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J. Biol. Chem., Vol. 275, Issue 30, 23398-23405, July 28, 2000

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Molecular Characterization of the S-Adenosyl-L-methionine:3'-Hydroxy-N-methylcoclaurine 4'-O-Methyltransferase Involved in Isoquinoline Alkaloid Biosynthesis in Coptis japonica^*

Takashi Morishige, Tetsuya Tsujita^§, Yasuyuki Yamada^¶, and Fumihiko Sato^**

From the  Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and the  Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan

Received for publication, March 23, 2000, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

S-Adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'-OMT) catalyzes the conversion of 3'-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, another O-methyltransferase, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4'-OMT preparation from cultured Coptis japonica cells. Heterologously expressed proteins had 6-OMT or 4'-OMT activities, indicative that each cDNA encodes a different enzyme. 4'-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe²⁺, Cu²⁺, Co²⁺, Zn²⁺, or Ni²⁺, but none by the SH reagent. 4'-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R,S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, an N-methylated substrate, was a much better substrate for 4'-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4'-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two related O-methyltransferases is discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reticuline is an important intermediate in the production of analgesic morphinan alkaloids (e.g. morphine), antimicrobial berberine alkaloids (e.g. berberine), and antimicrobial benzophenanthridine alkaloids (e.g. sanguinarine). The biosynthetic pathway to reticuline has been clarified, and nine enzymes that are involved in stepwise conversion of two molecules of the primary metabolite L-tyrosine to one molecule of reticuline have been identified (Fig. 1)  (Ref. 1 and references therein). Of the nine steps, three are methyl transfer reactions on the OH or NH group that are catalyzed by S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT)¹ (2-4); S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'-OMT) (5); and S-adenosyl-L-methionine:coclaurine N-methyltransferase (NMT) (6, 7).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Schematic biosynthetic pathway for a variety of isoquinoline alkaloids and the substrates for the routine O-methyltransferase assay. (R,S)-Norlaudanosoline and (R,S)-6-O-methylnorlaudanosoline (squared) were used as the substrates for the routine assay of 6- and 4'-OMT, respectively.

Methyltransferases are essential for directing intermediates to specific biosynthetic pathways (8). 6-OMT catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine (AdoMet) to the 6-hydroxyl group of norcoclaurine to form coclaurine. This is the first O-methylation step in the biosynthesis of benzylisoquinoline alkaloids. NMT catalyzes the N-methylation of coclaurine to form N-methylcoclaurine, and 4'-OMT transfers the methyl group to the 4'-hydroxyl group of 3'-hydroxy-N-methylcoclaurine to form reticuline. In addition S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase (SMT) (9-11) catalyzes the crucial step in the conversion of scoulerine, which is derived from reticuline, to tetrahydrocolumbamine in the berberine biosynthetic pathway (Fig. 1).

Each O-methyltransferase (OMT) in the biosynthesis of reticuline or berberine has strict substrate specificity, despite structural similarities of the various substrates. Because of their importance for the production of these pharmaceutically important alkaloids and their strict substrate recognition, these methyltransferases, especially OMTs, have been well characterized (2-5, 9-11). Characterizations, except for SMT, which has been purified to homogeneity, were done, however, with highly but incompletely purified enzymes, because of the high similarity of 4'- and 6-OMT.

In our previous attempt to purify 6-OMT from cultured Coptis japonica cells, two homologous 41- and 40-kDa polypeptides were isolated. Because the highly purified fraction showed both 6- and 4'-OMT activities, we speculated that these 41- and 40-kDa polypeptides encode 6- or 4'-OMT by themselves or with the aid of a heterodimer. To confirm whether this is so, we isolated the cDNAs corresponding to the internal amino acids of each polypeptide then characterized the enzyme activities using recombinant proteins heterologously expressed in Escherichia coli. The recombinant protein expressed from the cDNA of the 41-kDa polypeptide had 4'-OMT activity and that of the 40-kDa one 6-OMT activity. Enzymological properties of 4'-OMT purified without 6-OMT contamination were determined from its recombinant protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cultured Cells-- The original cultured cells were induced from rootlets of C. japonica Makino var. dissecta (Yatabe) Nakai. A cell line (156-1) that produces large amounts of alkaloids was subcultured as described elsewhere (12). Fourteen-day-old cultured cells were harvested and used for the extraction of mRNA.

Chemicals-- Berberine was purchased from Wako Pure Chemicals (Osaka, Japan). (R,S)-Norlaudanosoline and (R,S)-laudanosoline were from Aldrich. (R)- and (S)-Coclaurine were the gift of Dr. N. Nagakura of Kobe Women's College of Pharmacy. The other alkaloids used were gifts from Mitsui Petrochemical Industries, Ltd.

Amino Acid Sequence Analysis-- 6-OMT was purified from cultured Coptis cells, after which two 41- and 40-kDa polypeptides were isolated by reverse phase HPLC as described previously (4). The 41-kDa polypeptide was digested with trypsin, the 40-kDa one with the lysyl endopeptidase from Achromobacter or Staphylococcus aureus V8 protease using a Residue-specific Protease Kit (TaKaRa). The amino acid sequences of the peptides were determined with a protein sequencer (model 477A/120A; Applied Biosystems).

Screening of cDNAs and Nucleotide Sequencing-- A cDNA library of cultured Coptis cells was prepared with a ZAP-cDNA synthesis kit and Gigapack II Gold packaging extract (Stratagene) as described elsewhere (11). The oligonucleotide probes used to screen the cDNA library were based on the internal amino acid sequences of the 41- and 40-kDa polypeptides. The 41-kDa probe 1, 5'-ATGGTICCIATGATHYTIGGIATGACICARAA-3', corresponded to MVPMILGMTQK and the 41-kDa probe 2, 5'-ATHYTICAYGAYTGGAAYGA-3', to ILHDWND. The 40-kDa probe 1, 5'-GGIACIAATATTTGGGGITATATGGC-3', corresponded to GTNIWGYMA and the 40 kDa probe 2, 5'-TTTAATGAAGCIATGGCIAATAA-3', to FNEAMAND (in which probes I = inosine, H = A or C or T, Y = C or T, R = A or G). The first screening with the 41-kDa probe 1 yielded 18 positive signals, and the third screening yielded 9 independent phages. Similarly, 70 positive signals were obtained by the first screening with 40-kDa probe 1 and 18 independent phages by the third screening. The cDNA inserts of the plasmid vector (pBluescript II SK) were excised in vivo with a ZAP-cDNA synthesis kit and used for transformation to E. coli XL1-Blue. The cDNA clones (designated pBS41 for the 41-kDa polypeptide and pBS40 for the 40-kDa polypeptide) were analyzed by Southern blot hybridization using both probes and by digestion with the restriction enzyme.

cDNA sequences were determined with deletion clones prepared with ExoIII and an ALF DNA sequencer (Amersham Pharmacia Biotech) and an ABI 373A DNA sequencer (Applied Biosystems) with fluorescein isothiocyanate-labeled primers.

Construction of Expression Vectors for the 41- and 40-kDa Polypeptides-- Expression vectors for these polypeptides were constructed without the fused peptides derived from vector sequence in a pET-21d vector (Novagen). The 5'-fragment of the 41-kDa polypeptide cDNA (pBS41) was amplified by PCR by use of the following primers: the forward primer (5'-GAGAATTAACATGTCTTTCCATGG-3') was designed to introduce an AflIII site (ACPuPyGT) at the ATG start codon and the reverse primer (5'-TCCACCGACGTCAACAAGTGAGTC-3') to introduce an AatII site (GACGTC) with no modification of the conserved amino acid sequence in motif A of the AdoMet-dependent methyltransferase (13). The 3'-fragment of the 41-kDa polypeptide cDNA was amplified by use of the M13 primer and the forward primer 5'-TCACTTGTTGACGTCGGTGGAGG-3' to introduce an AatII site with no change in the amino acid sequence of motif A. The full-length cDNA that encoded the 40-kDa polypeptide (pBS40) was amplified by PCR with the M13 primer and the forward primer 5'-CAGAATCATAGACATGTTAGTGAAGAAGAAGG-3' to introduce an AflIII site at the start codon. Restriction sites were introduced by changing the underlined bases. pET-21d was digested with NcoI and XhoI, and the PCR products were ligated into the vector. The expression vectors were designated pE41 for the 41-kDa polypeptide and pE40 for the 40-kDa one. These constructs were completely sequenced to confirm that no changes were introduced by the subcloning process.

Heterologous Expression of the 41- and 40-kDa Polypeptides in E. coli-- The expression vector for the 41- or 40-kDa polypeptide was introduced into E. coli BL21 (DE3). Cells were induced with 1 mM isopropylthiogalactoside, incubated at 16 °C for 24 h, then harvested and sonicated in extraction buffer (0.1 M Tris-HCl, pH 7.5, containing 10 mM ascorbate and 20 mM 2-mercaptoethanol). The supernatant was desalted through an NAP-5 column (Amersham Pharmacia Biotech) and used to assay both 4'-OMT and 6-OMT activities.

Assay of Enzymatic Activity-- 4'-OMT and 6-OMT activities were detected by HPLC and liquid chromatography-mass spectroscopy (LC-MS). Whereas (S)-3'-hydroxy-N-methylcoclaurine and (S)-norcoclaurine are the true intermediates in reticuline biosynthesis (3), (R,S)-6-O-methylnorlaudanosoline (6-OMe-NLS) and (R,S)-norlaudanosoline (NLS) were used as the substrates for the routine assays of 4'-OMT and 6-OMT, respectively, due to the availability of these compounds and similar product formation in the reaction (Fig. 1) (4, 5). The standard 4'-OMT reaction mixture consisted of 0.3 M potassium phosphate (pH 7.4), 25 mM sodium ascorbate, 1 mM 6-OMe-NLS, 1 mM AdoMet, and the enzyme preparation. The standard 6-OMT reaction mixture consisted of 0.3 M CHES-NaOH (pH 9.0), 25 mM sodium ascorbate, 1 mM NLS, 1 mM AdoMet, and the enzyme preparation. The assay mixture was incubated at 30 °C for 1 h, after which the reaction was terminated by the addition of methanol. After protein precipitation, the reaction product was detected by reverse phase HPLC (mobile phase, 22% acetonitrile/H₂O for the 4'-OMT assay and 18% for the 6-OMT assay containing 1% acetic acid; column, LiChrospher 100 RP-18(e) (4 × 250 mm; Cica-Merck); flow rate, 0.8 ml/min; detection, absorbance measurement at 280 nm). Mass spectra were obtained with an API165 (Perkin-Elmer).

To quantify the enzymatic activity of 4'-OMT, the transfer of the ³H-labeled methyl group of S-adenosyl-L-[methyl-³H]methionine (NEN Life Science Products) to the product was measured. Reaction conditions have been described elsewhere (4).

Purification of 4'-OMT from E. coli Lysate-- Large scale production of 4'-OMT was obtained with 600-ml cultures of E. coli. Purification was done at 4 °C. All the buffers used contained 4 mM 2-mercaptoethanol and 10% glycerol. The crude bacterial lysate was applied to a Q-Sepharose Fast Flow column (2.5 × 10 cm; Amersham Pharmacia Biotech) equilibrated with 20 mM Tris-HCl (pH 7.5). Proteins were eluted with a linear NaCl gradient of 0-1 M in 20 mM Tris-HCl (pH 7.5) (total volume: 100 ml). Active fractions were applied to a Bio-Gel HTP column (2.5 × 10 cm; Bio-Rad) equilibrated with 10 mM potassium phosphate (pH 7.0). Proteins were eluted with a linear potassium phosphate gradient of 10-500 mM (total volume: 160 ml). Major enzyme activity-containing fractions were pooled and kept at 20 °C in the presence of approximately 40% glycerol, with no detectable activity loss over 5 months.

Other Methods-- The subunit molecular mass of the enzyme was ascertained by SDS-PAGE (10% polyacrylamide), and the molecular mass of the native enzyme by gel filtration chromatography through a Superose 12 column (Amersham Pharmacia Biotech) in fast protein liquid chromatography. Protein was determined according to Bradford (14) with bovine serum albumin as the standard.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of cDNAs of the 41- and 40-kDa Polypeptides-- A cDNA library prepared from C. japonica cells was screened with oligonucleotide probes designed for the internal amino acid sequences of the purified 41- and 40-kDa polypeptides. After approximately 200,000 plaque-forming units had been screened, 9 positive plaques were identified for the 41-kDa polypeptide and 18 for the 40-kDa one. cDNAs isolated in pBluescript SK (designated pBS41s and pBS40s) had inserts of 1.0-1.4 kilobase pairs.

Nucleotide Sequence and Predicted Amino Acid Sequences of pBS41 and pBS40-- The nucleotide sequences of the cDNA inserts of pBS41 and pBS40, which encoded the longest polypeptide, and of other cDNA inserts were determined (GenBank^TM accession numbers: D29812 for 41 kDa and D29811 for 40 kDa). Sequences respectively corresponding to the 41- and 40-kDa polypeptides carried 1,280 and 1,267 nucleotides, with open reading frames that encoded for 350 and 347 amino acids (Fig. 2). The calculated molecular masses of the pBS41 (38,731 Da) and pBS40 (38,655 Da) inserts were less than the observed molecular masses of the 41- and 40-kDa polypeptides from Coptis cells, but the deduced amino acid sequences had almost all the internal amino acid sequences determined from the purified polypeptides.

View larger version (73K): [in this window] [in a new window]   Fig. 2.   Amino acid sequence alignment of the O-methyltransferases in berberine biosynthesis. Motifs A, B, and C, conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferase. Boxes indicate identical amino acid residues.

Both polypeptide sequences had conserved putative AdoMet binding domains at the C-terminal end (Fig. 2, motif A-C) (13), and both had 52 amino acids between motifs A and B and 30 amino acids between motifs B and C. This spatial relationship is identical to that of the caffeic acid 3-O-methyltransferases. The 41- and 40-kDa polypeptides therefore belong to the Pl (plant)-OMT II group (13).

Their deduced amino acid sequences have a high degree of similarity (52% identity); whereas SMT, which also functions in berberine biosynthesis, showed much lower homology when its amino acid sequence was compared with that of the 41- or 40-kDa polypeptide (about 30% identity, see Fig. 2). Both polypeptides had a high degree of similarity (about 40% identity) to the isoflavone 7-O-methyltransferase of alfalfa (15), 6a-hydroxymaackiain 3-O-methyltransferase of pea (16), and hydroxycinnamic acids/hydroxycinnamoyl CoA esters O-methyltransferase of lobolly pine (17). SMT had greater similarity to the caffeic acid 3-O-methyltransferases and catechol O-methyltransferases of several plant species. Both the 41- and 40-kDa polypeptides had a somewhat low identity (24 and 35%, respectively) to catechol OMTs from Thalictrum tuberosum (Thatu 1 and 2, respective GenBank^TM accession numbers AF064693 and AF064694), even though these catechol OMTs from cultured T. tuberosum cells could methylate the 6-hydroxyl group of norcoclaurine (18). Phylogenic analysis clearly indicated that both the polypeptides belong to a different branch than the catechol OMT from Thalictrum (Thatu 2) (Fig. 3).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Phylogenic tree of plant S-adenosyl-L-methionine-dependent O-methyltransferase sequences. OMT protein sequences obtained from GenBankTM were used for tree building. Totally, 19 sequences were aligned by the multisequence alignment program in GeneWorks 2.5.1 (IntelliGenetics, Inc.) using the UPGMA (unweighted pair group maximum average) method. SMT, S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase; AEOMT, hydroxycinnamic acids/hydroxycinnamoyl CoA esters O-methyltransferase.

Expression of the 41- and 40-kDa Polypeptides in E. coli-- Expression vectors (pE41 and pE40, respectively) to produce recombinant proteins in E. coli were constructed to identify the enzymological activities of the 41- and 40-kDa polypeptides. We modified the first methionine codons of both cDNA inserts to fit the NcoI-XhoI sites of the E. coli expression vector pET-21d to obtain expression of nontagged polypeptides. These constructs then were introduced into E. coli cells, and recombinant protein production was induced. The crude E. coli lysate was used to identify the enzymatic activities of both 4'- and 6-OMT. HPLC analysis clearly showed that the cell lysate that expresses pE41 had methylation activity for 3'-hydroxy-N-methylcoclaurine, but not for norcoclaurine, whereas the lysate containing pE40 had methylation activity for norcoclaurine but not for 3'-hydroxy-N-methylcoclaurine (data not shown). The crude E. coli lysate carrying the pET-21d vector showed no enzymatic activity. LC-MS analysis confirmed that the respective reaction products of 41 kDa with 6-OMe-NLS and of 40 kDa with NLS were norreticuline and 6-OMe-NLS (Fig. 4), indicative that the 41-kDa polypeptide corresponds to 4'-OMT and the 40-kDa one to 6-OMT.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   LC-MS analysis of the 41-kDa reaction product (A), authentic norreticuline (B), 40-kDa reaction product (C), and authentic 6-O-methylnorlaudanosoline (D).

Purification of 4'-OMT and Its Characterization-- Because preparation of 4'-OMT without 6-OMT contamination is now possible, we tried to prepare highly purified 4'-OMT from E. coli. The final purification was 9.8-fold and the yield 35.9% (Table I and Fig. 5). SDS-PAGE analysis showed clearly that the purified fraction was almost homogeneous. The molecular mass of active 4'-OMT was estimated to be about 80 kDa by gel filtration chromatography, evidence that active 4'-OMT is a dimer of the 41-kDa subunit. Enzyme assays at various pH values indicated that the optimum pH for the methylation of 6-OMe-NLS was about 8.0. Half-maximal activity was found at pH 6.6 or 8.6. 

View larger version (87K): [in this window] [in a new window]   Fig. 5.   SDS-PAGE of 4'-OMT fractions in various stages of purification. Fractions from each purification step were separated by SDS-PAGE and stained with Coomassie Brilliant Blue G-250. Lane 1, molecular mass markers; lane 2, crude extract (10 µg); lane 3, Q-Sepharose column fraction (8 µg); lane 4, Bio-Gel HPT column fraction (1 µg).

4'-OMT, like Coptis 6-OMT, did not require divalent cations for activity. The addition of Fe²⁺, Cu²⁺, Co²⁺, Zn²⁺, or Ni²⁺ at 5 mM inhibited 4'-OMT activity, respectively, by 87%, 100%, 86%, 48%, or 96%. These cations also inhibited 6-OMT activity (4). Other cations (Ca²⁺, Mg²⁺, Mn²⁺) had no effect on 4'-OMT activity. The enzymatic activity of 4'-OMT, like that of 6-OMT, was negligibly inhibited by SH reagents (p-chloromercuribenzenesulfonate and iodacetamide) or EDTA at 1 mM. When, however, berberine (the end product of isoquinoline alkaloid biosynthesis in Coptis cells) was added to the assay mixture at 2.5 mM, 4'-OMT activity was not inhibited, whereas 1 mM berberine inhibited 6-OMT activity by 30% (4).

Purified 4'-OMT was used as the antigen to prepare anti-4'-OMT polyclonal antibody. In a Western blot analysis with this antibody, both the crude extract of E. coli-expressing 4'-OMT and the Coptis proteins had a major immunoreactive band of the same molecular mass (data not shown). This suggests that 4'-OMT is produced in mature form in E. coli, whereas the SMT expressed in E. coli apparently is longer than that purified from Coptis cells (10). Hydrophobicity analysis of full-length 4'-OMT, 6-OMT, and SMT showed a difference in the hydrophobicity of their N termini. There was no evidence that 4'- or 6-OMT is located in specific vesicles, whereas SMT had a hydrophobic signal sequence of 10 amino acids at its N terminus (10) (data not shown).

Substrate Specificity of 4'-OMT-- The incorporation of radioactivity from S-adenosyl-L-[methyl-³H]methionine to the products was used as a marker of substrate specificity (Fig. 6). When (R,S)-6-OMe-NLS was the control substrate (i.e. relative incorporation 100%), the respective relative activities with (R,S)-laudanosoline and (R,S)-norlaudanosoline were 767 and 118%, whereas no significant methylation was found for the other substrates. The preferential methylation of laudanosoline rather than norlaudanosoline suggests that N-methylation proceeds before 4'-O-methylation, as suggested by Stadler and Zenk (1).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Transfer by 6-OMT, 4'-OMT, and SMT of the [3H]methyl group of S-adenosyl-L-methionine to different substrates. Numerals under the structures indicate the incorporation of radioactivity from S-adenosyl-L-[methyl-3H]methionine into the product relative to the standard substrate (100%): (S)-norcoclaurine, (R,S)-6-O-methylnorlaudanosoline, (R,S)-scoulerine, respectively, for 6-OMT, 4'-OMT, and SMT. Values given are for 6-OMT (left), 4'-OMT (middle), and SMT (right). n.t., not tested.

Dependence of Initial Velocity on Substrate Concentrations and Product Inhibition Kinetics-- Whereas (S)-3'-hydroxy-N-methylcoclaurine is the true intermediate in reticuline biosynthesis (3), 6-OMe-NLS was used as the substrate for the kinetic assay of 4'-OMT due to the availability of this compound and similar product formation in the reaction (5). Substrate-saturation kinetics of the purified 4'-OMT for 6-OMe-NLS and AdoMet were the typical Michaelis-Menten type. Kinetic parameters therefore were estimated from double-reciprocal plots of the initial velocity versus the substrate concentration. By varying the concentration of 6-OMe-NLS and [³H]AdoMet in the range of 8-125 µM, a set of apparent K_m and V_max values could be calculated and replotted to determine the real K_m and V_max. The respective K_m values of 4'-OMT for (R,S)-6-OMe-NLS and AdoMet were 42 and 68 µM, with a V_max of 1.8 nkat/mg protein. The pattern of primary reciprocal plots was representative of a sequential substrate binding mechanism (data not shown).

Product inhibition analysis was used to determine the reaction mechanism. 4'-O-methylated 6-OMe-NLS (norreticuline) was used for inhibition by the methylated alkaloid. Double-reciprocal plots of the initial velocity versus the concentrations of 6-OMe-NLS, with respect to different fixed concentrations of S-adenosyl-L-homocysteine (AdoHcy), gave a series of lines intersecting to the left of the y axis (Fig. 7A). AdoHcy inhibition therefore was noncompetitive with respect to variations in 6-OMe-NLS as the substrate, and the K_i value was 43 µM. The inhibition constants were obtained as described elsewhere (19). AdoHcy inhibition with respect to varying AdoMet gave a series of lines that intersected at the y axis, indicative that the inhibition was competitive (Fig. 7B). The K_i value for AdoHcy versus AdoMet was 27 µM. Norreticuline inhibition with respect to varying the 6-OMe-NLS and AdoMet concentrations gave a series of lines intersecting to the left of the y axis (Fig. 7, C and D). Norreticuline inhibition with respect to 6-OMe-NLS and AdoMet therefore was noncompetitive. The respective K_i values for norreticuline versus 6-OMe-NLS and AdoMet were 103 and 115 µM. These findings indicate that 4'-OMT followed an ordered Bi Bi mechanism, in which AdoMet binds to the enzyme before the alkaloid substrate binds to the enzyme, after which the methylated alkaloid and AdoHcy are released sequentially.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Product inhibition of the 4'-OMT reaction presented as double-reciprocal plots. A, inhibition of the methylation reaction by AdoHcy with respect to 6-OMe-NLS. The concentration of AdoMet was fixed at 60 µM. B, inhibition of the methylation reaction by AdoHcy with respect to AdoMet. The concentration of 6-OMe-NLS was fixed at 40 µM. C, inhibition of the methylation reaction by norreticuline with respect to 6-OMe-NLS. The concentration of AdoMet was fixed at 60 µM. D, inhibition of the methylation reaction by norreticuline with respect to AdoMet. The concentration of 6-OMe-NLS was fixed at 40 µM.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We isolated full-length cDNA clones that encodes 3'-hydroxy-N-methylcoclaurine 4'-OMT and 6-OMT from cultured C. japonica cells. Clone identity was confirmed by the catalytic activities of the heterologously expressed polypeptides. Because these enzymes had very similar physical properties, it was not possible to separate them in the active form (4, 5). Our study provides the first evidence that 4'-OMT and 6-OMT are encoded by different polypeptides.

Characterization of the recombinant 4'-OMT indicated that previous data obtained for partially purified Berberis 4'-OMT was reliable. Coptis 4'-OMT has a substrate specificity similar to that of the Berberis enzyme; an adjacent 3'-hydroxyl group is essential for the 4'-O-methylation reaction, and N-methylation of the substrate enhances the reaction rate.

Previous reports suggested that 4'- and 6-OMT have similar physical properties (4, 5). Indeed, they have similar enzymological properties that are distinct from those of SMT: active 4'- and 6-OMT are dimers of the subunit, whereas SMT is a trimer. 4'- and 6-OMT activities are negligibly inhibited by SH reagents, whereas SMT activity is inhibited by the SH reagent, p-chloromercuribenzonate, evidence that the SH group(s) is involved in SMT activities. Effects of cations on the 4'- and 6-OMT activities were very similar, but each polypeptide has distinctive properties in terms of K_m values, substrate specificity, and the proposed reaction mechanism. Interestingly, 4'-OMT activity was not sensitive to berberine, the end product of alkaloid biosynthesis in Coptis, whereas the inhibitory effect of alkaloids on Berberis 4'-OMT (5) and Coptis 6-OMT and SMT has been reported (4, 8).

OMT characteristics are summarized in Table II. The deduced amino acid sequences of 4'- and 6-OMT have 52% identity, and SMT has 30% identity to both 4'- and 6-OMT. The enzymological similarities and differences are interesting in terms of molecular evolution and sequence similarities and diversities, in particular the aspects of substrate recognition.

The berberine bridge enzyme (20-22) and (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1) (23) utilize benzylisoquinoline alkaloids as substrate. The multiple sequence alignments of Coptis OMTs and these enzymes did not, however, show any sequence homology (data not shown). As with many proteins, the three-dimensional structure, rather than the primary sequence, is more important for substrate recognition. The C-terminal ends of 4'-OMT and 6-OMT are highly conserved (58% identity) for the putative AdoMet binding, but the N-terminal ends are more divergent even among these related OMTs (47% identity) (Fig. 2). These findings suggest that the N-terminal end constitutes the alkaloid-binding pocket and that these sequence diversities reflect the substrate specificities of the enzymes. X-ray diffraction studies of Coptis OMTs, characterization of the chimeric enzymes among the Coptis OMTs, or both, should prove useful for understanding the importance of the N-terminal end of OMT in substrate recognition.

Frick and Kutchan (19) reported that catechol OMTs (Thatu 1 and 2) isolated from T. tuberosum cells have very broad substrate specificity and catalyze the 6-hydroxyl methylation of norcoclaurine. These enzymes catalyzed the same reaction for 6-OMT, but showed fairly low identity with 6-OMT (35% identity). They obtained another catechol OMT (Thatu 4), which could not catalyze the methylation of isoquinoline alkaloid. Thatu 1 and Thatu 4 differ by only one amino acid; Thatu 1 has tyrosine, and Thatu 4 has cysteine at position 21. As shown in Fig. 3, the alkaloid and phenylpropanoid OMTs may be related evolutionarily, whereas the 4'- and 6-OMT obviously belong to a different branch of the phylogenic tree. OMTs provide us useful information on the molecular evolution of secondary plant metabolism.

	ACKNOWLEDGEMENTS

We thank Dr. N. Nagakura and Mitsui Petrochemical Industries Ltd. for their generous gifts of the alkaloids, and we are grateful to Dr. A. Ishihara of Kyoto University for his technical assistance in the LC-MS analysis.

	FOOTNOTES

^* This work was supported in part by Grant-in-aid B (08456172) from the Ministry of Education, Science, Sports and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBank^TM/EMBL Data Bank with accession number(s) D29811 (for 6-OMT) and D29812 (for 4'-OMT).

^§ Present address: Pharmaceutical Research Institute, Kyowa Hakko Kogyo, 1188 Shimotogari, Nagaizumi-cho, Suntou-gun, Shizuoka 411-8731, Japan.

^¶ Present address: Nara Institute of Science and Technology, Nara 630-0101, Japan.

^** To whom all correspondence should be addressed. Tel.: 81-75-753-6380; Fax: 81-75-753-6398; E-mail: fumihiko@kais.kyoto-u.ac.jp.

Published, JBC Papers in Press, May 12, 2000, DOI 10.1074/jbc.M002439200

	ABBREVIATIONS

The abbreviations used are: 6-OMT, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase; 4'-OMT, S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase; NMT, S-adenosyl-L-methionine:coclaurine N-methyltransferase; AdoMet, S-adenosyl-L-methionine; SMT, S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase; AdoHcy, S-adenosyl-L-homocysteine; OMT, O-methyltransferase; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography-mass spectroscopy; 6-OMe-NLS, 6-O-methylnorlaudanosoline; NLS, norlaudanosoline; PAGE, polyacrylamide gel electrophoresis; CHES, 2-(cyclohexylamino)ethanesulfonic acid.

	REFERENCES

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