JP2002291487A - Cocculaurine-n-methyl transferase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of efficiently producing an isoquinoline alkaloid useful as a drug using cocculaurine-N-methyl transferase(CNMT). SOLUTION: N-methyl cocculaurine and/or various kinds of alkaloids biosynthesized from the N-methyl cocculaurine are produced by using CNMT being N-methyl cocculaurine biosynthetic enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to cochraulin-N-methyltransferase (hereinafter sometimes abbreviated as "CNMT"), which is an N-methylcoclaurine biosynthesis enzyme.
The present invention relates to a method for producing N-methylcoclaurin and / or various alkaloids biosynthesized from the N-methylcoclaurin and an N-methylated compound produced by CNMT using the DNA.

[0002]

BACKGROUND ART Berberine is a kind of secondary metabolite of a plant classified as an isoquinoline alkaloid, and ceribauren (Coptis japonica Makino var. Di.
ssecta (Yatabe) Nakai) and Japanese larch (Thalictrum mi)
nus var. hypolencum), yellow citrus (Phelloden)
dron amurense Rupr), Barberry (Berber)
is wilsoniae) and has antibacterial, stomachic and anti-inflammatory activities. At present, berberine is produced by extraction from berberine-containing plant natural products such as the above plant species, and an industrial production method using cultured cells of the plant is being studied [K. Matsubara et
al., J. Chem. Tech. Biotechnol. 46,61-69 (1989)]
. Berberine biosynthesis in cultured cells has been well studied at the enzyme level as a source of biotechnology and because of the fundamental interest in the metabolic regulation of alkaloid biosynthesis [TM Kutchan, In The alkaloid
s. Vol 50 (G. Cardell, ed.), San Diego, Academic Pr
ess, pp257-316 (1998); F. Sato et al., Phytochemist
ry 32, 659-664 (1993); F. Sato et al., European Jo
urnal of Biochemistry 225, 125-131 (1994)]. Berberine uses tyrosine as a starting compound in biosynthesis,
It is biosynthesized through 13 different enzymatic reactions via (S) -norcoclaurin (FIG. 1). One N-methyltransferase (NMT) [T. Fren
zel and MH Zenk, Phytochemistry 29, 3491-3497
(1990); CK Wat and MH Zenk, Zeitschrift fuer
Naturforschung 41c, 126-134 (1986)], three O-methyltransferases (OMTs) [M. Ruffer et al., Planta
Medica 49, 131-137 (1983); S. Muemmler et al., Pla
nt Cell Reports 4, 36-39 (1985); T. Frenzel and M.
H. Zenk, Phytochemistry 29, 3505-3511 (1990); F. Sa
to etal., Phytochemistry 32, 659-664 (1993); F. Sa
to, et al. European Journal of Biochemistry 225, 1
25-131 (1994)], one hydroxylase [S. Loeffle
r and MH Zenk, Phytochemistry 29, 3499-3503 (19
90)] One berberine cross-linking enzyme [P. Steffens et al.,
Tetrahedron Letters 25, 951-952 (1984)], one methylenedioxy ring-forming enzyme [M. Rueffer and MH Zenk,
Tetrahedron Letters 26, 201-202 (1985)], one tetrahydroprotoberberine oxidase [Y. Yamada and
N. Okada, Phytochemistry 24, 63-65 (1985); E. Gal
nder et al., Plant Cell Reports 7, 1-4 (1988)]. However, among these enzymes, those highly purified and whose enzymological properties were revealed were 6-O-methyltransferase [M. Ruffer et al., Planta Me
dica 49, 131-137 (1983); F. Sato, et al. European
Journal of Biochemistry 225, 125-131 (1994)], 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase [T. Frenzel and MH Zenk, Phytochemi
stry 29, 3505-3511 (1990)], (S) -Skourelin 9-O-
Methyltransferase [F. Sato et al., Phytoche
mistry 32, 659-664 (1993)]. It is useful to clarify the enzymological properties of the enzymes involved in biosynthesis and the enzyme genes and to apply biotechnology in the bioconversion of fine chemicals. However, the difficulty in purifying enzymes on the berberine biosynthetic pathway lies in the fact that the characteristics of the enzymes are similar due to the similar reaction mechanisms and substrates of these enzymes.

Recent studies on the isolation of methyltransferase cDNAs and their expression in E. coli have
It provides further information on O-methyltransferase [Frick et al., Plant J. 17 (4), 329-339 (199
9); Morishige et al., J. Biol. Chem. 275 (30), 2339
8-23405 (2000)]. However, there are few reports on cokraulin N-methyltransferase, a unique N-methyltransferase in berberine biosynthesis. In N-methyltransferase,
Only putrescine N-methyltransferase in nicotine biosynthesis has been well studied and cDNA has been isolated [N. Hibi et al., Plant Cell 6, 723-735 (1994)].
CNMT, which catalyzes the N-methylation reaction of berberine biosynthesis,
S-adenosyl-L-methionine (hereinafter abbreviated as "SAM")
Is an enzyme that methylates the amino group of (S) -coclaurine using N as a methyl group donor to produce N-methylcoclaurine. Berberis is an enzyme that catalyzes the same reaction as this enzyme
It has already been isolated from koeineana and its enzymatic properties have been investigated [T. Frenzel and MH Zenk, Phyt
ochemistry 29, 3491-3497 (1990)], and the enzyme is norlaudanosoline, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, methyl-6,7-dihydroxy-1,2,
3,4-Tetrahydroisoquinoline is not recognized as a substrate and does not catalyze its N-methylation reaction. On the other hand, there has been no research to elucidate the isolation, purification, enzymatic chemical properties and gene of CNMT derived from spinach cells.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to efficiently produce isoquinoline alkaloids useful as pharmaceuticals by using CNMT. In addition, DNA encoding CNMT was cloned, its nucleotide sequence was determined, and CNM was performed using cells in which the cloned recombinant DNA was expressed.
T, production of DNA encoding the enzyme and N-methylcoclaurine and / or various alkaloids biosynthesized from this N-methylcoclaurin, and various N-methylated compounds catalyzed by CNMT. Aim.

[0005]

Means for Solving the Problems As a result of sincerity research, the present inventors found that CNMT enzyme activity (production of reticuline from norreticulin and S-adenosylmethionine) from cultured cells of spinach was used as an index. Isolation and purification were attempted, and ammonium sulfate precipitation and various column chromatography
A 0-fold purified enzyme was obtained in a yield of 1% of the sparse extract (Table 1).
1). The enzymological properties of this purified enzyme were examined. CNMT obtained from cultured spinach cells showed that norlaudanosoline, 6,7-dimethoxy-, which did not serve as a substrate for the enzymatic reaction in CNMT isolated from Berberis. 1,2,3,4-tetrahydroisoquinoline, methyl-6,7-dihydroxy-1,2,3,
4-Tetrahydroisoquinoline is also used as a substrate and catalyzes the methylation reaction.
Substrate specificity was lower than that of CNMT, and it was confirmed that more compounds could be methylated. In addition, the N
Based on the terminal amino acid sequence and the frequency of use of the codons of ours, which we have analyzed so far,
The CNMT cDNA was cloned by the R method. Full length of this cDNA
The ORF was incorporated into an Escherichia coli expression vector, transformed, and analyzed for function.
Generation of reticuline from S-adenosylmethionine, that is, N-methyltransferase activity was confirmed, confirming that the present cDNA encodes CNMT.

That is, a first aspect of the present invention has an enzyme activity of coclaurin N-methyltransferase,
Further, norlaudanosoline, 6,7-dimethoxy-1,2,
A wide range of compounds, including isoquinolines such as 3,4-tetrahydroisoquinoline and methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, and phenolamines such as phenylethanolamine, as substrates for enzyme reactions An enzyme that catalyzes a methylation reaction and / or a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. Further, a second embodiment is a DNA comprising a nucleotide sequence encoding a polypeptide having CNMT enzyme activity and / or a DNA comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. DNA comprising the nucleotide sequence of No. 1
Is mentioned. A third aspect of the present invention is a vector comprising the DNA according to the second aspect. A fourth aspect of the present invention is a cell transformed with the above vector. Also,
A fifth aspect of the present invention is a method for producing a secondary metabolite of a plant using the above transformed cell. This secondary plant metabolite may be an alkaloid biosynthesized from N-methylcoclaurine as well as N-methylated compounds produced by CNMT.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The present inventors isolated CNMT from cultured cells of spinach, which is one of the berberine-type alkaloid-producing plants, but SA
Any plant having a reaction of converting coclaurine to N-methylcoclaurin using M as a methyl group donor is presumed to contain substantially the same enzyme as the CNMT of the present invention, and is described herein. By using the method described above, substantially the same CNMT of the present invention can be isolated from these plants. Plants having a reaction of converting cocolaurine to N-methylcoclaurin as a SAM as a methyl group donor include plants of the genus Coptis belonging to the family Ranunculaceae such as Seribaouren,
Rutaceae Thalictrum genus plants such as Japanese larch, Rutaceae Phellodendron genus plants such as yellowfin, Barberry Berberis genus plants such as barberry, and poppies Papa such as poppies
Examples include plants of the genus ver.

Further, the CNMT of the present invention is shown in Table 2 and FIG.
As shown in the above, since structurally similar compounds such as norlaudanosoline and norreticulin can also be methylated in addition to coclaurin, plants containing these structurally similar compounds of coclaurine even if they are other than the above plant species If so, the method of the present invention can be applied.

[0009]

[Table 1] Substrate specificity of Seribaouren CNMT ─────────────────────────────────── Substrate specific activity ── ───────────────────────────────── (R) -Cokraurin 122 (S) -Cokraurin 100 (R, S) -Norreticulin 55 (R, S) -Norlaudanosoline 48 (R, S) -6-O-Methylnorlaudanosoline 38 (R, S) -Scorelin 0 6,7-Dimethoxy-1,2 , 3,4-tetrahydroisoquinoline 39 1,2,3,4-tetrahydroisoquinoline 0 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline 10 1,2,3,4-tetrahydro- 3-isoquinolinecarboxylic acid 0 (+)-emetine 0 ─────────────────────────────────── Enzyme As a result of a chemical study, it was found that the CNMT according to the present invention has the following basic properties. It has become. ─────────────────────────────────── tetramer molecular weight: 160,000 (gel filtration chromatography) Molecular weight: 45,000 (SDS / PAGE) Isoelectric point: 4.2 (Mono-P) Optimum pH: 7.0 Km value (Fig. 3): 0.65 mM (S-adenosyl-L-methionine) 0.38 mM ((R, S)- Norreticulin) Inhibitor (Table 3): Co ²⁺ , Cu ²⁺ , Mn ²⁺ Reaction mechanism (Fig. 4): Typical Michaels-Menten type ─────────────── ─────────────────────

Next, cDNA encoding CNMT was isolated and its nucleotide sequence was determined. First, the N-terminal amino acid sequence of a CNMT sample isolated and purified from cultured berberine-producing spinach cells was determined, and a degenerate primer for this sequence was used to perform PCR using the alkaloid-producing spinach cultured cell cDNA library as a template. The cDNA corresponding to this amino acid sequence was amplified and isolated, and its nucleotide sequence was determined. Furthermore, by obtaining a nucleotide sequence corresponding to the C-terminal and N-terminal portions of the enzyme by PCR using a primer designed from the determined internal nucleotide sequence and a primer for the adapter region of the cDNA library, the full-length nucleotide sequence was obtained. It was determined. The full-length ORF of the present cDNA was integrated into an E. coli expression vector, and transformed into E. coli.Functional analysis revealed that N-methyltransferase activity, which was not originally present in the E. coli, was confirmed. It was confirmed that it encodes transferase. In addition, the present recombinant enzyme has an N-methyltransferase activity for the above-mentioned substrate possessed by the isolated and purified CNMT, and additionally has 4-O-methyldopamine and norepinephrine.
-Has weak methylating activity.

[0011]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples. [Example 1] Extraction and purification of CNMT First, high-cultured berberine-producing cultured cells were derived from seribaouren as follows.

After sterilizing the leaves and petiole of the squirrel spinach with 70% ethanol and antiformin solution, sections were prepared and rinsed with Rinsmeier Skoog containing 10 ⁻⁵ M α-naphthaleneacetic acid and 10 ⁻⁸ M 6-benzyladenine. The plate was placed on an agar medium and cultured at 25 ° C. in a dark place. About three weeks after the start of the culture, yellow calli generated at the cut end of the section were collected, transplanted to the same agar medium as described above, and further grown. The callus thus obtained was suspended in a liquid medium having the same composition as described above, and cultured at 25 ° C in the same manner as described above in the dark at 100 rpm using a rotary rotary shaker for shaking.
The transplant was repeated every ~ 3 weeks.

The obtained liquid culture cells have a diameter of several tens μm.
Since the cells consist of cell clusters of a few mm in diameter, a berberine high-producing strain was selected using these cell clusters as a unit. That is,
A part of the cell mass was uniformly spread on the agar medium and cultured, and colonies were grown from each cell mass. The obtained colonies were individually grown on the same agar medium, while the content of berberine was measured using liquid chromatography, and those with a high content were selected as high-producing cells, and this strain was used as a new parent strain. , And the selection by the above operation was repeated to obtain a high production strain.

The berberine high-producing cultured cells are subcultured every three weeks in a Linsmeier Skoog liquid medium containing 10 μM α-naphthaleneacetic acid and 0.01 μM 6-benzyladenine, and swirled at 25 ° C. in the dark. The cells were cultured with shaking at 100 rpm using a rotary shaker. Cells were harvested on day 14 of culture, immediately frozen in liquid nitrogen, and stored at -80 ° C.

Using these cells, CNMT was isolated and purified. Unless otherwise noted, all operations are performed at 0-4 ° C,
20 mM β-mercaptoethanol and 10% in all buffers
Glycerol was added. Approximately 200 g of frozen cultured spinach cells were disrupted in 450 ml of 0.2 M Tris-HCl buffer using a Waring blender at maximum speed for 2 minutes, followed by 2 min.
The enzyme was extracted by sonication for 0 minutes. The suspension was filtered and the filtrate was centrifuged at 10,000 xg for 50 minutes. Supernatant
Desalting was performed using a NAP-10 column (Parmacia) to obtain a sparse extract.
The enzyme was precipitated with 30-50% saturated ammonium sulfate. The precipitated protein
Dissolved in 60 ml of extraction buffer and centrifuged. Protein solution is PD-1
The mixture was desalted with a 0 column (Pharmacia) and loaded on a phenyl sepharose CL-4B column equilibrated with an initial buffer (200 mM Tris-HCl buffer containing 30% ammonium sulfate (pH 7.5)). After thoroughly washing the column with the same buffer to remove yellow alkaloids, CNMT was eluted with 120 ml of a 30% to 0% linear gradient ammonium sulfate solution. CNMT fraction was pre-equilibrated with 200 mM Tris-HCl buffer (pH 7.5)
The sample was subjected to ion exchange chromatography using a Q-Sepharose FF column (Pharmacia). After washing the column with the same buffer, the CNMT was eluted with 120 ml of a 0 to 0.5 M linear gradient of sodium chloride solution. The active fractions were collected, desalted and 20 mM Tris-HCl buffer (pH 7.
Mono-Q column pre-equilibrated in 5) (HR5 / 5, Pharmacia)
On an FPLC system. CNMT is 40ml 0 ~ 0.
Elution was performed stepwise with a 35M gradient of sodium chloride solution. Further, active fractions were collected and chromatofocused on an FPLC system using a Mono-P column (HR5 / 5, Pharmacia) (initial buffer: 25 mM Bis Tris).
-Iminodiacetic acid buffer (pH 7.1), elution buffer; 10% poly buffer 74 adjusted to pH 4 with iminodiacetic acid 74). The pH of the eluted fraction was immediately adjusted to 100 mM Tris-HCl buffer (pH
Adjusted in 7.5). The purified enzyme was stored at -20 ° C in 50% glycerol. In addition, due to repeated freezing and thawing, C
Thawed samples were not re-frozen because NMT activity was significantly reduced. When the purified enzyme was stored in 50% glycerol at -20 ° C, it retained CNMT activity even after one year. All FPLC steps were performed at room temperature.

The CNMT activity was measured under the following conditions. Table 2
The purification process is summarized below.ア ッ セ イ Assay solution Norreticulin 0.1 mM SAM (S-adenosyl-L-methionine) 1 mM ascorbic acid Sodium 25 mM potassium-phosphate buffer (pH 7.0) 100 mM enzyme solution 20 μl Total 50 μl Enzyme reaction conditions Temperature ： 30 ° C. Time ： 60 min Stop reaction ： Add 50 μl methanol HPLC analysis conditions Sample ： Supernatant centrifuged at 10,000 × g for 50 minutes Column: LiChrosper 100RP-18 (250 mm × 4 mm, Cica Merck) Detection: UV 280 nm Flow rate: 1.0 ml / min Mobile phase: 22% acetonitrile + 1% acetic acid 酢 酸─────────────────────────

[0017]

[Table 2]

Example 2 Enzyme Properties of CNMT It is the first time that coclaurine N-methyltransferase was purified with high purity and its enzyme properties were examined. The molecular weight of the CNMT monomer in SDS-PAGE analysis was 45 kDa. On the other hand, gel filtration chromatography on a Superose 12 column revealed that the molecular weight of native CNMT was about 160 kDa. From these results, CNMT is a tetrameric enzyme. M
The isoelectric point of CNMT was 4.2 from the chromatofocusing on the ono-P column. The optimum pH for enzyme activity is 7.0,
The pH at 50% activity was 6.0 and 9.0. CNMT did not require divalent cations for activity expression and EDTA addition did not inhibit CNMT activity (Table 3). The addition of 5 mM Co ²⁺ , Cu ²⁺ , Mn ²⁺
The activity was inhibited by 75, 47 and 57%, respectively. Some O-
The SH-inhibitors p-chloromericuribenzoate and iodoacetamide, which inhibit methyltransferase activity, did not inhibit CNMT activity at 5 mM.

[0019]

[Table 3]

Example 3 Substrate Specificity of CNMT The substrate characteristics of the enzyme were examined for a wide range of isoquinoline alkaloids. Enzyme activity is S-adenosyl-L- [methyl- ³
The incorporation of radioactivity from the [H] methionine into the product was measured. The enzyme reaction was performed under the following conditions. ───────────────────────────── assay solution Substrate ^{1 mM [methyl- 3 H] SAM} (7.4dpm / μmol) 1 mM ascorbic Sodium acid 25 mM potassium-phosphate buffer (pH 7.0) 100 mM enzyme solution 20 μl Total 50 μl Enzyme reaction conditions Temperature ： 30 ℃ Time ： 20 minutes ─────────────────後 After the enzymatic reaction, the reaction was stopped by adding 150 μl of disodium carbonate and 400 μl of isoamyl alcohol. After mixing vigorously, the mixture was centrifuged at 10,000 xg for 5 minutes at room temperature, and 150 µl of the organic layer was removed and the radioactivity was measured. An assay solution containing no enzyme solution was used as a control.

The results are shown in Table 1. Assuming that the activity of (S) -coclaurine is 100%, the specific activities of (R) -coclaurine, norreticulin, norlaudanosoline and 6-O-methylnorlaudanosoline are 122%, 55%, 48%,
And 38%. Interestingly, 6,7-dimethoxy
-1,2,3,4-tetrahydroisoquinoline was methylated with a specific activity of 39%, whereas 1,2,3,4-tetrahydroisoquinoline was not methylated. Our CNMT transferred methyl groups to a fairly wide range of substrates and showed no stereospecificity.

The substrate specificity of Ouren CNMT is as described by Berberis.
Very similar to the substrate specificity of N-methyltransferase, neither enzyme shows stereospecificity and (R)-
Kokraulin, (S) -kokraulin, norreticulin,
Shows a wide substrate specificity for 6-O-methylnorlaudanosoline. However, Ourn's CNMT converts norlaudanosoline to N-
Although methylated, Berberis N-methyltransferase does not N-methyl norlaudanosoline. Spinach-derived CNMT has a wider substrate specificity than Berberis-derived CNMT.

[Example 4] Enzyme kinetics of CNMT The optimal substrate of CNMT is (R) -coclaurin, but the most available norreticulin was used to examine the enzyme specificity of CNMT. Our cell CNMT showed Michaels-Menten type kinetics with norreticulin and SAM. Apparent Km value is 0.38 mM for norreticulin, SAM
Was 0.68 mM. This is due to the Km value of the Berberis CNMT (0.02 mM for norreticulin, 0.04 mM for SAM) [T. Frenzel a
nd MH Zenk, Phytochemistry 29, 3491-3497 (199
0)] and the Sanguinaria CNMT Km value (0.02 mM for (R, S) -tetrahydroberberine, 0.012 mM for SAM) [BR O'keefe and
CWW Beecher, Plant Physiology, 105, 395-403
(1994)], but the Km value of norcoclaurin 6-O-methyltransferase (2.23 mM for (R, S) -norlaudanosoline and 3.95 mM for SAM) [F. Sato, et al. Europe
an Journal of Biochemistry, 225, 125-131 (1994)].

[Example 5] Determination of the N-terminal amino acid sequence of CNMT Cocraulin N-methyltransferase isolated and purified from the cultured berberine-producing celibaouren culture cells described in Example 1 was separated by SDS-PAGE and subjected to Bio-Rad blotting.
It was electrophoretically transferred to a PVDF blotting membrane filter (manufactured by Millipore) using an apparatus. After washing the membrane filter, the position of the protein was detected by color development with the CBB reagent. After cutting out the target band and drying, the amino acid sequencer 477A / 120A (Applied Biosy
stems). The result is shown in SEQ ID NO: 2.

Example 6 Preparation of cDNA Library Total RNA was extracted by the guanidine thiocyanate / hot phenol method from 10 days of cultivated celiva uren culture cells obtained as in Example 1, followed by mRNA purification kit. (Pharmacia) and according to the manufacturer's instructions
y (A) ⁺ RNA was purified. Using purified Poly (A) ⁺ RNA,
According to the method of ler and Hoffman (Gene 25: 263 (1983)), a cDNA library of cultured serauba urenium was prepared.

[Example 7] Obtaining a cDNA fragment corresponding to the N-terminal amino acid sequence of CNMT Based on the N-terminal amino acid sequence determined in Example 5, the base sequence described in SEQ ID NO: 3 was deduced. Was amplified by PCR using degenerate primers predicted by the codon usage of ours. The PCR was carried out using the cDNA library of cultured squirrel spinach described in Example 6 as a template, degenerate primers having the nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 at 94 ° C. for 30 seconds, and at 50 ° C. for 30 seconds. A cycle of annealing for 72 seconds and an extension reaction at 72 ° C. for 45 seconds was defined as one cycle, and a reaction was performed for 30 cycles. P obtained
The CR product was subcloned into the pGEM-T vector. As a result of sequencing several clones, SEQ ID NO: 6
A cDNA fragment of interest consisting of the nucleotide sequence represented by was obtained.

[Example 8] Determination of the full-length nucleotide sequence encoding CNMT Based on the nucleotide sequence shown in SEQ ID NO: 6, a cDNA portion corresponding to the C-terminal amino acid sequence of the present enzyme was replaced with celiba uren cD.
Amplification was performed by nested PCR using the NA library as a template. Initially, using a primer having the nucleotide sequence shown in SEQ ID NO: 7 and a primer for the adapter region of the cDNA library shown in SEQ ID NO: 8, a denature at 94 ° C. for 30 seconds, annealing at 50 ° C. for 30 seconds, 72 ° C. The extension reaction for 45 seconds was regarded as one cycle, and the reaction was carried out for 30 cycles. In the second cycle, using a primer having the nucleotide sequence of SEQ ID NO: 9 and an oligo dT primer, a cycle of denaturation at 94 ° C. for 30 seconds, annealing at 50 ° C. for 30 seconds, and extension reaction at 72 ° C. for 45 seconds was one cycle. For 30 cycles. The obtained PCR product was subcloned into a pGEM-T vector.
As a result of sequencing several clones, SEQ ID NO:
A target cDNA fragment consisting of the nucleotide sequence represented by 10 was obtained. Based on the nucleotide sequence shown in SEQ ID NO: 10, a cDNA portion corresponding to the N-terminal amino acid sequence of the present enzyme was amplified by nested PCR using a Seriva urenium cDNA library as a template. Initially, using a primer of the nucleotide sequence represented by SEQ ID NO: 11 and a primer for the adapter region of the cDNA library represented by SEQ ID NO: 12, a denature at 94 ° C. for 30 seconds, annealing at 50 ° C. for 30 seconds, annealing at 72 ° C. In ° C
The extension reaction for 45 seconds was defined as one cycle, and the reaction was performed for 30 cycles. The second time was performed using a primer having the nucleotide sequence shown in SEQ ID NO: 13 and the primer shown in SEQ ID NO: 12 used in the first PCR, and a denature at 94 ° C. for 30 seconds, 50
Annealing at 30 ° C. for 30 seconds and elongation at 72 ° C. for 45 seconds were defined as one cycle, and the reaction was performed for 30 cycles. The obtained PCR product was subcloned into a pGEM-T vector. As a result of sequencing several clones, a target cDNA fragment consisting of the nucleotide sequence represented by SEQ ID NO: 14 was obtained. Together with the sequence already determined, 1274 shown in SEQ ID NO: 1
A base sequence consisting of bases was obtained, and the amino acid sequence was deduced from the sequence.

Example 9 Confirmation of Function To clarify the function of the polypeptide having the amino acid sequence determined in Example 8, the full-length ORF of cDNA encoding the polypeptide was amplified by PCR and expressed in Escherichia coli. It was integrated into plasmid pET-21d (Novagen). PCR is used as a template of Seriba ouren cDNA library, using primers of the nucleotide sequence shown in SEQ ID NO: 15 and SEQ ID NO: 16, 94 ° C.
For 30 seconds, annealing at 60 ° C for 30 seconds,
The extension reaction at 72 ° C. for 90 seconds was defined as one cycle, and the reaction was performed for 30 cycles. The obtained PCR product was treated with restriction enzymes NcoI and EcoRI, and integrated into the NcoI and EcoRI sites of the expression plasmid pET-21d, which was designated as pET-21d-CNMT. This allows CNMT
Is introduced downstream of the T7 promoter in the sense direction. The obtained pET-21d-CNMT plasmid was further transformed into E. coli strain BL21 (DE
3) was introduced. Culture conditions, expression induction by IPTG, and a method for adjusting the expressed enzyme were performed in the same manner as described in Morishige, T. et al. Literature [J. Boil. Chem. 275, 233]
98-23405 (2000)]. The culture after induction was performed at 30 ° C. for 3 hours. According to Example 1 using the expressed protein,
MT activity was measured. The alkaloid fraction formed was analyzed by HPLC. The identity of the compound produced was determined by LC-MS (manufactured by Shimadzu Corporation).

As a result of LC-MS analysis of the methylated compound,
It was confirmed that reticuline (m / z 330, m / z 192) was produced from norreticulin (m / z 316, m / z 178).
Therefore, the present cDNA is cocouraurin-N-, which is positioned as one of N-methyltransferases obtained from higher plants.
It was concluded that it encodes methyltransferase (CNMT).

Further, when the recombinant CNMT was purified from the above Escherichia coli according to Example 1, the specific activity was much higher than that of the purified product from the spinach cells (18 nkat / mg), and this enzyme was norreticuline When the activity when using as a substrate is expressed as 100, 1-methyl-6,7-dihydor
xy-1,2,3,4-tetrahydroisoqauinoline is methylated by 180, phenylethanolamine is converted to 20, 3-hydroxy-4-methoxyp
Henylethylamine was added to 11, norphenylephrine (noradrena
rine) by 16-methylation, not only for isoquinoline alkaloids but also for general phenylamine
It was revealed to show methylation activity.

[0031]

As described above, the present invention provides CNMT and DNA encoding CNMT. It is possible to efficiently produce isoquinoline alkaloids useful as pharmaceuticals by using the enzyme of the present invention, and furthermore, using cells expressing cloned recombinant DNA, CNMT,
It is also possible to produce DNA encoding the enzyme and N-methylcoclaurin and / or various alkaloids biosynthesized from this N-methylcoclaurin, or N-methylated phenylamine analogs.

[0032]

[Sequence List] SEQUENCE LISTING <110> MITSUI CHEMICALS, INC <120> A coclaurine N-methyltransferase and a gene encodes the coclaurine N-methyltransferase <130> coclaurine N-methyltransferase <160> 16 <170> PatentIn Ver. 2.1 <210 > 1 <211> 1274 <212> DNA <213> Coptis japonica <220> <221> CDS <222> (1) .. (1077) <400> 1 atg gct gtg gaa gca aag caa aca aag aag gca gcc ata gta gag ttg 48 Met Ala Val Glu Ala Lys Gln Thr Lys Lys Ala Ala Ile Val Glu Leu 1 5 10 15 tta aaa cag ttg gag ctg ggc ttg gtt cca tat gat gat att aag cag 96 Leu Lys Gln Leu Glu Leu Gly Leu Val Pro Tyr Asp Asp Ile Lys Gln 20 25 30 ctc ata agg agg gaa ctg gca agg cgc ctg caa tgg ggt tat aaa cct 144 Leu Ile Arg Arg Glu Leu Ala Arg Arg Leu Gln Trp Gly Tyr Lys Pro 35 40 45 act tat gaa gaa caa ata gct gaa atc caa aac tta act cat tct ctg 192 Thr Tyr Glu Glu Gln Ile Ala Glu Ile Gln Asn Leu Thr His Ser Leu 50 55 60 cga caa atg aaa att gca aca gag gtt gag acc ttg gat tca caa ttg 240 Arg Gln Met Lys Ile Ala Thr Glu Val Glu Thr Leu Asp Ser Gln Leu 65 70 75 80 tac gag att cct att gag ttt cta aag att atg aat gga agt aac tta 288 Tyr Glu Ile Pro Ile Glu Phe Leu Lys Ile Met Asn Gly Ser Asn Leu 85 90 95 aaa gga agt tgt tgc tac ttc aaa gaa gat tca aca aca tta gat gaa 336 Lys Gly Ser Cys Cys Tyr Phe Lys Glu Asp Ser Thr Thr Leu Asp Glu 100 105 110 gct gag ata gcg atg ctg gat tta tac tgc gag aga gct caa atc caa 384 Ala Glu Ile Ala Met Leu Asp Leu Tyr Cys Glu Arg Ala Gln Ile Gln 115 120 125 gat gga cag agt gtt ctt gat ctt gga tgt ggg caa gga gct ctt aca 432 Asp Gly Gln Ser Val Leu Asp Leu Gly Cys Gly Gln Gly Ala Leu Thr 130 135 140 tta cat gtt gca cag aaa tat aaa aac tgt cgc gta aca gca gta aca 480 Leu His Val Ala Gln Lys Tyr Lys Asn Cys Arg Val Thr Ala Val Thr 145 150 155 160 aat tca gtt tca caa aaa gag tac att gaa gaa gaa tca agg aga cgt 528 Asn Ser Val Ser Gln Lys Glu Tyr Ile Glu Glu Glu Ser Arg Arg Arg 165 170 175 aat ttg ttg aat gtg gaa gtc aaa ttg gca gac ata acc aca cat gag 576 Asn Leu Leu Asn Val Glu Val Lysu Ala Asp Ile Thr Thr His Glu 180 185 190 atg gct gag aca tac gat cgt att ttg gta ata gag ttg ttt gag cac 624 Met Ala Glu Thr Tyr Asp Arg Ile Leu Val Ile Glu Leu Phe Glu His 195 200 205 atg aag aac tat gaa ctt ctc ctg agg aaa atc tca gag tgg ata tcg 672 Met Lys Asn Tyr Glu Leu Leu Leu Arg Lys Ile Ser Glu Trp Ile Ser 210 215 220 aaa gat ggg ctt ctc ttt cta gag cac ata tgc cac aag acc ttt gct 720 Lys Asp Leu Leu Phe Leu Glu His Ile Cys His Lys Thr Phe Ala 225 230 235 240 tac cac tat gag cct cta gac gac gac gat tgg ttt aca gag tac gtg 768 Tyr His Tyr Glu Pro Leu Asp Asp Asp Asp Trp Phe Thr Glu Tyr Val 245 250 255 ttt cct gct ggg act atg atc ata cca tct gca tcg ttc ttt ttg tat 816 Phe Pro Ala Gly Thr Met Ile Ile Pro Ser Ala Ser Phe Phe Leu Tyr 260 265 270 ttc cag gat gac gtt tcg gtt gtg aac cat tgg act ctt agt ggg aag 864 Phe Gln Asp Asp Val Ser Val Val Asn His Trp Thr Leu Ser Gly Lys 275 280 285 cac ttt tcg cgt acc aat gag gaa tgg ttg aag aga ttg gac gca aac 912 His Phe Ser Arg Thr Asn Glu Glu Trp Leu Lys Arg Leu Asp Ala Asn 290 295 300 ctt gat gtt att aaa cca atg ttt gag act tta atg gga aat gag gaa 960 Leu Asp Val Ile Lys Pro Met Phe Glu Thr Leu Met Gly Asn Glu Glu 305 310 315 320 gag gca gtg aag ttg att aac tat tgg aga gga ttt tgt tta tct gga 1008 Glu Ala Val Lys Leu Ile Asn Tyr Trp Arg Gly Phe Cys Leu Ser Gly 325 330 335 atg gaa atg ttt gga tat aac aat ggt gaa gaagtg atgca 1056 Met Glu Met Phe Gly Tyr Asn Asn Gly Glu Glu Trp Met Ala Ser His 340 345 350 gtt ctg ttc aag aaa aaa tga ttttgcccaa cagtgtattt ctttcattag 1107 Val Leu Phe Lys Lys Lys 355 tagcattgca tgca tgca tgca tgca tg tgag ttttggagat 1227 gaaataaaat atatctttgg atgggcaaaa aaaaaaaaaa aaaaaaa 1274 <210> 2 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: N-terminal amino acid sequences of CNMT <400> 2 Ala Val Glu Ala Lys Gln Thr Lys Lys Ala Ala Ile Val Glu Leu Leu 1 5 10 15 Ly s Gln Leu Glu Leu Gly Leu Val Pro Tyr Asp Asp Ile Lys Gln Leu 20 25 30 Ile <210> 3 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Nucleotide sequences corresponing to N-terminal amino acid sequences of CNMT <400> 3 gcngtngarg cnaarcarac naaraargcn gcnathgtng arytnytnaa rcarytngar 60 ytnggnytng tnccntayga ygayathaar carytnath 99 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> of Artificial Sequence: Forward degenerate primer for PCR to amplify cDNA fragment corresponding to N-terminal amino acid sequences of CNMT <400> 4 gcngtngarg cnaarcarac 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Reverse degenerate primer for PCR to amplify cDNA fragment corresponding to N-terminal amino acid sequences of CNMT <400> 5 arytgyttda trtcrtcrta 20 <210> 6 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Nucleotide sequences of cDNA fragment obtained from PCR <400> 6 gctgtggaag caaagcaaac aaagaaggca gctatagtag agttgttaaa acagttggag 60 ctgggcttgg ttccatatga tgatattaag cagct 95 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: for the first PCR to amplify 3'-end of CNMT gene <400> 7 acgactcact atagggcgaa ttgg 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Reverse primer corresponding to adaptor region <400> 8 acgactcact atagggcgaa ttgg 24 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Forward primer for the second PCR to amplify 3 '-end of CNMT gene <400> 9 gttgttaaaa cagttggagc tgggc 25 <210> 10 <211> 1230 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Nucleotide sequences of 3'-end cDNA <400> 10 gttgttaaaa cagttggagc tgggcttggt tccatatgat gatattaagc agctcataag 60 gagggaactg gcaaggcgcc tgcaatgggg ttataaacct a cttatgaag aacaaatagc 120 tgaaatccaa aacttaactc attctctgcg acaaatgaaa attgcaacag aggttgagac 180 cttggattca caattgtacg agattcctat tgagtttcta aagattatga atggaagtaa 240 cttaaaagga agttgttgct acttcaaaga agattcaaca acattagatg aagctgagat 300 agcgatgctg gatttatact gcgagagagc tcaaatccaa gatggacaga gtgttcttga 360 tcttggatgt gggcaaggag ctcttacatt acatgttgca cagaaatata aaaactgtcg 420 cgtaacagca gtaacaaatt cagtttcaca aaaagagtac attgaagaag aatcaaggag 480 acgtaatttg ttgaatgtgg aagtcaaatt ggcagacata accacacatg agatggctga 540 gacatacgat cgtattttgg taatagagtt gtttgagcac atgaagaact atgaacttct 600 cctgaggaaa atctcagagt ggatatcgaa agatgggctt ctctttctag agcacatatg 660 ccacaagacc tttgcttacc actatgagcc tctagacgac gacgattggt ttacagagta 720 cgtgtttcct gctgggacta tgatcatacc atctgcatcg ttctttttgt atttccagga 780 tgacgtttcg gttgtgaacc attggactct tagtgggaag cacttttcgc gtaccaatga 840 ggaatggttg aagagattgg acgcaaacct tgatgttatt aaaccaatgt ttgagacttt 900 aatgggaaat gaggaagagg cagtgaagtt gattaactat tggagaggat tttgtttatc 960 tggaatggaa atgtttggat ataacaatgg tgaagaatgg atggcaagtc atgttctgtt 1020 caagaaaaaa tgattttgcc caacagtgta tttctttcat tagtagcatt acttgaataa 1080 gtttggaaga gtcttcatat atctgctaca tccagaaagg actagcagca cagtttgtag 1140 atcgattgtc ccctgctaca tttgtatgag ttattttgga gatgaaataa aatatatctt 1200 tggatgggca aaaaaaaaaa aaaaaaaaaa 1230 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Reverse primer for the first PCR to amplify 5'-end of CNMT gene <400> 11 caacttcctt ttaagttact tcc 23 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Forward primer corresponding to adaptor region <400> 12 gaaagaaaaa aaatataccc cagc 24 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Reverse primer for the second PCR to amplify 5'-end of CNMT gene <400> 13 caattgtgaa tccaaggtct caacctctgt tgc 33 <210> 14 <211> 299 <212> DNA <213> Artificial Sequence <220> <223 > Des cription of Artificial Sequence: Nucleotide sequences of 5'-end cDNA <400> 14 atggctgtgg aagcaaagca aacaaagaag gcagccatag tagagttgtt aaaacagttg 60 gagctgggct tggttccata tgatgatatt aagcagctca taaggaggga actggcaagg 120 cgcctgcaat ggggttataa acctacttat gaagaacaaa tagctgaaat ccaaaactta 180 actcattctc tgcgacaaat gaaaattgca acagaggttg agaccttgga ttcacaattg 240 tacgagattc ctattgagtt tctaaagatt atgaatggaa gtaacttaaa aggaagttg 299 <210> 15 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Forward primer for PCR (CNMT) <400> 15 gttgccatgg ctgtggaagc aaagcaaaca aagaaggc 38 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Reverse primer for PCR (CNMT) <400> 16 gcggaattca cgactcacta tagggcgaat tgg 33

[Brief description of the drawings]

FIG. 1 shows the berberine biosynthesis pathway. In the figure,
1 to 13 represent enzymes that catalyze the reaction shown. Each enzyme is as follows. 1: L-tyrosine decarboxylase, 2: phenolase, 3: L-tyrosine transaminase, 4: p-hydroxyphenylpyruvate decarboxylase, 5: (S) -norcoclaurin synthase, 6: norcoclaurin-6-O -Methyltotransferase (6-OMT), 7: coclaurine N-methyltransferase (NMT), 8: phenolase, 9: (S)-
3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'-OMT), 10: berberine cross-linking enzyme (B
BE), 11: (S) -scourelin 9-O-methyltransferase (SMT), 12: methylenedioxy ring-forming enzyme, 1
3: Tetrahydroberberine oxidase (THBO)

FIG. 2 is a view showing a chemical structure of a group of compounds for which the substrate specificity of CNMT was examined.

FIG. 3 is a graph showing the relationship between the CNMT reaction rate and the concentrations of norreticulin and SAM.

FIG. 4 is a graph showing a double reciprocal plot of CNMT activity. (a) shows a plot of 1 / v vs. 1 / [SAM] at each concentration of norreticulin, and (b) shows a plot of 1 / v vs. 1 / [norreticulin] at each concentration of SAM.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/10 C12R 1:19 C12P 17/12 C12N 15/00 ZNAA // (C12N 9/10 5/00 A C12R 1:19) F term (reference) 4B024 AA01 AA03 BA10 CA04 DA06 EA04 GA11 4B050 CC03 DD13 EE10 LL01 LL05 4B064 AE49 CA02 CA19 CA21 CC24 DA01 DA16 4B065 AA26X AA88Y AB01 AC14 BA02 CA29 CA44

Claims (12)

[Claims] A cocolaurine having the following physicochemical properties:
N-methyltransferase. (1) optimum pH 7.0 (2) isoelectric point 4.2 (3) tetramer (4) molecular weight 140 kDa (gel filtration chromatography) (5) subunit molecular weight 45 kDa (SDS-PAGE) 2. A method for producing a secondary metabolite of a plant using the enzyme of claim 1. 3. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1. 4. A DNA comprising a nucleotide sequence encoding the polypeptide according to claim 1. 5. The DNA according to claim 4, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1. 6. The DNA according to claim 5, comprising the nucleotide sequence of SEQ ID NO: 1. 7. The DNA according to claim 5, comprising the nucleotide sequence of positions 1 to 1074 of SEQ ID NO: 1 in the sequence listing. 8. The DNA according to any one of claims 4 to 7,
Vector containing. 9. A cell transformed with the vector according to claim 8. [10] A method for producing a polypeptide having an enzyme activity of coclaurin-N-methyltransferase using the cell according to [9]. 11. A method for producing a plant-related secondary metabolite using the cell according to claim 9. 12. The method according to claim 11, wherein the plant secondary metabolite is an alkaloid biosynthesized from coclaurin-N-methyltransferase.