rapid purification. Full-length CNMT was hydrophilic, as

predicted from a hydropathy plot (data not shown).

Since the specific activity was considerably different, we re-

examined the enzymological properties in comparison with those of

native CNMT purified from cultured Coptis cells. Enzyme assays at

various pH values indicated that the optimum pH for recombinant

CNMT activity was about 7.0. Half-maximal activity was found at pH

6.5 or 8.0. Recombinant CNMT, like native CNMT, did not require

divalent cations for activity. The addition of Cu2+, Co2+, or Mn2+ at 5

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mM severely inhibited the activity by 52 %, 37 %, and 52 %

respectively. Other cations (Ca2+, Mg2+, and Fe2+) had no effect on

CNMT activity.

Substrate-saturation kinetics of purified recombinant CNMT for

norreticuline and AdoMet were the typical Michaelis-Menten. Therefore,

kinetic parameters were estimated from double-reciprocal plots of the

initial velocity versus the substrate concentration (data not shown). By

varying the concentration of norreticuline and [methyl-3H] AdoMet in

the range of 0.125 - 1 mM, a set of apparent Km and Vmax values could

be calculated and replotted to determine the actual Km and Vmax. The

respective Km values of recombinant CNMT for norreticuline and

AdoMet were 0.16 and 0.39 mM. The pattern of primary reciprocal plots

was representative of a sequential substrate binding mechanism. These

findings for recombinant CNMT indicated that the previous data

obtained for purified native CNMT (1) were reliable even though the

specific activity was considerably low. Our data also indicated that

recombinant CNMT should be quite useful for biotransformation of the

intermediates of alkaloid biosynthesis to N-methyltransfered products.

Substrate specificity of recombinant CNMT- Previously, we found

that Coptis CNMT had relatively broad substrate specificity. Since

heterologous expression in E. coli provided a sufficient amount of

recombinant enzyme, we examined the substrate specificity. The

incorporation of radioactivity from S-adenosyl-L-[methyl-3H]

methionine to the products was used to determine substrate specificity.

When norreticuline was the control substrate (i.e. relative incorporation

100 %), the respective relative activities with (R)-coclaurine and (S)-

coclaurine were 240 % and 153 %, which were comparable to values

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reported previously. Recombinant CNMT methylated 6,7-dimethoxy-

1,2,3,4-tetrahydroisoquinoline as much as coclaurine and the

methylation product was confirmed by LC-MS (data not shown, Table

2). However, the methylation of 1,2,3,4-tetrahydroisoquinoline,

tryptamine, and purine was not detected either by measuring

radioactivity incorporation or by HPLC analysis (data not shown).

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Discussion:

Based on the N-terminal amino acid sequence of purified CNMT and

information on codon usage in Coptis genes, a full-length cDNA

encoding CNMT was successfully cloned and its enzymatic activity was

confirmed in a heterologous expression system. Recombinant CNMT

purified from transgenic E. coli showed a significantly higher activity

than that of CNMT purified from cultured Coptis cells. CNMT deduced

from the cDNA sequence was 358 amino acids long, with a calculated

molecular mass of 41 kDa, which was slightly lower than the apparent

molecular mass (45 kDa) of purified CNMT determined by SDS-PAGE.

SDS-PAGE analysis of purified recombinant CNMT revealed a similar

mobility for CNMT purified from Coptis cells, indicating that this

discrepancy between calculated molecular mass and apparent molecular

mass was not due to post-translational modification, but rather to

experimental variation, probably associated with the surface charge or

conformation of CNMT.

Since this CNMT sequence was the first reported NMT sequence in

isoquinoline alkaloid biosynthesis and the third reported NMT in

alkaloid biosynthesis in plants (13, 14), the sequences of these plant

alkaloid NMTs and other methyltransferases were compared to get more

information about the diversity of methyltransferases. Whereas several

plant O-methyltransferases have relatively high sequence homology,

ranging from 32 to 71 %, the diversity of N-methyltransferases is quite

high (Fig. 3); the sequence identity among NMTs is about 5-15 %. This

means that each NMT may have evolved from an independent origin.

CNMT showed relatively high homology to a hypothetical Arabidopsis

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protein (T05192) and cyclopropane-fatty-acyl-phospholipid synthase of

Mesorhizobium (21), whereas the functional similarity is not clear.

The recent determination of the 3-dimensional structures of plant O-

methyltransferases suggested the structural conservation of an AdoMet

binding site among methyltransferases (22). As Chandrashekhar and

Vincent (23) have deduced from 56 plant enzyme sequences, motif A

(V/I/L)(V/L)(D/K)(V/I)GGXX(G/A) is present in all plant O-

methyltransferases with 0-2 mismatches. Motifs B and C,

(V/I/F)(A/P/E)X(A/P/G)DAXXXK(W/Y/F) and

(A/P/G/S)(L/I/V)(A/P/G/S)XX(A/P/G/S)(K/R)(V/I)(E/I)(L/I/V)

respectively, (X is any amino acid), are conserved 98 % with 0-3

mismatches. On the other hand, the AdoMet recognition domain was not

as highly conserved in NMTs, even though motif A was found among

almost all OMTs. In CNMT, only motif A is present with 2 mismatches

(Fig. 6). The lower conservation of the AdoMet binding domain in NMT

might be due to structural differences between NMTs and OMTs.

Whereas many OMTs have an AdoMet binding site in their C-terminal

half, motif A in CNMT and other NMTs is located at the N-terminal end

(24-26). Interestingly, a putative Arabidopsis protein (T05192) also has

this motif A, and cyclopropane-fatty-acyl-phospholipid synthase also

shows a similar identity in this region. Some enzymes in secondary

metabolism such as putrescine N-methyltransferase and tropinone

reductase have been postulated to have evolved from enzymes in

primary metabolism such as spermine synthase or the short-chain

dehydrogenase gene family (13, 27). Further characterization of the

enzyme activity of Arabidopsis T05192 and site-directed mutagenesis of

CNMT should provide clues for understanding the evolution of CNMT.

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Acknowledgments:

We greatly appreciate Dr. Fang-Sik Che of Nara Institute of Science

and Technology for N-terminal amino acid sequencing of CNMT. We

also thank Dr. N. Nagakura and Mitsui PetroChemical Industries Ltd. for

their generous gifts of the alkaloids. We are grateful to Dr. W. Frommer

of the University of Tuebingen for the gift of pDR196.

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References:

1. Choi, K.B., Morishige, T., and Sato, F. (2001) Phytochemistry 56,

649-655

2. Frenzel, T., and Zenk, M.H. (1990) Phytochemistry 29, 3491-3497

3. Wat, C.K., Steffens, P., and Zenk, M.H. (1986) Z. Naturforschung

41c, 126-134

4. Morishige, T., Tsujita, T., Yamada, Y., and Sato, F. (2000) J. Biol.

Chem. 275, 23398-23405

5. Stadler, R., and Zenk, M.H. (1993) J. Biol. Chem. 268, 823-831

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

(1993) Phytochemistry 32, 659-664

7. Sato, F., Tsujita, T., Katagiri, Y., Yoshida, S., and Yamada, Y.

(1994) Eur. J. Biochem. 225, 125-131

8. Yamada, Y., and Okada, N. (1985) Phytochemistry 24, 63-65

9. Takeshita, N., Fujiwara, H., Mimura, H., Fitchen, J.H., Yamada, Y.,

and Sato, F. (1995) Plant Cell Physiol. 36, 29-36

10. Ibrahim, R.K. (1997) Trends Plant Sci. 2, 249-250

11. Ibrahim R.K., Bruneau, A., and Bantignies, B (1998) Plant Mol. Biol.

36, 1-10

12. Frick, S., and Kutchan, T.M. (1999) Plant J. 17, 329-339

13. Hibi, N., Higashiguchi, S., Hashimoto, T., and Yamada, Y. (1994)

Plant Cell 6, 723-735

14. Kato, M., Mizuno, K., Crozier, A. Fujimura, T., and Ashihara, H.

(2000) Nature 406, 956-7

15. Sato, F., and Yamada, Y. (1984) Phytochemistry 23, 281-285

by guest on July 14, 2018http://w

ww

.jbc.org/D

ownloaded from

16. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor

Laboratory, New York

17. Gubler, U., and Hoffman, B.J. (1983) Gene 25, 263-269

18. Rentsch, D., Laloi, M., Rouhara, I., Schmelzer, E., Delrot, S., and

Frommer, W.B. (1995) FEBS Lett. 370, 264-268

19. Frohman, M.A. (1990) PCR Protocols: a Guide to Methods and

Applications. (Gelfand, D.H., Sninsky, J., and White, T.J. eds) pp.28-38,

Academic Press, San Diego

20. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254

21. Kaneko, T., Nakamura, Y., Sato, S., Asamizu, E., Kato, T.,

Sasamoto, S., Watanabe, A., Idesawa, K., Ishikawa, A., Kawashima, K.,

Kimura, T., Kishida, Y., Kiyokawa, C., Kohara, M., Matsumoto, M.,

Matsuno, A., Mochizuki, Y., Nakayama, S., Nakazaki, N., Shimpo, S.,

Sugimoto, M., Takeuchi, C., Yamada, M., and Tabata, S. (2000) DNA

Res. 7, 331-338

22. Zubieta, C., He X.Z., Dixon, R.A., and Noel, J.P. (2001) Nature

Struct. Biol. 8, 271-279

23. Chandrashekhar, P.J., and Vincent, L.C. (1998) Plant Mol. Biol. 37,

663-674

24. Quaife, C.J., Hoyle, G.W., Froelick, G.J., Findley, S.D., Baetge, E.E.,

Behringer, R.R., Hammang, J.P., Brinster, R.L., and Palmiter, R.D.

(1994) Transgenic Res. 3, 388-400

25. Ying, Z., Mulligan, R.M., Janney, N., and Houtz, R.L. (1999) J. Biol.

Chem. 274, 36750-36756

26. Scoilas, A., Black, M.H., Talieri, M., and Diamandis, E.P. (2000)

Biochem. Biophys. Res. Commun. 278, 349-359

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27. Nakajima K., Hashimoto, T., and Yamada, Y. (1993) Proc. Natl.

Acad. Sci. U.S.A. 90, 9591-9595

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Footnotes:

*This research was supported in part by a Grant-in-Aid B (08456172) for

Scientific Research from the Ministry of Education, Science, Culture and

Sports (Japan) and a Research for the Future Program grant (JSPS-RFTF

00L01607) from the Japan Society for Promotion of Science (F.S.).

1Abbreviations: AdoMet, S-adenosyl-L-methionine; CNMT,

coclaurine N-methyltransferase; LC-MS, liquid chromatography-mass

spectrometry; NMT, N-methyltransferase; OMT, O-methyltransferase;

PCR, polymerase chain reaction; 5’RACE, 5’ rapid amplification of

cDNA end; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel

electrophoresis

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Figure legends:

Fig. 1

Schematic biosynthetic pathway of berberine and the structure of

isoquinoline alkaloid tested as CNMT substrate (squared). 6OMT;

norcoclaurine 6-O-methyltransferase, CNMT; coclaurine N-

methyltransferase, 4’OMT; 3’-hydroxy-N-methylcoclaurine 4’-O-

methyltransferase, SMT; scoulerine 9-O-methyltransferase

Fig. 2

Nucleotide and deduced amino acid sequences of S-adenosyl-L-

methionine: coclaurine N-methyltransferase. The N-terminal amino

acid sequence determined from purified CNMT is boxed. Bold arrows

(USP, UAP) above the nucleotides indicate the sense and antisense

primers for N-terminal cloning of 100 bp. Thin arrows (UFSP, UESP-

45N) with dotted lines are PCR primers for the C-terminal fragment.

Thin arrows (5RAP1, 5RAP2) indicate the sense primers for 5’RACE.

Asterisk shows the stop codon. Motif A, a conserved sequence motif in

plant AdoMet-dependent methyltransferase, is shadowed.

Fig. 3

Phylogenic tree of N- and O-methyltransferases. Amino acid sequences

obtained from GenBankTM were used for tree building. Nine sequences were

aligned by the multisequence alignment program in GENETYX-MAX Ver.11

(Software Development Inc., Japan) using the UPGMA (unweighted pair

group maximum average) method.

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Fig. 4

LC-MS analysis of recombinant CNMT reaction product (a), authentic

reticuline (b), and authentic norreticuline (c).

Fig. 5

SDS-PAGE analysis of purified CNMT.

(a), (b), and (c) show the analysis of crude extract of transformed E. coli,

purified recombinant CNMT, and native CNMT purified from Coptis cells,

respectively. Fractions purified on a Mono P column were analyzed by SDS-

PAGE in 10 % polyacrylamide gel and stained with Coomassie Brilliant Blue.

Fig. 6

Amino acid sequence alignment of the motif A domains of the N- and O-

methyltransferases in Coptis japonica, a hypothetical protein in

Arabidopsis thaliana, and cyclopropane-fatty-acyl-phospholipid synthase

in Mesorhizobium loti.

Motifs A is conserved sequence motif in plant S-adenosyl-L-methionine-

dependent methyltransferases. Amino acids conserved in all of the sequences

are boxed and similar ones are shadowed. CNMT; coclaurine N-

methyltransferase, cyclopropane; cyclopropane-fatty-acyl-phospholipid

synthase, 4’OMT; 3’-hydroxy-N-methylcoclaurine 4’-O-methyltransferase,

6OMT; norcoclaurine 6-O-methyltransferase, SMT; scoulerine 9-O-

methyltransferase

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Table 1. Purification of coclaurine N-methyltransferase from recombinant E.coli.

PurificationSteps

TotalProtein(mg)

TotalActivity(nkat)

SpecificActivity

(nkat/mg)

PurificationFold

Yield(%)

Crudeextract

85.0 15.36 0.18 1 100

30~50%(NH4) 2SO4

precipitation

41.0 8.20 0.20 1.1 53

Phenyl-Sepharose

4.78 6.90 1.44 8 44

Q-Sepharose 0.25 3.30 13.2 73.3 21

Mono P 0.05 0.90 18.0 100 5.8

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Table 2. Substrate specificity of purified recombinant coclaurine N- methyltransferasea.

(R)-Coclaurine

(S)-Coclaurine

Relative

activity R1b R2 R3 R4

240 OMe OH H OH

153 OMe OH H OH

OMe OH OH OMe

OH OH OH OH

OMe OH OH OH

(R,S)-Norreticuline

(R,S)-Norlaudanosoline

(R,S)-6-O-

Methylnorlaudanosoline

(R,S)-Scoulerine

6,7-Dimethoxy-1,2,3,4-

tetrahydroisoquinoline

1-Methyl-6,7-dihydroxy-

1,2,3,4-tetrahydroisoquinoline

1,2,3,4-Tetrahydroisoquinoline

1,2,3,4-Tetrahydro-3-

isoquinoline carboxylic acid

(+)-Emetine

100

49

69

0

180

15

0

0

0

aThe enzyme reaction mixture was incubated for 10 min at 30 ˚C; total volume 50

µl containing 100 mM potassium phosphate (pH 7.0), 2.5 mM sodium ascorbate,

1 mM 3H-AdoMet and 20 µl of the purified Mono P fraction (ca. 0.2 µg protein).bSee Fig.1 for the numbering system.

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NHHO

HO

OH

NHH3CO

HO

OH

NCH3

H3CO

HO

OH

NCH3

H3CO

HO

OH

NCH3

H3CO

HO

OCH3

NH3CO

HOH

H H H

H HOH

NH3CO

HO NR1

R2

OH OH

H

R4

OCH3

OCH3

OCH3

HR3

Berberine

(S )-Scoulerine

SMT

(S )-Tetrahydrocolumbamine

(S )-N-Methylcoclaurine

(S )-3'-Hydroxy-N-methylcoclaurine

4'OMT

(S )-Reticuline

CNMT

(S )-Coclaurine

6OMT

(S )-Norcoclaurine

L-Tyrosine

Fig. 1

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M A V E A K Q T K K A A I V E L L

K Q L E L G L V P Y D D I K Q L I

R R E L A R R L Q W G Y K P T Y

E E Q I A E I Q N L T H S L R Q M

K I A T E V E T L D S Q L Y E I P

I E F L K I M N G S N L K G S C

C Y F K E D S T T L D E A E I A M

L D L Y C E R A Q I Q D G Q S V L

D L G C G Q G A L T L H V A Q K

Y K N C R V T A V T N S V S Q K E

Y I E E E S R R R N L L N V E V K

L A D I T T H E M A E T Y D R I

L V I E L F E H M K N Y E L L L R

K I S E W I S K D G L L F L E H I

C H K T F A Y H Y E P L D D D D

W F T E Y V F P A G T M I I P S A

S F F L Y F Q D D V S V V N H W T

L S G K H F S R T N E E W L K R

L D A N L D V I K P M F E T L M G

N E E E A V K L I N Y W R G F C L

S G M E M F G Y N N G E E W M A

S H V L F K K K *

ATGGCTGTGG AAGCAAAGCA AACAAAGAAG GCAGCCATAG TAGAGTTGTT

AAAACAGTTG GAGCTGGGCT TGGTTCCATA TGATGATATT AAGCAGCTCA

TAAGGAGGGA ACTGGCAAGG CGCCTGCAAT GGGGTTATAA ACCTACTTAT

GAAGAACAAA TAGCTGAAAT CCAAAACTTA ACTCATTCTC TGCGACAAAT

GAAAATTGCA ACAGAGGTTG AGACCTTGGA TTCACAATTG TACGAGATTC

CTATTGAGTT TCTAAAGATT ATGAATGGAA GTAACTTAAA AGGAAGTTGT

TGCTACTTCA AAGAAGATTC AACAACATTA GATGAAGCTG AGATAGCGAT

GCTGGATTTA TACTGCGAGA GAGCTCAAAT CCAAGATGGA CAGAGTGTTC

TTGATCTTGG ATGTGGGCAA GGAGCTCTTA CATTACATGT TGCACAGAAA

TATAAAAACT GTCGCGTAAC AGCAGTAACA AATTCAGTTT CACAAAAAGA

GTACATTGAA GAAGAATCAA GGAGACGTAA TTTGTTGAAT GTGGAAGTCA

AATTGGCAGA CATAACCACA CATGAGATGG CTGAGACATA CGATCGTATT

TTGGTAATAG AGTTGTTTGA GCACATGAAG AACTATGAAC TTCTCCTGAG

GAAAATCTCA GAGTGGATAT CGAAAGATGG GCTTCTCTTT CTAGAGCACA

TATGCCACAA GACCTTTGCT TACCACTATG AGCCTCTAGA CGACGACGAT

TGGTTTACAG AGTACGTGTT TCCTGCTGGG ACTATGATCA TACCATCTGC

ATCGTTCTTT TTGTATTTCC AGGATGACGT TTCGGTTGTG AACCATTGGA

CTCTTAGTGG GAAGCACTTT TCGCGTACCA ATGAGGAATG GTTGAAGAGA

TTGGACGCAA ACCTTGATGT TATTAAACCA ATGTTTGAGA CTTTAATGGG

AAATGAGGAA GAGGCAGTGA AGTTGATTAA CTATTGGAGA GGATTTTGTT

TATCTGGAAT GGAAATGTTT GGATATAACA ATGGTGAAGA ATGGATGGCA

AGTCATGTTC TGTTCAAGAA AAAATGA

USP

UFSP UESP-45N

UAP

5RAP1

5RAP2

Motif A

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1077

Fig. 2

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4'OMT(D29812;Coptis japonica)

putrescine NMT(AB004324;Nicotiana sylvestris)

phosphoethnolamine NMT(AAG59874;Lycopersicon esculentum)

hypothetic protein(T05192;Arabidopsis thaliana)

CNMT(AB061863;This work)

SMT(D29809;Coptis japonica)

6OMT(D29811;Coptis japonica)

caffeine synthase(AB031280;Camellia sinensis)

phenylethanolamine NMT(X52730-1;Human)

0.3308

0.3308

0.2562

0.5870

0.5740

0.1033

0.9199

0.9199

0.4216

1.1192

1.1192

0.1451

0.0772

0.8349

0.3261

0.3261

Figure 3.

0.3308

1.1192

0.1451

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192299

330

(a)

330

299192

(b)

N

H3CO

HO

OH

OCH3

CH3

Reticuline m/z=330

N

H3CO

HOCH3

+

m/z=192

178299

316

(c)

N

H3CO

HO

OH

OCH3

HN

H3CO

HOH

+

Norreticuline m/z=316

m/z=178

Fig. 4

m/z

m/z

m/z

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kDa

111

73

48

33

28

22

(a) (b) (c)

Fig. 5

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hypothetical(T05192) VLDIGCGWGCNMT(AB061863) VLDLGCGQGSMT(D29809) LVDVGGGIG6OMT(D29811) LVDVGGGTG4'OMT(D29812) LVDVGGGNG

cyclopropane(BAB53730) ILELGCGWG

Fig. 6

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SatoKum-Boo Choi, Takashi Morishige, Nobukazu Shitan, Kazufumi Yazaki and Fumihiko

cultured cells of Coptis japonicaMolecular cloning and characterization of coclaurine N-methyltransferase from

published online October 26, 2001J. Biol. Chem. 

  10.1074/jbc.M106405200Access the most updated version of this article at doi:

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