Molecular cloning of columbamine O -methyltransferase fromTakashi Morishige1, Emilyn Dubouzet2, Kum-Boo Choi2, Kazufumi Yazaki1,2and Fumihiko Sato1,2 1 Division of Applied Life Sciences,
Trang 1Molecular cloning of columbamine O -methyltransferase from
Takashi Morishige1, Emilyn Dubouzet2, Kum-Boo Choi2, Kazufumi Yazaki1,2and Fumihiko Sato1,2
1
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan;2Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Japan
To identify all of the O-methyltransferase genes involved in
isoquinoline alkaloid biosynthesis in Coptis japonica cells, we
sequenced 1014 cDNAclones isolated from
high-alkaloid-producing cultured cells of C japonica A mong them, we
found all three reported O-methyltransferases and an
O-methyltransferase-like cDNAclone (CJEST64) This cDNA
was quite similar to S-adenosyl-L-methionine:coclaurine
6-O-methyltransferase and S-adenosyl-L
-methionine:iso-flavone 7-O-methyltransferase As S-adenosyl-L
-methion-ine:columbamine O-methyltransferase, which catalyzes the
conversion of columbamine to palmatine, is one of the
remaining unelucidated components in isoquinoline alkaloid
biosynthesis in C japonica, we heterologously expressed the
protein in Escherichia coli and examined the activity of
columbamine O-methyltransferase The recombinant protein clearly showed O-methylation activity using columbamine, as well as (S)-tetrahydrocolumbamine, (S)-, (R,S)-scoulerine and (R,S)-2,3,9,10-tetrahydroxypro-toberberine as substrates This result clearly indicated that EST analysis was useful for isolating the candidate gene in a relatively well-characterized biosynthetic pathway The relationship between the structure and substrate recognition
of the O-methyltransferases involved in isoquinoline alka-loid biosynthesis, and a reconsideration of the biosynthetic pathway to palmatine are discussed
Keywords: alkaloid biosynthesis; methyltransferase; Coptis japonica; palmatine; columbamine
Higher plants produce a wide variety of chemicals, including
more than 25 000 terpenoids, approximately 12 000
alka-loids and approximately 8000 phenolic compounds [1]
Thus, secondary metabolism in plant cells could be a useful
source of these chemicals, especially alkaloids, many of
which have high biological activities and are used as
medicines
Isoquinoline alkaloid biosynthesis is the most
well-characterized pathway in plant cells and some biosynthetic
genes have been isolated [2,3 and references cited therein]
However, the conventional method for isolating the
corres-ponding biosynthetic genes based on the purified enzymes is
a rate-limiting step for characterization, even though this
approach can be very useful when we have no information about the desired enzyme(s) Although a useful approach is
to isolate a group of genes based on their structural similarity, such as O-methyltransferase (OMT) or P450 motifs, this approach faces the problem of identifying biological activity due to the high redundancy of related genes
On the other hand, recent scientific and technological advances now enable the high-through-put sequencing of cDNAclones, and we can analyze these expressed sequence tag (EST) using on-line databases and search tools These ESTs are also useful for evaluating mRNAtranscripts to estimate their biological function
To evaluate the effectiveness of this technology, we sequenced the cDNAclones isolated from high-alkaloid-producing cultured cells of Coptis japonica A s we previ-ously characterized some of the OMTs in isoquinoline alkaloid biosynthesis in Coptis cells, we focused on the isolation of the another unelucidated OMT in this biosyn-thetic pathway; i.e S-adenosyl-L-methionine:columbamine O-methyltransferase (CoOMT), which catalyzes the transfer
of the S-methyl group of S-adenosyl-L-methionine (Ado-Met) to the 2-hydroxyl group of columbamine to form palmatine [4] (Fig 1)
Methyltransferases are essential for directing inter-mediates to specific biosynthetic pathways [5] Each meth-yltransferase in the biosynthesis of palmatine (i.e S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransf-erase (6OMT) [6–10]; S-adenosyl-L-methionine:coclaurine N-methyltransferase (CNMT) [11–14]; S-adenosyl-L -methi-onine:3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltrans-ferase (4¢OMT) [10,15]; S-adenosyl-L-methionine:scoulerine 9-O-methyltransferase (SMT) [16–18]; and CoOMT [4]) has strict substrate specificity, despite the structural
Correspondence to F Sato, Division of Integrated Life Sciences,
Graduate School of Biostudies, Kyoto University, Kyoto,
606-8502, Japan Fax: + 81 75 753 6398, Tel.: + 81 75 753 6381,
E-mail: fumihiko@kais.kyoto-u.ac.jp
Abbreviations: OMT, O-methyltransferase; EST, expressed sequence
tag; CoOMT, S-adenosyl- L -methionine:columbamine
O-methyl-transferase; AdoMet, S-adenosyl- L -methionine; AdoHcy,
S-adenosyl- L -homocysteine; 6OMT, S-adenosyl- L
-methionine:norco-claurine 6-O-methyltransferase; CNMT, S-adenosyl- L -methionine:
coclaurine N-methyltransferase; 4¢OMT, S-adenosyl- L
-methionine:3¢-hydroxy-N-methylcoclaurine 4¢-O-methyltransferase; SMT,
S-adenosyl- L -methionine:scoulerine 9-O-methyltransferase; IOMT,
S-adenosyl- L -methionine:isoflavone 7-O-methyltransferase; LC-MS,
liquid chromatography-mass spectrometry.
Note: The nucleotide sequence for columbamine O-methyltransferase
has been deposited in the GenBank database under GenBank
Accession Number AB073908.
(Received 13 March 2002, revised 2 July 2002,
accepted 20 September 2002)
Eur J Biochem 269, 5659–5667 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03275.x
Trang 2similarities of the various substrates Due to their
importance in the production of pharmaceutically
important alkaloids and their strict substrate recognition,
these methyltransferases, especially OMTs, have been
well characterized [6–17] and their cDNAhas been
isolated [10,14,18] However, CoOMT was characterized
using only partially purified enzymes [4] and its cDNA
has not yet been isolated
We report here the first isolation of CoOMT from
C japonica ESTs, the expression of functionally active
recombinant enzyme, and its characterization
Characteri-zation of its specificity showed that Coptis CoOMT could
methylate tetrahydrocolumbamine as well as columbamine,
and suggests that the biosynthetic pathway to palmatine
should be reconsidered
E X P E R I M E N T A L P R O C E D U R E S
Cultured cells
The original cultured cells were induced from rootlets of
C japonicaMakino var dissecta (Yatabe) Nakai Acell line
(156–1) that produces large amounts of alkaloids was
subcultured as described elsewhere [19]
Ten-day-old-cul-tured cells were harvested and used for the extraction of mRNA
Chemicals (R,S)-Norlaudanosoline and (R,S)-laudanosoline were purchased from Aldrich, and berberine was from Wako Pure Chemical Industries, Ltd (R)- and (S)-coclaurine were the gift of Dr Nagakura of Kobe Women’s College of Phar-macy, and (S)-reticuline was a gift from Dr Facchini of the University of Calgary The other alkaloids were generous gifts from Mitsui Petrochemical Industries, Ltd
Chemical synthesis of columbamine Columbamine was synthesized and purified from berberine
by two-step reactions according to the method of Cava and Reed [20,21]
Construction and sequencing of cDNA library
ofC japonica Poly(A)+RNAwas isolated, and a cDNAlibrary was constructed as described elsewhere [14] The cDNA
Fig 1 Schematic biosynthetic pathway of palmatine Dotted lines and question marks indicate the possible pathway based on the present findings.
Trang 3fragments were ligated into pDR196 vector [22]
Sequen-cing of the cDNAlibrary was performed using a
MegaBACE 1000 DNA Sequencing System (Amersham
Pharmacia Biotech) in accordance with the manufacturer’s
instructions Overall, 1014 ESTs were obtained in
suffi-cient length and these sequences were annotated using a
BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) (E
Dubouzet et al in preparation) During theBLASTsearch,
we found an unidentified OMT cDNA(CJEST64) in
addition to the sequences of reported Coptis OMTs To
obtain more information about the sequence of this
CJEST64, the complete cDNAsequence was determined
with deletion clones of CJEST64 and a DSQ-2000 L
DNAsequencer (Shimadzu) with fluorescein
isothiocya-nate-labelled primers
Isolation of full-length cDNA
To isolate full-length cDNAof the unknown OMT
(CJEST64), rapid amplification of the 5¢ end of cDNA
(5¢RACE) was carried out using a Marathon cDNA
Amplification Kit (Clontech) Gene-specific antisense primer
(5¢-CTCCAAACTGAGAACTCTTCCG-3¢) was designed
based on the nucleotide sequence of CJEST64 PCR
fragments were isolated and subcloned into pT7Blue vector
(Novagen), and their nucleotide sequences were determined
Construction of an expression vector for full-length
CJEST64
An expression vector was constructed for full-length
CJEST64 cDNAwithout the fused peptide derived from
the vector sequence in pET-21d vector (Novagen) The
full-length cDNAwas first prepared by PCR with a Marathon
adaptor primer (Clontech) and a forward primer
5¢-TTGTTCTAAGGCCATGGTATCTCCG-3¢ to
intro-duce a NcoI site (CCATGG) at the start codon A
restriction site was introduced by changing the underlined
base pET-21d was digested with NcoI and XhoI, and the PCR product was ligated into the vector This construct was completely sequenced to confirm that no changes were introduced by the subcloning process
Heterologous expression of full-length CJEST64
inEscherichia coli The expression vector for full-length CJEST64 cDNAwas introduced into Escherichia coli BL21 (DE3) After induc-tion with 1 mMisopropylthiogalactoside, E coli cells were incubated at 25C for 5 h, and then harvested and extracted in extraction buffer (0.1M Tris/HCl, pH 8.0, containing 10% glycerol, 5 mM EDTAand 10 mM 2-mercaptoethanol) After centrifugation at 12 000 g for
10 min, the supernatant was desalted through an NAP-5 column (Amersham Pharmacia Biotech) and used to measure CoOMT activity
Measurement ofO-methylation activity The standard CoOMT reaction mixture (50 lL) consisted
of 100 mMpotassium phosphate (pH 7.8), 25 mMsodium ascorbate, 1 mM columbamine, 3 mM AdoMet and the enzyme preparation (c 100 lg protein) The assay mixture was incubated at 30C for 1 h, after which the reaction was terminated by the addition of methanol After protein precipitation, the reaction product was detected by reversed-phase HPLC (mobile reversed-phase, 35% acetonitrile/H2O contain-ing 1% acetic acid; column, TSKgel ODS-80Tm (4.6 ¥
250 mm; TOSOH); flow rate, 0.5 mLÆmin)1; detection, absorbance measurement at 280 nm Mass spectra were obtained with an LCMS-2010 (Shimadzu)
Tetrahydrocolumbamine was prepared from scoulerine viaa scoulerine 9-O-methyltransferase (SMT) reaction The SMT reaction was carried out as described previously [18] with a modification In brief, the expression of SMT in E coli was induced using a pET expression system and the E coli
Fig 2 Amino acid sequence alignment of the
O-methyltransferases in palmatine biosynthesis.
Motifs A, B, C; conserved sequence motifs in
plant S-adenosyl- L -methionine-dependent
methyltransferase Amino acids conserved in
all of the sequences are boxed and similar ones
are shaded.
FEBS 2002 Coptiscolumbamine O-methyltransferase (Eur J Biochem 269) 5661
Trang 4lysate was prepared The SMT reaction mixture consisted of
100 mMTris/HCl (pH 8.0), 25 mMsodium ascorbate, 1 mM
(S)-scoulerine, 5 mMAdoMet and the enzyme preparation
The reaction mixture was incubated at 30C for 3 h In this
reaction, scoulerine was converted into
tetrahydrocolumb-amine After the enzyme reaction was terminated by the
addition of 0.5Msodium carbonate, the reaction product
was extracted twice with ethyl acetate The extracted product
was dried and used for further characterization HPLC
analysis showed that tetrahydrocolumbamine prepared was
80% pure and contained unconverted scoulerine Because
scoulerine was a poor substrate for CoOMT, we analyzed
substrate affinity of CoOMT for tetrahydrocolumbamine
using this 80% pure tetrahydrocolumbamine
To quantify the enzymatic activity of CoOMT, the transfer of the3H-labeled methyl group of S-adenosyl-L -[methyl-3H]methionine (0.5 MBqÆlmol)1) (PerkinElmer Life Sciences) to the product was measured as described elsewhere [7] The kinetic constants of the crude enzyme were determined by varying the concentration of alkaloid substrates, but keeping the concentration of 3H-labeled AdoMet fixed at 1 mMthen apparent Kmand Vmaxvalues were calculated
Other methods The subunit molecular mass of the enzyme was deter-mined by SDS/PAGE (12.5% polyacrylamide) Protein
Fig 3 Phylogenic tree of plant
S-adenosyl-L -methionine-dependent O-methyltransferase sequences OMT protein sequences obtained from GenBankTMwere used to build the tree Accession numbers are indicated in paren-thesis Overall, 20 sequences were aligned by the multisequence alignment program in
GENETYX - MAC ver.1.1 (Software Develop-ment, Inc Tokyo, Japan) using the UPGMA
(unweighted pair group maximum average) method A EOMT, hydroxycinnamic acids/ hydroxycinnamoyl CoAesters O-methyl-transferase.
Trang 5concentration was determined according to Bradford [23]
with bovine serum albumin as a standard
R E S U L T S
Sequence analysis of the OMT-like clone
AcDNAlibrary was prepared from C japonica cells that
produce large amounts of alkaloids, and its 1014 ESTs were
obtained (E Dubouzet et al in preparation) A BLAST
search (http://www.ncbi.nlm.nih.gov/blast/) showed that
these sequenced clones included 4 clones of 4¢OMT, 2
clones of 6OMT and 4 clones of SMT This result indicated
that this cDNAlibrary was highly enriched with
biosyn-thetic genes involved in isoquinoline alkaloid biosynthesis
Thus, we speculated that this library also included the gene
for the additional unelucidated OMT in isoquinoline
alkaloid biosynthesis in C japonica, i.e CoOMT In fact,
aBLASTsearch identified a clone (CJEST64) which was quite
similar to isoflavone 7-O-methyltransferase (IOMT) and
6OMT
Whereas CoOMT is found in the last step of palmatine
biosynthesis and 6OMT is found at an early step, we
examined the possibility that this CJEST64 was CoOMT
As only a partial sequence was determined for the EST
project, full-length of CJEST64 was determined The
sequence obtained further confirmed that CJEST64 was
similar to 6OMT, 4¢OMT and IOMT (data not shown),
although the sequence was not full-length based on
sequence alignment Thus, the 5¢-fragment of CJEST64
was obtained by 5¢RACE and sequenced Finally,
full-length cDNAwas re-cloned from the cDNAlibrary and the
expression vector was constructed as described below
This full-length cDNAcarried 1378 nucleotides, with an
open reading frame that encoded 351 amino acids (Fig 2)
The deduced amino acid sequences had conserved putative
AdoMet binding domains at the C-terminal end (Fig 2,
motifs A–C), like other OMTs [24] This polypeptide had a
high degree of similarity (approximately 40% identity) to
Coptis6OMT, Coptis 4¢OMT, IOMT of alfalfa [25], and
6a-hydroxymaackiain 3-O-methyltransferase of pea [26]
The deduced polypeptide showed a rather low identity
(21%) to SMT Phylogenetic analysis clearly indicated that
this polypeptide belongs to the same branch as 6OMT and
4¢OMT (Fig 3)
Expression of the full-length cDNA inE coli
We constructed the expression vector to produce
recom-binant proteins in E coli to examine the activity of CoOMT
We introduced an NcoI site into the cDNAto fit the
initiation codon in the E coli expression vector pET-21d to
produce nontagged polypeptide This construct was then
introduced into E coli cells, and production of the
recom-binant protein was induced The crude E coli lysate was
used to detect CoOMT activity SDS/PAGE analysis clearly
showed that transgene expression was successfully induced
and the subunit molecular mass was estimated to be
approximately 40 kDa (Fig 4)
HPLC analysis of the reaction mixture clearly showed
that recombinant E coli lysate had CoOMT activity (data
not shown) The control E coli lysate with the pET-21d
vector showed no enzymatic activity LC-MS analysis
Fig 4 SDS/PAGE of E coli crude extract expressing CJEST64 Crude extracts of E coli transformed with expression vector for CJEST64 or control vector were separated by SDS/PAGE and stained with Coomassie Brilliant Blue G-250 Lane 1, molecular mass markers; lane 2, pET-CJEST64 (10 lg); lane 3, control vector (pET-21d) (10 lg) Arrow indicates production of the expected recombinant CoOMT.
Fig 5 LC-MS analysis of the reaction product (A), authentic columb-amine (B), authentic palmatine (C) and the control reaction (pET-21d) (D) TIC, total ion chromatogram.
FEBS 2002 Coptiscolumbamine O-methyltransferase (Eur J Biochem 269) 5663
Trang 6confirmed that palmatine was produced from columbamine
by recombinant E coli lysate (Fig 5), and that the
CJEST64 product was CoOMT
Characterization of recombinant CoOMT
As CoOMT activity was identified, we further characterized
the enzyme properties of CoOMT using this crude
recom-binant enzyme First, we optimized the pH conditions for
the CoOMT reaction using columbamine as a substrate
Enzyme assays at various pH values indicated that the
optimum pH for the methylation of columbamine was
approximately 8.4 Whereas product inhibition of 6OMT
and SMT has been reported [8,17], berberine, one of the
end-products of isoquinoline alkaloid biosynthesis in Coptis
cells, did not inhibit CoOMT activity in the assay mixture at
2.5 mM
Next, we determined the substrate specificity of CoOMT
using the incorporation of radioactivity from
S-adenosyl-L-[methyl-3H]methionine into the products as an index
(Table 1) When columbamine was used as the control
substrate (i.e relative incorporation 100%), the respective
relative activities with (S)- and (R,S)-scoulerine and
2,3,9,10-tetrahydroxyprotoberberine were 31, 22 and 14%,
whereas no significant methylation was found for other
substrates The methylation of scoulerine and 2,3,9,10-tetrahydroxyprotoberberine by CoOMT was further confirmed by LC-MS (Fig 6) Interestingly, mono- and di-methylated products were formed from 2,3,9,10-tetra-hydroxyprotoberberine by reacting with CoOMT This result indicated that the regio-specificity of CoOMT was rather low
Above result suggested that tetrahydroprotoberberine should also be a good substrate for CoOMT To confirm this idea, tetrahydrocolumbamine was produced from scoulerine by an SMT reaction, and the reaction pro-duct, tetrahydrocolumbamine, was used in the successive CoOMT reaction Figure 6E,F show that a new reaction product with a 14-m/z greater mass was detected
To compare the substrate specificity, we determined the kinetic constants of CoOMT for columbamine, tetrahydro-columbamine and (S)-scoulerine Substrate-saturation kin-etics of the recombinant CoOMT prepared from E coli for these alkaloids were the typical Michaelis–Menten type Then, we calculated apparent Kmand Vmaxvalues by varying the concentration of these alkaloids in the presence of 1 mM AdoMet The respective Kmvalues of CoOMT for columb-amine, tetrahydrocolumbamine and (S)-scoulerine were
66 ± 18, 35 ± 18 and 173 ± 51 lM, and respective Vmax were 125 ± 12, 21 ± 7, and 6.9 ± 1.3 pkatÆmg protein)1
Table 1 Transfer of [ 3 H]-methyl group of S-adnosyl- L -methionine into different substrates by columbamine OMT Values indicate the incorporation
of radioactivity from S-adenosyl- L -[methyl- 3 H]methionine into the product relative to columbamine Other assay conditions are indicated in Materials and methods ND, not detected.
Columbamine OCH 3 OH OCH 3 OCH 3 - 100%
2,3,9,10-Tetrahydroxyprotoberberine OH OH OH OH - 14%
(S)-Scoulerine OCH 3 OH OH OCH 3 - 31%
(R,S)-Scoulerine OCH 3 OH OH OCH 3 - 22%
(R,S)-2,3,9,10-Tetrahydroxy-tetrahydroprotoberberine
(R,S)-6-O-Methylnorlaudanosoline OCH 3 OH OH OH H ND (R,S)-Laudanosoline OH OH OH OH CH 3 ND (R,S)-Norlaudanosoline OH OH OH OH H ND (R,S)-Reticuline OCH 3 OH OH OCH 3 CH 3 ND (S)-Reticuline OCH 3 OH OH OCH 3 CH 3 ND (R,S)-Norreticuline OCH 3 OH OH OCH 3 H ND (S)-Coclaurine OCH 3 OH H OH H ND (R)-Coclaurine OCH 3 OH H OH H ND
4-Hydroxy-3-methoxycinnamic acid OCH 3 - - - - ND
Trang 7D I S C U S S I O N
The present results indicate that high-through-put
sequen-cing of a cDNAlibrary of high-metabolite-produsequen-cing cells
and computer-assisted sequence analysis can be useful for
isolating a desired but not yet identified gene(s) Using this
strategy, we successfully identified cDNAof CoOMT
without purification of the enzyme from plant material
Similar approaches have been reported to isolate enzyme in
peppermint oil gland cells [27] Heterologously expressed
enzyme clearly showed CoOMT activity and provided a
sufficient amount of material for characterization, whereas
such characterization was only partial due to substrate
limitations Further computer-assisted analyses for the
hydropathy and localization of CoOMT using theSIGNAL
P (http://www.cbs.dtu.dk/services/SignalP/) and PSORT
(http://psort.nibb.ac.jp/) programs indicated that CoOMT
was a cytosolic enzyme, whereas Berberis CoOMT has been
reported to be vesicle-bound [4]
This CoOMT is the fourth OMT isolated from C japonica
cells to play a role in isoquinoline alkaloid biosynthesis As
each enzyme shows distinct substrate specificity, we
com-pared the sequences of these OMTs Surprisingly, CoOMT
was much more similar to 6OMT and 4¢OMT than SMT,
even though SMT catalyzes the O-methylation of a
proto-berberine alkaloid, like CoOMT CoOMT showed 40%
identity to 4¢OMT However, we did not detect recombinant
CoOMT in the crude extract of E coli by an immunoblot
analysis with anti4¢OMT polyclonal antibodies Multiple
sequence alignment of CoOMT and the biosynthetic enzymes involved in benzylisoquinoline alkaloid biosynthe-sis, including berberine bridge enzyme [28–30] and (S)-N-methylcoclaurine 3¢-hydroxylase (CYP80B1) [31], also failed
to indicate any sequence homology (data not shown) A comparison of CoOMT and CNMT only showed limited sequence similarity in motif A, as seen between CNMT and other OMTs [14] It is not clear why the sequence of CoOMT
is so different from that of SMT
The three-dimensional structures of IOMT and chalcone OMT in flavonoid biosynthesis have recently been charac-terized [32] Multiple sequence alignment of Coptis OMTs, including CoOMT, with OMTs in flavonoid biosynthesis showed that the residues involved in AdoMet binding and the catalytic residues were highly conserved, and the methionine residues that interact with methyl group accep-tor were also conserved However, other substrate-binding residues were not conserved (data not shown) The sequence diversity in the substrate-binding site, which is expected to
be located in the N-terminal end of Coptis OMTs (T Morishige & F Sato, unpublished data), is considerably high The sequence of CoOMT may be useful for under-standing substrate binding and the enzyme structures in isoquinoline alkaloid biosynthesis
Our recombinant CoOMT also provides additional information about the branching point in the late steps in palmatine biosynthesis Columbamine was once thought to
be the main substrate for palmatine biosynthesis in Berberis wilsoniae and Berberis aggregata using partially purified
Fig 6 LC-MS analysis of the CoOMT reaction products for (S)-scoulerine (A), 2,3,9,10-tetrahydroxyprotoberberine (C) and tetrahydrocolumbamine (E), respectively (B) (D) and (F) show the respective control reactions (pET-21d) for (S)-scoulerine, 2,3,9,10-tetrahydroxyprotoberberine and tetrahydrocolumbamine Products with a m/z increase of 14 mass units were observed only in the CoOMT reaction Tetrahydrocolumbamine was prepared by the SMT reaction and used in a successive CoOMT reaction without further purification TIC, total ion chromatogram.
FEBS 2002 Coptiscolumbamine O-methyltransferase (Eur J Biochem 269) 5665
Trang 8enzyme, as Berberis CoOMT could not methylate
tetra-hydrocolumbamine [4] However, our findings clearly
indicate that tetrahydrocolumbamine can be a substrate
for the formation of tetrahydropalmatine (Fig 6), which
can be easily converted to palmatine by
(S)-tetrahydropro-toberberine oxidase [33] Our preliminary analyses also
showed that CoOMT had rather smaller Km value for
tetrahydrocolumbamine than columbamine, while the Vmax
value for tetrahydrocolumbamine was also smaller than
that for columbamine These data suggested that both
pathways might operate in Coptis cells Differences in
substrate specificity have also been reported for the CNMT
of Coptis and Berberis, i.e Coptis enzyme could
N-methy-late norlaudanosoline, whereas Berberis enzyme could not
[11,13] These results suggest that the late biosynthetic
pathway should be re-examined in various plant species,
and that the pathway in secondary metabolism may vary
depending on the enzyme(s) that each plant has acquired
during its evolution
A C K N O W L E D G E M E N T S
We thank Dr N Nagakura, Dr P Facchini and Mitsui Petrochemical
Industries Ltd for their generous gifts of the alkaloids We also thank
Ms L Huang for her technical assistance with preparing columbamine.
We thank Dr W Frommer of the University of Tuebingen for the gift
of pDR196 vector.
This research was supported in part by a Research for the Future
Program Grant (JSPS-RFTF00L01607) from the Japan Society for the
Promotion of Science (to F S), and a fellowship from the Japan Society
for the Promotion of Science (to T M).
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