Three new O-methyltransferases are sufficient for all O-methylation reactions of ipecac alkaloid biosynthesis in root culture of Psychotria ipecacuanha

Article abstract of DOI:10.1074/jbc.M109.086157

The medicinal plant Psychotria ipecacuanha produces ipecac alkaloids, a series of monoterpenoid-isoquinoline alkaloids such as emetine and cephaeline, whose biosynthesis derives from condensation of dopamine and secologanin. Here, we identified three cDNAs, IpeOMT1-IpeOMT3, encoding ipecac alkaloid O-methyltransferases (OMTs) from P. ipecacuanha. They were coordinately transcribed with the recently identified ipecac alkaloid β-glucosidase Ipeglu1. Their amino acid sequences were closely related to each other and rather to the flavonoid OMTs than to the OMTs involved in benzylisoquinoline alkaloid biosynthesis. Characterization of the recombinant IpeOMT enzymes with integration of the enzymatic properties of the IpeGlu1 revealed that emetine biosynthesis branches off from N-deacetylisoipecoside through its 6-O-methylation by IpeOMT1, with a minor contribution by IpeOMT2, followed by deglucosylation by IpeGlu1. The 7-hydroxy group of the isoquinoline skeleton of the aglycon is methylated by IpeOMT3 prior to the formation of protoemetine that is condensed with a second dopamine molecule, followed by sequential O-methylations by IpeOMT2 and IpeOMT1 to form cephaeline and emetine, respectively. In addition to this central pathway of ipecac alkaloid biosynthesis, formation of all methyl derivatives of ipecac alkaloids in P. ipecacuanha could be explained by the enzymatic activities of IpeOMT1-IpeOMT3, indicating that they are sufficient for all O-methylation reactions of ipecac alkaloid biosynthesis.

Full text of DOI:10.1074/jbc.M109.086157

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 10, pp. 7722–7738, March 5, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Three New O-Methyltransferases Are Sufficient for All
O-Methylation Reactions of Ipecac Alkaloid Biosynthesis in
□
S
Root Culture of Psychotria ipecacuanha
Received for publication, November 17, 2009, and in revised form, January 3, 2010 Published, JBC Papers in Press, January 8, 2010, DOI 10.1074/jbc.M109.086157
Taiji Nomura1 and Toni M. Kutchan2
From the Donald Danforth Plant Science Center, St. Louis, Missouri 63132
The medicinal plant Psychotria ipecacuanha produces ipecac
alkaloids, a series of monoterpenoid-isoquinoline alkaloids
such as emetine and cephaeline, whose biosynthesis derives
from condensation of dopamine and secologanin. Here, we
identified three cDNAs, IpeOMT1–IpeOMT3, encoding ipecac
alkaloid O-methyltransferases (OMTs) from P. ipecacuanha.
They were coordinately transcribed with the recently identified
ipecac alkaloid ␤-glucosidase Ipeglu1. Their amino acid se-
quences were closely related to each other and rather to the
flavonoid OMTs than to the OMTs involved in benzylisoquino-
line alkaloid biosynthesis. Characterization of the recombinant
IpeOMT enzymes with integration of the enzymatic properties
of the IpeGlu1 revealed that emetine biosynthesis branches off
from N-deacetylisoipecoside through its 6-O-methylation by
IpeOMT1, with a minor contribution by IpeOMT2, followed by
deglucosylation by IpeGlu1. The 7-hydroxy group of the iso-
quinoline skeleton of the aglycon is methylated by IpeOMT3
prior to the formation of protoemetine that is condensed with a
second dopamine molecule, followed by sequential O-methyla-
tions by IpeOMT2 and IpeOMT1 to form cephaeline and eme-
tine, respectively. In addition to this central pathway of ipecac
alkaloid biosynthesis, formation of all methyl derivatives of ipe-
cac alkaloids in P. ipecacuanha could be explained by the enzy-
matic activities of IpeOMT1–IpeOMT3, indicating that they are
sufficient for all O-methylation reactions of ipecac alkaloid
biosynthesis.
maceutical sources. Alkaloids are one of the better studied
classes of plant natural products due to their pharmacological
activities. Psychotria ipecacuanha Stokes (Rubiaceae), which is
native to South and Central America, has been used in therapy
since the early 17th century as an emetic and expectorant and
later as a medication for amebic dysentery (1). Such medicinal
effects of the root extracts derive from the principal alkaloid
emetine. P. ipecacuanha also produces many kinds of emetine-
related alkaloids such as cephaeline and ipecoside, known as
ipecac alkaloids (Fig. 1) (2). Alangium lamarckii Thwaites
(Alangiaceae), a medicinal plant indigenous to India (2), also
produces ipecac alkaloids.
Structurally, ipecac alkaloids possess a monoterpenoid-iso-
quinoline skeleton that originates from secologanin, a gluco-
sidal monoterpenoid, and dopamine. Secologanin and dop-
amine are condensed in a Pictet-Spengler manner to form the
(1␣S)-diastereomer N-deacetylisoipecoside and the (1␤R)-di-
astereomer N-deacetylipecoside (Fig. 1). The reaction proceeds
nonenzymatically under weakly acidic conditions, but it was
demonstrated that two enzymes, each of which stereospecifi-
cally catalyzes the formation of each diastereomer, are involved
in the reaction in Alangium (3, 4). The (1S)-diastereomer
N-deacetylisoipecoside is deglucosylated and converted to pro-
toemetine, which is then condensed with a second molecule of
dopamine and further converted to cephaeline and emetine
(Fig. 1). The (1R)-diastereomer N-deacetylipecoside is con-
verted to alkaloidal glucosides, such as ipecoside and alangiside,
through N-acetylation and spontaneous lactamization (Fig. 1)
(5, 6). They are accumulated as main alkaloidal glucosides in
Psychotria and Alangium.
Plants produce a diverse array of secondary metabolites,
whose number has been estimated to be over 200,000. In plants,
a large portion of those natural products are considered to be
synthesized for chemical defense against microbial attack and
herbivore predation because of their biological activities.
Because some of them exhibit a wide range of pharmacological
activities, they have also been studied from the aspect of phar-
The early stage of the ipecac alkaloid biosynthetic pathway is
analogous to that of terpenoid-indole alkaloid formation, which
begins a Pictet-Spengler condensation of secologanin and
tryptamine instead of dopamine. The condensation reaction is
catalyzed by strictosidine synthase that stereospecifically pro-
duces the (3␣S)-diastereomer strictosidine but not the (3␤R)-
diastereomer in Rauvolfia serpentina (7, 8), Catharanthus
roseus (9–11), Cinchona robusta (12), and Ophiorrhiza pumila
(13). Strictosidine is then deglucosylated by strictosidine
␤-glucosidase, and the pathway branches off into the biosyn-
thesis of over 1800 members of terpenoid-indole alkaloids
from strictosidine aglycon (14). Recently, we have identified
an ipecac alkaloid ␤-glucosidase (IpeGlu1) from P. ipecacu-
anha root culture that catalyzes the deglucosylation of the
(1S)-diastereomer N-deacetylisoipecoside and the (1R)-di-
astereomer N-deacetylipecoside (15). In contrast to the ste-
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. S1 and Table S1.
The nucleotide sequence(s) reported in this paper has been submitted to the
DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB527082 to
AB527084.
1 Supported by a Japan Society for the Promotion of Science Postdoctoral
Fellowship for Research Abroad. Present address: Dept. of Biotechnology,
Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa,
Imizu, Toyama 939-0398, Japan.
2 To whom correspondence should be addressed: Dept. of Biotechnology,
Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa,
Imizu, Toyama 939-0398, Japan. Tel.: 81-766-56-7500 (ext. 517); Fax:
81-766-56-2498; E-mail: tnomura@pu-toyama.ac.jp.
7722 JOURNAL OF BIOLOGICAL CHEMISTRY
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Ipecac Alkaloid O-Methyltransferases
FIGURE1.Schematicrepresentationofthebiosyntheticpathwayofemetine,cephaeline,andrelatedalkaloidalglucosidesinP.ipecacuanha.Probable
O-methylation steps in ipecac alkaloid biosynthesis are shown. IpeGlu1 has been identified by Ref. 15. The alkaloidal glucosides having an R-configuration,
methylalangiside and 6,7-O,O-dimethylipecoside, both of which have two methoxy groups on the isoquinoline skeleton, have not yet been identified in
P. ipecacuanha.
reospecific strictosidine ␤-glucosidase, IpeGlu1 accepts not syl-L-methionine (AdoMet)3-dependent O-methyltransferases
(OMTs). Several OMTs catalyzing these reactions have been
identified in opium poppy Papaver somniferum, Coptis japon-
ica, Thalictrum tuberosum, and Eschscholzia californica as fol-
lows: norcoclaurine 6-OMT (16–18), reticuline 7-OMT (16),
norreticuline 7-OMT (19), scoulerine 9-OMT (20), and colum-
bamine 2-OMT (21). In addition to these OMTs, 3Ј-hydroxy-
N-methylcoclaurine 4Ј-OMT (22), coclaurine N-methyltrans-
ferase (23), and stylopine cis-N-methyltransferase (24) catalyze
O- and N-methyl transfer reactions specific to benzylisoquino-
line alkaloid biosynthesis.
only the (1S)-diastereomer but also the (1R)-diastereomer,
including the main alkaloidal glucoside, ipecoside. Deglu-
cosylation of the (1S)-diastereomer is one of the steps of
emetine biosynthesis. From a physiological point of view, it
is inferred that (1R)-configured glucoalkaloids function in
defense when toxic ipecoside aglycon is released by IpeGlu1
in response to pathogen and herbivore attack (15).
Although N-deacetylisoipecoside aglycon is highly reactive
and is converted into multiple forms through nonenzymatic
intramolecular reactions in vitro, we have proposed that the
iminium cation formed by dehydration is the intermediate for
emetine biosynthesis in vivo (see Fig. 4 in Ref. 15). With respect
to the first condensation and the following deglucosylation
reactions, the ipecac alkaloid pathway is similar to the terpe-
noid-indole alkaloid pathway. It is expected that modification
of the isoquinoline moiety of ipecac alkaloids that derives from
dopamine is analogous to that of benzylisoquinoline alkaloids,
the biosynthesis of which begins with the condensation of
dopamine and 4-hydroxyphenylacetaldehyde. Two hydroxy
groups of the isoquinoline skeleton are methylated by S-adeno-
Ipecac alkaloids possess one isoquinoline moiety in the car-
bon skeleton prior to condensation with the second molecule of
dopamine to form cephaeline and emetine, which contain two
isoquinoline groups (Fig. 1). At completion of emetine biosyn-
3 The abbreviations used are: AdoMet, S-adenosyl-L-methionine; OMT,
O-methyltransferase; GFP, green fluorescence protein; YFP, yellow fluores-
cence protein; RT, reverse transcription; HPLC, high performance liquid
chromatography; LC-MS/MS, liquid chromatography/tandem mass spec-
trometry; N-Boc, N-tert-butoxycarbonyl; EST, expressed sequence tag;
DIBOA-Glc, 2-O-␤-D-glucopyranosyl-4-hydroxy-1,4-benzoxazin-3-one.
MARCH 5, 2010•VOLUME 285•NUMBER 10
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Ipecac Alkaloid O-Methyltransferases
thesis, four hydroxy groups are methylated. Two hydroxy
Heterologous Expression and Purification of IpeOMT En-
groups of the first isoquinoline skeleton are methylated during zymes—The entire coding region of each of the three IpeOMTs
protoemetine formation. IpeGlu1 is involved in the deglucosy- was amplified by PCR and ligated between the NdeI and EcoRI
lation step, but it is unknown whether O-methylations occur restriction sites of a pET28a vector (Novagen). The resulting
before or after deglucosylation (Fig. 1). In addition, O-methyl- vector was introduced into the Escherichia coli strain BL21
ation reactions of the second isoquinoline skeleton to form the CodonPlus(DE3) RIL (Stratagene) for protein expression. The
final product emetine have not been determined. Moreover, a recombinant E. coli was grown in 500 ml of LB medium con-
large part of the chemical diversity of ipecac alkaloids is attrib- taining 30 ␮g/ml kanamycin and 35 ␮g/ml chloramphenicol at
utable to the distinct O-methylation patterns with respect to 37 °C until the A600 reached 0.6–0.8. After cooling, isopropyl
the four hydroxy moieties. Thus, OMTs are essential targets to 1-thio-␤-D-galactopyranoside was added at 1 mM, and the cul-
be identified for the elucidation of the order of transformations ture was incubated for 18 h at 18 °C on an orbital shaker at 200
occurring along the ipecac alkaloid biosynthetic pathway. In rpm. The cells were harvested (6000 ϫ g, 10 min) and resus-
this study, we report the identification of three cDNAs encod- pended in 20 ml of 50 mM Tris-HCl buffer (pH 7.5) containing
ing ipecac alkaloid OMTs in P. ipecacuanha through EST anal- 20% (v/v) glycerol, 10 mM 2-mercaptoethanol, and 0.5% (v/v)
ysis coupled with the characterization of the substrate specific- protease inhibitor mixture (Sigma). After sonication (20 s at 50
ity and kinetic parameters of recombinant enzymes. The watts, six times) and centrifugation (20,000 ϫ g, 20 min), the
substrate specificities of the three recombinant OMTs are suf- supernatant containing soluble protein was collected for the
ficient to explain all O-methylation reactions of ipecac alkaloid purification of the N-terminal His-tagged IpeOMT proteins.
biosynthesis. Based on their functional characterization, we
The recombinant protein was purified with TALON His tag
propose the detailed biosynthetic network of ipecac alkaloids in purification resin (Clontech). All operations were done at 4 °C.
P. ipecacuanha.
The resin (1 ml of bed volume per 20 ml of the supernatant)
equilibrated in 50 mM Tris-HCl buffer (pH 7.5) containing 0.5 M
KCl, 20% glycerol, and 10 mM 2-mercaptoethanol was mixed
EXPERIMENTAL PROCEDURES
Plant Materials—P. ipecacuanha root cultures were main- with the supernatant for 1 h. After the resin was washed with
tained as described by Ref. 15.
equilibration buffer, the recombinant IpeOMT protein was
Cloning of O-Methyltransferase cDNAs—1050 ESTs from a eluted in a stepwise manner by increasing imidazole concentra-
␭-ZAP cDNA library constructed from mRNA of P. ipecacua- tions (5 and 200 mM) in equilibration buffer (5 ml for each
nha in vitro roots were recently sequenced (15). Homology elution). The 200 mM imidazole fraction was subjected to a
searches of the ESTs using the BLASTX algorithm revealed that PD10 column (GE Healthcare) equilibrated with storage buffer
one of the contigs (contig-213) was similar to known plant (50 mM Tris-HCl (pH 7.5) containing 0.1 M KCl, 20% glycerol,
OMTs. The contig consisted of four EST members (ph1903-35, and 5 mM dithiothreitol) and was concentrated appropriately
ph1903-62, ph1803-II-61, and ph3011-41), of which ph1903-62 by ultrafiltration using a Centriprep YM-10 (Millipore). Puri-
and ph1803-II-61 were identical in the overlapped region, but fied protein was stored at Ϫ80 °C. The protein profile was ana-
they were different from ph1903-35 and ph3011-41. Oligonu- lyzed by 12% SDS-PAGE. Protein concentration was measured
cleotide primers were designed from common sequences by the method of Ref. 25 using bovine serum albumin as a
among them for the isolation of full-length coding sequences by standard.
RT-PCR, 5Ј-AACTTGGCAAATGGAAACTG-3Ј (forward)
Substrates for Enzyme Assays—Of the 52 compounds listed
and 5Ј-CTTGAATTCAAGGAGAAAGCTC-3Ј (reverse). Root in supplemental Fig. S1, ipecoside, cephaeline, 21 benzyliso-
mRNA was purified as described in Ref. 15, and the cDNA syn- quinoline alkaloids, kaempferol, and myricetin were from
thesized using a SMART rapid amplification of cDNA ends the chemical stock of Dr. Meinhart H. Zenk (Donald Dan-
cDNA amplification kit (Clontech) was used for the PCR as forth Plant Science Center). Quercetin and resveratrol were
follows: 30 s at 98 °C, followed by 35 cycles of amplification (10 s gifts from Dr. Oliver Yu (Donald Danforth Plant Science
at 98 °C, 30 s at 55 °C, and 1 min at 72 °C) in a 50-␮l reaction Center). 7Ј-O-Demethylcephaeline was a gift from Dr. Takao
mixture containing 3 ng of cDNA, 0.5 ␮M primers, 0.2 mM Tanahashi (Kobe Pharmaceutical University, Japan). Dopam-
dNTPs, 1ϫ reaction buffer, and 1 unit of Phusion HS-HF poly- ine, 3-hydroxy-4-methoxyphenethylamine, 4-hydroxy-3-me-
merase (Finnzymes). The PCR products were inserted into thoxyphenethylamine, caffeic acid, guaiacol, catechol, vanillin,
pCR-BluntII-TOPO vector (Invitrogen) and sequenced to vanillic acid, and isovanillic acid were purchased from Sigma
obtain IpeOMT1 and IpeOMT2. IpeOMT1 corresponded to the and Acros Organics. N-Deacetylisoipecoside, N-deacetyli-
EST ph3011-41, and IpeOMT2 to the ph1903-62 and ph1803- pecoside, 6-O-methyl-N-deacetylisoipecoside, 6-O-methyl-
II-61. Because the full-length cDNA clone corresponding to the N-deacetylipecoside, 7-O-methyl-N-deacetylisoipecoside,
EST ph1903-35 was not obtained, 3Ј-rapid amplification of 7-O-methyl-N-deacetylipecoside, demethylisoalangiside, and
cDNA ends was performed using ph1903-35-specific forward demethylalangiside were prepared according to Ref. 15.
primer (5Ј-GTCTAGATTAATAGCATCATAAGTTAGG-3Ј) Isoalangiside, alangiside, 3-O-demethyl-2-O-methylisoalangiside,
and the adapter primer under the same conditions as described and 3-O-demethyl-2-O-methylalangiside were synthesized from
above except for the annealing temperature at 60 °C. The PCR 6-O-methyl-N-deacetylisoipecoside, 6-O-methyl-N-deacetylipe-
products were inserted into pCR-BluntII-TOPO and se- coside, 7-O-methyl-N-deacetylisoipecoside, and 7-O-methyl-N-
quenced to obtain IpeOMT3, which corresponded to the EST deacetylipecoside, respectively, according to the method for syn-
ph1903-35.
thesis of lactams (15).
7724 JOURNAL OF BIOLOGICAL CHEMISTRY
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Ipecac Alkaloid O-Methyltransferases
Redipecamine (reduced ipecac amine, as designated in the
For the determination of pH optimum, the same buffer sys-
present study) was prepared by deglucosylating N-deacetyli- tems were used as described in Ref. 15. Temperature optimum
soipecoside with ipecac alkaloid ␤-glucosidase (IpeGlu) under was determined by the reactions at 25–55 °C. N-Deacetyli-
reducing conditions (15). The deglucosylation reaction was soipecoside was used as substrate for IpeOMT1 and IpeOMT2
performed in 2 ml of 0.1 M citrate, 0.2 M phosphate buffer and (S)-reticuline for IpeOMT3.
(pH 5.0) containing 10 mM N-deacetylisoipecoside, 10 mM
Enzyme reactions for the determination of kinetic parame-
NaBH3CN, and 108 ␮g of IpeGlu9 enzyme. After 1 h of incuba- ters were performed in 0.1 M KPi buffer at the optimum pH for
tion at 55 °C, the reaction was stopped by adding 400 ␮l of 1 N each IpeOMT at 30 °C. Kinetic parameters were calculated
HCl, and the denatured enzyme was removed by centrifugation from three replicates by nonlinear fitting of the data to the
(16,000 ϫ g, 2 min). To the supernatant was added 400 ␮l of 10% Michaelis-Menten equation using SigmaPlot 2001.
(w/v) Na2CO3, followed by the extraction with ethyl acetate (2.8
Identification of Enzymatic Products—The enzymatic prod-
ml two times). The organic layer was dried under N2 flow, and ucts that are dependent on the presence of AdoMet were iden-
the residue was dissolved in 1 ml of 50% (v/v) methanol con- tified by comparing the retention time and the fragmentation
taining 0.1% (v/v) trifluoroacetic acid and subjected to prepar- pattern in liquid chromatography (LC)-tandem mass spec-
ative HPLC (column, LiChrosorb RP18, 25 ϫ 250 mm, 7 ␮m, trometry (MS/MS) analysis with authentic compounds. For
Merck; eluent, A ϭ 0.1% (v/v) trifluoroacetic acid and B ϭ ace- LC-MS/MS analysis, HPLC separation was conducted with a
tonitrile, gradient ϭ 10–40% (v/v) B/(A ϩ B) in 50 min; flow Shimadzu LC20A system, and mass spectra were obtained
rate, 8 ml/min; detection, 230 nm). The collected fraction was using a 4000 Q-TRAP triple stage quadruple mass spectrome-
concentrated in vacuo and freeze-dried.
ter equipped with a TurboIonSpray ionization source (Applied
3-O-Methylredipecamine was prepared from N-deacetyli- Biosystems) in a positive ion mode. The HPLC separation was
soipecoside via 6-O-methyl-N-deacetylisoipecoside. The reac- performed under the following conditions: column, Nova-Pak
tion mixture (30 ml) containing 0.1 M citrate, 0.2 M phosphate C18, 3.9 ϫ 300 mm, 4 ␮m, Waters; eluent, solvent A ϭ 0.2%
buffer (pH 6.5), 1 mM N-deacetylisoipecoside, 10 mM AdoMet, formic acid, solvent B ϭ acetonitrile; flow rate, 0.8 ml/min.
and 4.6 mg of IpeOMT1 enzyme was incubated at 40 °C. After a Solvent gradient conditions basically followed that described
7-h incubation, AdoMet (65 mg) and 0.9 mg of IpeOMT1 above but were changed appropriately when the 6-O-meth-
enzyme were added and further incubated for 2 h. The reaction ylate and 7-O-methylate of isoquinolines needed to be sepa-
was stopped by adding 6.2 ml of 1 N HCl, and the precipitation rated. For the analyses of IpeOMT1 and IpeOMT2 reaction
formed was removed by filtration (0.2 ␮m, Whatman), and the mixtures with cephaeline and 7Ј-O-demethylcephaeline to
pH was adjusted to 5.0 with 0.2 M Na2HPO4. To the solution (55 detect emetine and cephaeline, respectively, the column was
ml) was added 1.8 ml of 10 mM NaBH3CN dissolved in 0.1 M changed to Lichrospher 60 RP-select B (4 ϫ 250 mm, 5 ␮m,
citrate, 0.2 M phosphate buffer (pH 5.0), and 360 ␮g of IpeGlu9 Merck) for better peak shape of those substrates and prod-
enzyme. After a 1-h incubation at 55 °C, the reaction was ucts. To identify the molecular ion of the reaction products,
stopped by adding 12 ml of 1 N HCl. The pH was adjusted to 9.0 MS detection was done by enhanced mass scan (collision
with 10% Na2CO3, and ethyl acetate extraction was performed energy, 10 V; declustering potential, 60 V), and then the
(100 ml three times). The organic layer was washed with satu- fragmentation pattern of the molecular ion detected was
rated NaCl solution and evaporated in vacuo to dryness. The analyzed by enhanced product ion scan, for which the
residue was dissolved in 1 ml of acetonitrile containing 0.1% declustering potential was kept at 80 V while the collision
trifluoroacetic acid and subjected to preparative HPLC as energy was adjusted appropriately.
described above, and the collected fraction was concentrated in
To identify the enzymatic reaction products of N-deacetyli-
vacuo and freeze-dried. 2-O-Methylredipecamine was pre- soipecoside by IpeOMT1 and IpeOMT2, the products were
pared from 7-O-methyl-N-deacetylisoipecoside using the same derivatized with di-tert-butyldicarbonate before LC-MS/MS
procedure.
analysis, because 6-O-methyl-N-deacetylisoipecoside and 7-O-
Enzyme Assay—In a series of screening of substrates for the methyl-N-deacetylisoipecoside could not be separated by
IpeOMT1, IpeOMT2, and IpeOMT3 enzymes, standard HPLC under any conditions tested. For the analysis of
enzyme reaction was performed in 0.1 M KPi buffer (pH 7.5) IpeOMT1 reaction product, the enzyme reaction was per-
containing 10 ␮g of enzyme, 1 mM AdoMet, and 1 mM substrate formed in 0.1 M KPi (pH 7.5) containing 1.7 mM N-deacetyli-
(varied between 0.05 and 1 mM with solubility and availability) soipecoside, 2 mM AdoMet, and 200 ␮g of IpeOMT1 enzyme in
in a total volume of 100 ␮l. After incubation at 30 °C for 2 h, the a total volume of 1 ml. After a 4-h incubation at 30 °C, the
reaction was terminated by the addition of 20 ␮l of 1 N HCl. reaction was terminated by addition of 0.2 ml of 1 N HCl, fol-
After centrifugation (16,000 ϫ g, 2 min), the supernatant was lowed by centrifugation (16,000 ϫ g, 2 min). The supernatant
subjected to HPLC analysis to detect the reaction products (col- was subjected to preparative HPLC under the same conditions
umn, Nova-Pak C18, 3.9 ϫ 300 mm, 4 ␮m, Waters; eluent, A ϭ as described above except for the gradient condition (10–21%
0.1% trifluoroacetic acid, B ϭ acetonitrile, gradient ϭ 12–80% B/(A ϩ B) in 60 min). The collected fraction was evaporated in
B/(A ϩ B) in 30 min; flow rate, 0.8 ml/min; detection, 230 nm). vacuo and freeze-dried. The residue was treated with 1 ␮mol of
For the analysis of dopamine, 3-hydroxy-4-methoxyphenethyl- di-tert-butyldicarbonate in methanol containing 1 ␮mol of tri-
amine, 4-hydroxy-3-methoxyphenethylamine, guaiacol, and ethylamine for 1 h at room temperature. The reaction mixture
catechol, conditions for UV detection and solvent gradient, was dried under N2 flow and dissolved in 500 ␮l of 50% meth-
were changed appropriately.
anol. The chromatographic behavior of the N-Boc derivative
MARCH 5, 2010•VOLUME 285•NUMBER 10
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Ipecac Alkaloid O-Methyltransferases
was compared with those of standard 6-O-methyl-N-Boc- of the target cDNAs as template. Specificity of the amplifi-
deacetylisoipecoside and 7-O-methyl-N-Boc-deacetylisoipeco- cation was verified by a melt-curve analysis at the end of each
side synthesized according to Ref. 15. To identify the enzymatic PCR.
reaction products of N-deacetylisoipecoside by IpeOMT2, the
RESULTS
enzyme reaction was performed in 0.1 M citrate, 0.2 M phos-
phate buffer (pH 6.5) containing 0.5 mM N-deacetylisoipeco-
Isolation of O-Methyltransferase cDNAs from P. ipecacuanha—
side, 1 mM AdoMet, 1 mM EDTA, 1 mg/ml bovine serum albu- RT-PCR from mRNA of P. ipecacuanha root culture resulted in
min and 400 ␮g of IpeOMT2. After the overnight incubation at the isolation of two full-length cDNAs IpeOMT1 (ipecac alka-
40 °C, the reaction was terminated by 0.2 ml of 1 N HCl. Purifi- loid O-methyltransferase 1, GenBankTM accession number
cation and N-Boc derivatization of the products were con- AB527082) and IpeOMT2 (GenBankTM accession number
ducted in the same procedure as described above and subjected AB527083). IpeOMT1 corresponded to the EST ph3011-41 and
to LC-MS/MS analysis.
IpeOMT2 to the ph1903-62 and ph1803-II-61. The 3Ј-rapid
Subcellular Localization of IpeOMTs and IpeGlu1—The amplification of cDNA ends PCR-specific to the EST
coding sequences of IpeOMT1, IpeOMT2, IpeOMT3, and ph1903-35 resulted in the isolation of IpeOMT3 (GenBankTM
Ipeglu1 (GenBankTM accession number AB455576) were accession number AB527084).
amplified by PCR and inserted into pBA103 vector (gift from
IpeOMT1, IpeOMT2, and IpeOMT3 encoded polypeptides of
Brian Kelly, Donald Danforth Plant Science Center), which pos- 350, 350, and 358 amino acids with calculated molecular masses
sesses an enhanced cauliflower mosaic virus 35S promoter, an of 38.7, 39.0, and 39.7 kDa, respectively. The deduced amino
enhanced green fluorescence protein gene, and a nopaline syn- acid sequences were highly similar to each other, where
thase terminator, to express C-terminal GFP-fused proteins, IpeOMT1 showed 82 and 72% identities to IpeOMT2 and
IpeOMT1-GFP, IpeOMT2-GFP, IpeOMT3-GFP, and Ipe- IpeOMT3, respectively, and IpeOMT2 showed 73% identity
Glu1-GFP. DNA-coated gold particles (0.6 mm, Bio-Rad) were to IpeOMT3. IpeOMT1, IpeOMT2, and IpeOMT3 amino acid
prepared according to the manufacturer’s instructions and sequences exhibited the highest identity (55, 59, and 55%,
bombarded into a small piece (ϳ2 ϫ 2 cm) of onion with a respectively) to the flavonoid OMTs of C. roseus (27), which
PDS1000 He Biolistic Particle Delivery System (Bio-Rad) catalyzes O-methylation of 3Ј- and 5Ј-hydroxy groups of myric-
according to manufacturer’s instructions. The bombarded etin and dihydromyricetin. IpeOMT3 also shared comparable
onion was wrapped with a wet paper towel and incubated at identity (53%) to resveratrol OMT that catalyzes methylations
room temperature overnight in a Petri dish. Cytosol- and vac- of 3- and 5-hydroxy groups of resveratrol to form pterostilbene
uole-targeted enhanced yellow fluorescence protein constructs in Vitis vinifera (28). Phylogenetic analysis of the IpeOMTs
(gift from Dr. Isabel Ordiz, Donald Danforth Plant Science with selected members of plant OMTs, including benzyliso-
Center), endoplasmic reticulum-targeted enhanced GFP con- quinoline alkaloid OMTs (Fig. 2), showed that three IpeOMTs
struct (gift from Dr. Nigel Taylor, Donald Danforth Plant Sci- are closely related to each other, and the clade exhibited the
ence Center), and pBA103 without insert were also bombarded closest relationship to the flavonoid OMTs and terpenoid-in-
as controls. After the incubation, the epidermis was peeled off dole alkaloid 16-hydroxytabersonine 16-OMT in C. roseus (29)
and analyzed using an LSM 510 confocal/multiphoton micro- but not to the OMTs of benzylisoquinoline alkaloids such as
scope (Zeiss). Fluorescence of GFP and YFP was visualized by 3Ј-hydroxy-N-methylcoclaurine 4Ј-OMT in C. japonica (22),
excitation at 488 nm and a bandpass emission filter (505–550 norcoclaurine 6-OMTs in P. somniferum (16), T. tuberosum
nm). Confocal images were analyzed using the Imaris software (18), and C. japonica (22), reticuline 7-OMT (16), norreticuline
(Bitplane).
7-OMT (19), columbamine 2-OMT (21), and scoulerine
Quantitative RT-PCR Analysis—P. ipecacuanha in vitro 9-OMT (20).
roots precultured for 8 weeks were transferred to new LS liquid
Enzymatic Characterization of Recombinant IpeOMT En-
medium (26), and the roots were harvested in duplicate 1 and 3 zymes—N-terminal His-tagged IpeOMT enzymes were puri-
days and 1, 2, 4, 6, and 8 weeks after transfer. Total RNA was fied to apparent homogeneity from the soluble protein fraction
purified using an RNeasy plant mini kit (Qiagen), followed by by metal chelation chromatography (Fig. 3), yielding 6 mg
DNase treatment and re-purification. The synthesis of cDNA (IpeOMT1), 16 mg (IpeOMT2), and 4 mg (IpeOMT3) of
was performed with a SuperScript III reverse transcriptase recombinant enzymes from 1 liter of E. coli culture. Enzymatic
(Invitrogen) (0.5 ␮g of RNA in 20 ␮l of reaction mixture). activities were first tested with seven glucosidal ipecac alkaloids
Duplicates were further pooled, and 5 ng of cDNA was used for (Table 1, substrates 1–7, and supplemental Fig. S1), including
PCR. PCR was performed in triplicate on a StepOnePlus real N-deacetylisoipecoside, N-deacetylipecoside, and their 6- and
time PCR system (Applied Biosystems) under the following 7-O-monomethylates. In the reaction of IpeOMT1 using
conditions: 5 min at 95 °C, followed by 40 cycles of amplifica- N-deacetylisoipecoside as substrate, product dependent upon
tion (15 s at 95 °C and 1 min at 60 °C) in a 20-␮l reaction mix- the presence of IpeOMT1 and the methyl donor AdoMet was
ture containing template cDNA, 0.5 ␮M primers, and 10 ␮l of detected by HPLC analysis. Because the possible products 6-O-
PerfeCTa SYBR Green FastMix Reaction Mixes (Quanta Bio- methyl-N-deacetylisoipecoside and 7-O-methyl-N-deacetyli-
sciences). Primers specific to each of IpeOMT1, IpeOMT2, soipecoside could not be separated by reverse-phase HPLC
IpeOMT3, and Ipeglu1 were used (supplemental Table S1). A under conditions tested (Fig. 4A), the product was purified and
standard curve was made for every experiment with the dilu- chemically converted to the N-Boc derivative. LC-MS/MS pro-
tion series of the known quantities of a plasmid having each files of the derivative were the same as those of authentic 6-O-
7726 JOURNAL OF BIOLOGICAL CHEMISTRY
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Ipecac Alkaloid O-Methyltransferases
FIGURE 2. Unrooted phylogenetic tree of the ipecac alkaloid O-methyltransferases (IpeOMT1, IpeOMT2 and IpeOMT3) with selected members of
plant O-methyltransferases. Full-length amino acid sequences were aligned using the ClustalW (version 1.83). The phylogenetic tree was built based
on calculation by the neighbor-joining method (40) with bootstrap analysis of 1000 replicates and was visualized with the Treeview (version 1.66). The
numbers at each node are bootstrap values per 1000 trials (values over 70% are shown). The scale bar indicates the substitutions per site. The accession
numbers for the proteins listed are as follows: AAM97497 (C. roseus, flavonoid OMT) ; ABR20103 (C. roseus, 16-hydroxytabersonine 16-OMT); AAR02419
(C. roseus, flavonoid 4Ј-OMT); CAQ76879 (V. vinifera, resveratrol OMT); AAB71213 (Prunus armeniaca, OMT); AAM23004 (Rosa hybrid cultivar, orcinol
OMT); BAA86059 (Pyrus pyrifolia, OMT); AAL30423 (Ocimum basilicum, chavicol OMT); BAB08005 (C. japonica, 3Ј-hydroxy-N-methylcoclaurine 4Ј-OMT);
BAB08004 (C. japonica, norcoclaurine 6-OMT); AAQ01669 (P. somniferum, norcoclaurine 6-OMT); ACN88562 (P. somniferum, norreticuline 7-OMT);
AAC49708 (Pinus taeda, caffeic acid OMT); AAQ01668 (P. somniferum, reticuline 7-OMT); BAA06192 (C. japonica, scoulerine 9-OMT); AAA33032 (Masem-
bryanthemum crystallinun, myo-inositol OMT); CAA50561 (Nicotiana tabacum, catechol OMT); CAA13175 (Saccharum officinarum, caffeic acid OMT);
AAQ01670 (P. somnuferum, catechol OMT); AAB46623 (Medicago sativa, caffeic acid OMT); AAD38189 (O. basilicum, caffeic acid OMT), AAA86718 (Zinnia
violacea, caffeic acid OMT); AAC01533 (Clarkia breweri, (iso)eugenol OMT); AAB96879 (Arabidopsis thaliana, caffeic acid/5-hydroxyferulic acid OMT);
AAA80579 (Chrysosplenium americanum, flavonoid 3Ј-OMT); AAD29841 (T. tuberosum, norcoclaurine 6-OMT1); AAD29842 (T. tuberosum, norcoclaurine
6-OMT2); AAB48059 (M. sativa, isoliquiritigenin 2Ј-OMT); BAA13683 (Glycyrrhiza echinata, OMT); BAC22084 (C. japonica, columbamine 2-OMT);
AAC49856 (Pisum sativum, 6a-hydroxymaackiain OMT); AAC49928 (M. sativa, isoflavone OMT); CAA54616 (Hordeum vulgare, flavonoid 7-OMT);
ABY59051 (Zea mays, TRIBOA-glucoside 7-OMT); AAD10485 (Triticum aestivum, OMT); and P47917 (Z. mays, OMT).
methyl-N-Boc-deacetylisoipecoside (Fig. 4B), showing that showed no activity toward (1R)-diastereomer N-deacetylipeco-
IpeOMT1 catalyzed 6-O-methylation of N-deacetylisoipeco- side, IpeOMT2 reacted with N-deacetylipecoside as well as
side regiospecifically to form 6-O-methyl-N-deacetylisoipeco- (1S)-diastereomer N-deacetylisoipecoside. By LC-MS/MS
side. IpeOMT2 also reacted with N-deacetylisoipecoside, and analysis, the products were identified to be 6-O-methyl-N-
AdoMet-dependent products were detected (Fig. 4A). Product deacetylipecoside and 7-O-methyl-N-deacetylipecoside (Fig.
identification was performed after N-Boc derivatization. Two 4C). In the reaction, 7-O-methylation occurred approximately
peaks were detected, and they showed the same LC-MS/MS eight times more efficiently than 6-O-methylation. In addition
profiles as authentic 6-O-methyl-N-Boc-deacetylisoipecoside to the substrate and the products, their lactams formed by non-
and 7-O-methyl-N-Boc-deacetylisoipecoside (Fig. 4B). Thus, enzymatic intramolecular reaction were also detected (Fig. 4C).
it was found that IpeOMT2 catalyzed both a 6- and 7-O- The enzyme assay with O-monomethylates of N-deacetyli-
monomethylation of N-deacetylisoipecoside, where 6-OMT soipecoside and N-deacetylipecoside revealed that IpeOMT1
activity was approximately three times higher than 7-OMT also catalyzed 6-O-methylation of 7-O-methyl-N-deacetyli-
activity, as determined from the chromatogram of the N-Boc soipecoside to form 6,7-O,O-dimethyl-N-deacetylisoipecoside
derivatives (Fig. 4B). In contrast to IpeOMT1 enzyme that (Fig. 4D).
MARCH 5, 2010•VOLUME 285•NUMBER 10
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Ipecac Alkaloid O-Methyltransferases
Enzymatic activities for lactam substrates (Table 1, sub-
strates 8–13, and supplemental Fig. S1) were also tested.
IpeOMT1 did not show any activity for the lactam substrates.
IpeOMT2 catalyzed O-methylation of the 2-hydroxy group of
demethylalangiside, which corresponds to the 7-hydroxy group
with respect to the isoquinoline skeleton, to form 3-O-dem-
ethyl-2-O-methylalangiside (Fig. 4E). Its activity was regio- and
stereoselective, in contrast to the activities for non-lactam glu-
cosidal ipecac alkaloids as described above. IpeOMT3 accepted
none of the 13 glucosidal ipecac alkaloids (Table 1, substrates
1–13) tested as substrate.
To examine the involvement of IpeOMT enzymes in 6Ј- and
7Ј-O-methylation of the second isoquinoline moiety in emetine
biosynthesis (Fig. 1), their activities toward 7Ј-O-demethyl-
cephaeline, the penultimate intermediate for emetine biosyn-
thesis, and toward cephaeline (Table 1, substrates 14 and 15,
and supplemental Fig. S1) were tested. As a result, IpeOMT2
methylated the 7Ј-hydroxy group of 7Ј-O-demethylcephaeline
to form cephaeline (Fig. 4F), and IpeOMT1 methylated the
FIGURE 3. SDS-PAGE of the recombinant IpeOMT enzymes expressed in
E. coli. Proteins were separated on 12% SDS-PAGE and stained with Coomas-
sie Brilliant Blue G-250. Lane M, molecular size marker; lane C, crude extract
from the recombinant E. coli; lane P, purified recombinant enzyme by metal
chelation chromatography.
TABLE 1
Substrates tested for enzyme assay and the activities catalyzed by IpeOMT1-IpeOMT3 enzymes
Substrates 40–52 (phenolics 40–48, flavonols 49-51, and stilbene 52) are not listed due to absence of enzymatic activity toward all of them. See supplemental Fig. S1 for
chemical structures of each substrate. Number in parentheses is the specific activity (nmolꢀminϪ1ꢀmg proteinϪ1) at 0.2 mM substrate. AdoMet concentration was 1 mM for
all assays.
Substrate
Stereo-chemistrya
IpeOMT1
IpeOMT2
IpeOMT3
Ipecac alkaloids
c
1 N-Deacetylisoipecoside
2 N-Deacetylipecoside
(1S)
(1R)
6-OMT (11)
6-OMT Ͼ 7-OMT (0.14)b
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6-OMT Ͻ 7-OMTd
3 Ipecoside
(1R)
–
–
4 6-O-Methyl-N-deacetylisoipecoside
5 6-O-Methyl-N-deacetylipecoside
6 7-O-Methyl-N-deacetylisoipecoside
7 7-O-Methyl-N-deacetylipecoside
8 Demethylisoalangiside
(1S)
–
–
(1R)
–
–
(1S)
6-OMT (21)
–
(1R)
–
–
(13aS)
(13aR)
(13aS)
(13aR)
(13aS)
(13aR)
(11bS)
(11bS)
(13aS)
(13aS)
(13aS)
–
–
9 Demethylalangiside
–
2-OMT (1.7)
10 Isoalangiside
–
–
11 Alangiside
–
–
12 3-O-Demethyl-2-O-methylisoalangiside
13 3-O-Demethyl-2-O-methylalangiside
14 7Ј-O-Demethylcephaeline
15 Cephaeline
–
–
–
–
–
7Ј-OMT (51)
6Ј-OMT (0.31)
–
16 Redipecaminee
–
–
–
2-OMT (2.9)
17 2-O-Methylredipecaminee
–
–
18 3-O-Methylredipecaminee
2-OMT (12)
Benzylisoquinoline alkaloids
19 Coclaurine
(1S)
(1R,S)
(1R,S)
(1R,S)
(1R,S)
(1R,S)
(1R,S)
(1S)
–
–
7-OMT (1.9)
20 Isococlaurine
6-OMT (32)
6-OMT (0.35)
–
21 N-Methylcoclaurine
22 4Ј-O-Methylcoclaurine
23 Norcoclaurine
24 Laudanine
–
–
7-OMT (0.88)
–
–
7-OMT (1.0)
6-OMT (15)
6-OMT (0.60)
–
–
–
–
25 6-O-Methyllaudanosoline
26 4Ј-O-Methyllaudanosoline
27 Nororientaline
28 Isoorientaline
–
–
7-OMT (0.85)
6-OMT (0.23)
–
–
(1R,S)
(1R,S)
(1S)
4Ј-OMT (1.0)
–
7-OMT (2.2)
6-OMT (3.5)
6-OMT (0.94)
–
29 Norprotosinomenine
30 Norprotosinomenine
31 Protosinomenine
32 Norreticuline
6-OMT (11)
–
–
(1R)
6-OMT (7.0)
–
–
(1R,S)
(1S)
6-OMT (0.72)
–
–
–
–
–
–
–
–
–
–
–
7-OMT (4.2)
33 Norreticuline
(1R)
–
–
34 Reticuline
(1S)
–
7-OMT (0.42)
35 Reticuline
(1R)
–
–
36 Coreximine
(13aS)
–
2- or 11-OMT (0.88)
37 Oripavine
3-OMT (9.0)
–
–
–
38 Codeine
–
–
39 Morphine
a See supplemental Fig. S1 for corresponding chiral center.
b Activity was calculated from total amount of the two products (6-O-methyl-N-deacetylisoipecoside and 7-O-methyl-N-deacetylisoipecoside), which were not separated under
HPLC conditions tested. Their identification was conducted by separating the N-Boc derivatives.
c – means activity was not detected.
d Activity was not determined due to nonenzymatic lactamization of substrate and products.
e Synthetic ipecac alkaloid aglycon mimic.
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Ipecac Alkaloid O-Methyltransferases
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Ipecac Alkaloid O-Methyltransferases
FIGURE 4—continued
7730 JOURNAL OF BIOLOGICAL CHEMISTRY
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Ipecac Alkaloid O-Methyltransferases
FIGURE 4—continued
FIGURE4. LC-MS/MSanalysesoftheenzymaticreactionmixtureofIpeOMT1-IpeOMT3withipecacalkaloids. Totalionchromatogram(leftpanel)andthe
MS/MS fragmentation (right panel(s)) of the reaction product(s) are shown with those of standard compounds. A, reaction mixtures of IpeOMT1 and IpeOMT2
with N-deacetylisoipecoside, where 6-O-methyl-N-deacetylisoipecoside and 7-O-methyl-N-deacetylisoipecoside are not separated. B, N-Boc derivatives of the
reactionproductsofN-deacetylisoipecosidebyIpeOMT1andIpeOMT2, where6-O-methyl-N-Boc-deacetylisoipecosideand7-O-methyl-N-Boc-deacetylisoipe-
coside are separated. C, reaction mixture of IpeOMT2 with N-deacetylipecoside, where demethylalangiside, 3-O-demethyl-2-O-methylalangiside, and alangi-
side are lactams nonenzymatically formed from substrate and products. D, reaction mixture of IpeOMT1 with 7-O-methyl-N-deacetylisoipecoside. E, reaction
mixture of IpeOMT2 with demethylalangiside. F, reaction mixture of IpeOMT2 with 7Ј-O-demethylcephaeline. G, reaction mixture of IpeOMT1 with cephaeline.
H, reaction mixture of IpeOMT2 and IpeOMT3 with redipecamine and 3-O-methylredipecamine, respectively.
MARCH 5, 2010•VOLUME 285•NUMBER 10
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Ipecac Alkaloid O-Methyltransferases
6Ј-hydroxy group of cephaeline to form emetine (Fig. 4G). 2-hydroxy group that corresponds to 7-hydroxy group of iso-
IpeOMT3 was active neither with 7Ј-O-demethylcephaeline quinoline skeleton is most likely, judging from the regiospeci-
nor cephaeline.
ficity of IpeOMT3 enzyme for other benzylisoquinoline
Although an endogenous substrate for IpeOMT3 was not alkaloids.
known at this stage, because (S)-reticuline, one of the benzyli-
Although IpeOMT3 was found to catalyze 7-O-methylation
soquinoline alkaloids, was found to serve as its substrate as of 7-hydroxy-6-methoxybenzylisoquinoline alkaloids, they are
described below, pH and temperature optima were determined not the endogenous substrates, because none of these benzyli-
using N-deacetylisoipecoside as substrate for IpeOMT1 and soquinoline alkaloids have been found in P. ipecacuanha. How-
IpeOMT2 and (S)-reticuline for IpeOMT3. The pH optimum ever, the results encouraged us to examine the ipecac alkaloid
of IpeOMT1 was 8.0, whereas the pH optima of IpeOMT2 and aglycon as a possible endogenous substrate. As shown in Ref.
IpeOMT3 were 7.0. Temperature optima of IpeOMT1, 15, deglucosylation of N-deacetylisoipecoside gives multiple
IpeOMT2, and IpeOMT3 were 40, 45, and 40 °C, respectively. products formed by nonenzymatic intramolecular reactions of
Substrate Specificity of the IpeOMT Enzymes—To reveal the highly reactive aglycon. Redipecamine (supplemental Fig.
structural feature of substrates for each IpeOMT enzyme, S1) that is formed by the deglucosylation reaction under reduc-
enzyme reactions were performed using various substrates, ing conditions could, however, be stably prepared (15). There-
including benzylisoquinoline alkaloids (Table 1, substrates fore, we used redipecamine, 2-O-methylredipecamine, and
19–39, and supplemental Fig. S1) and simple phenolics (sub- 3-O-methylredipecamine (Table 1, substrates 16–18, and sup-
strates 40–48, supplemental Fig. S1). Simple phenolics were plemental Fig. S1) as mimics of endogenous ipecac alkaloid
not accepted as substrate by the IpeOMT1, IpeOMT2, and aglycons. IpeOMT1 reacted with none of them. IpeOMT2
IpeOMT3 enzymes. IpeOMT1 catalyzed 6-O-methylation of methylated the 2-hydroxy group of redipecamine (Fig. 4H) that
(R,S)-isococlaurine, (R,S)-norcoclaurine, (S)-4Ј-O-methyllau- corresponds to the 7-hydroxy group of the isoquinoline skele-
danosoline, (R,S)-isoorientaline, (R)-norprotosinomenine, (S)- ton, whereas IpeOMT3 catalyzed 2-O-methylation of 3-O-
norprotosinomenine, and (R,S)-protosinomenine, and an methylredipecamine (Fig. 4H). These results strongly suggested
exceptional 4Ј-O-methylation of (R,S)-nororientaline (Table 1). that IpeOMT2 and IpeOMT3 accept endogenous ipecac
The results indicated that IpeOMT1 dominantly catalyzes 6-O- alkaloid aglycons that are formed by deglucosylation of
methylation of the 6,7-dihydroxy- and 6-hydroxy-7-methoxy- N-deacetylisoipecoside and 6-O-methyl-N-deacetylisoipeco-
isoquinolines. This is consistent with the IpeOMT1 activities side, respectively.
for ipecac alkaloids as described above (Table 1, substrates 1, 6,
and 15).
Because IpeOMT sequences exhibited the highest identities
to the flavonoid OMT of C. roseus, and IpeOMT3 also exhibited
IpeOMT2 catalyzed 6-O-methylation of (R,S)-isococlaurine, comparably high identity to the resveratrol OMT of V. vinifera,
(R,S)-norcoclaurine, and (R,S)-isoorientaline (Table 1), show- IpeOMT activities toward kaempferol, quercetin, myricetin,
ing that IpeOMT2 catalyzes 6-O-methylation of 6,7-dihydroxy- and resveratrol (Table 1, substrates 49–52, and supplemental
and 6-hydroxy-7-methoxyisoquinolines. This is different from Fig. S1) were examined, but none were accepted by IpeOMTs as
the IpeOMT2 activities for ipecac alkaloids described above, i.e. substrate.
6-O-monomethylation and 7-O-monomethylation activities
Kinetic parameters of the IpeOMT enzymes for ipecac alka-
toward the 6,7-dihydroxyisoquinoline moiety. Considering loids, synthetic mimics of the ipecac alkaloid aglycons, and the
that 6- and 7-O-methylation of N-deacetylipecoside was cata- methyl donor AdoMet were determined (Table 2). Apparent
lyzed only by IpeOMT2 and that 7-O-methyl-N-deacetyli- Km and apparent kcat values of IpeOMT1 for N-deacetylisoipe-
Ϫ3
soipecoside was synthesized only by IpeOMT2 (Figs. 7 and 8), coside were 107 ␮M and 11.3 ϫ 10
s
Ϫ1, respectively. The
this relaxed regiospecificity appears to be an essential feature in apparent Km value for 7-O-methyl-N-deacetylisoipecoside (105
ipecac alkaloid biosynthesis. It is notable, however, that ␮M) was equal to that for N-deacetylisoipecoside, but the
IpeOMT2 regiospecifically catalyzed the methylation of the apparent kcat value for 7-O-methyl-N-deacetylisoipecoside
7Ј-hydroxy group, but not the 6Ј-hydroxy group, of 7Ј-O-dem- (20.4 ϫ 10Ϫ3 sϪ1) was 2-fold higher than that for N-deacetyli-
ethylcephaeline to form cephaeline, which is considered to be soipecoside. The catalytic efficiency of IpeOMT1 for cephae-
the primary reaction of IpeOMT2 judging from the catalytic line (3.5 sϪ1ꢀMϪ1) was remarkably lower than those for
parameters (Table 2). Although 3-OMT activity toward oripa- N-deacetylisoipecoside (106 sϪ1ꢀMϪ1) and 7-O-methyl-N-
vine was detected, because the 3-hydroxy group of oripavine is deacetylisoipecoside (194 sϪ1ꢀMϪ1) mainly due to the lower
not bound to an isoquinoline skeleton, this activity seems to be
fortuitous.
k
cat value (0.28 ϫ 10Ϫ3 sϪ1) despite the comparable Km value
(80 ␮M).
IpeOMT3 catalyzed the 7-O-methylation of (S)-coclaurine,
Kinetic parameters of IpeOMT2 for N-deacetylisoipecoside
(R,S)-N-methylcoclaurine, (R,S)-4Ј-O-methylcoclaurine, (R,S)- were evaluated based on the total amount of the two products,
6-O-methyllaudanosoline, (R,S)-nororientaline, (S)-norreticu- 6-O-methyl-N-deacetylisoipecoside and 7-O-methyl-N-de-
line, and (S)-reticuline (Table 1). IpeOMT3 accepted only acetylisoipecoside, because they could not be separated by
7-hydroxy-6-methoxyisoquinolines. Although the methylated HPLC analysis. The apparent reaction efficiency (0.81 sϪ1ꢀM
)
Ϫ1
position of the enzymatic product of coreximine could not be was ϳ130-fold lower than that of IpeOMT1 (106 sϪ1ꢀMϪ1) due
Ϫ3
assigned due to the lack of authentic 2-O-methylcoreximine to larger Km value (420 ␮M) and lower kcat value (0.34 ϫ 10
and 11-O-methylcoreximine, and possibly due their identical
s
Ϫ1). Kinetic parameters of IpeOMT2 for N-deacetylipecoside
fragmentation pattern in MS/MS analysis, methylation of the were not determinable, because considerable amounts of sub-
7732 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 10•MARCH 5, 2010

Ipecac Alkaloid O-Methyltransferases
TABLE 2
Kinetic parameters of the IpeOMT1-IpeOMT3 enzymes
Enzyme
Substrate
Km
␮M
Vmax
kcat
k
s
cat/Km
Ϫ1
Ϫ3
Ϫ1
nmolꢀminϪ1ꢀmg
s
Ϫ1 (ϫ10
)
Ϫ1ꢀM
IpeOMT1
N-Deacetylisoipecosidea
7-O-Methyl-N-deacetylisoipecosidea
Cephaelinea
107 Ϯ 4
105 Ϯ 4
80 Ϯ 7
42 Ϯ 6
420 Ϯ 17
NDc
16.6 Ϯ 0.3
30.0 Ϯ 0.8
0.41 Ϯ 0.01
10.5 Ϯ 0.5
0.50 Ϯ 0.01
ND
11.3 Ϯ 0.2
106
194
20.4 Ϯ 0.5
0.28 Ϯ 0.007
7.1 Ϯ 0.3
3.5
AdoMetb
169
0.81
ND
8.4
IpeOMT2
N-Deacetylisoipecosidea
N-Deacetylipecosidea
Demethylalangisidea
Redipecaminea
0.34 Ϯ 0.007
ND
450 Ϯ 6
35 Ϯ 1
1.0 Ϯ 0.5
20 Ϯ 1
64 Ϯ 4
34 Ϯ 3
5.6 Ϯ 0.1
3.4 Ϯ 0.06
47.6 Ϯ 2.7
3.8 Ϯ 0.07
2.3 Ϯ 0.04
32.6 Ϯ 1.9
0.26 Ϯ 0.004
10.5 Ϯ 0.1
8.9 Ϯ 0.3
66
7Ј-O-Demethylcephaelinea
AdoMetb
32600
13
0.38 Ϯ 0.006
15.0 Ϯ 0.2
12.8 Ϯ 0.4
IpeOMT3
3-O-Methylredipecaminea
AdoMetd
159
262
a Reaction was performed at 1 mM AdoMet.
b Reaction was performed at 0.7 mM N-deacetylisoipecoside.
c ND means not determined due to nonenzymatic lactamization of substrate and products.
d Reaction was performed at 0.5 mM 3-O-methylredipecamine.
strate and the enzymatic products (6-O-methyl-N-deacetylipe-
coside and 7-O-methyl-N-deacetylipecoside) were nonenzy-
matically converted to their lactams under the reaction
conditions used (pH 7.0) (see Fig. 4C). The apparent reaction
efficiency for demethylalangiside (8.4 sϪ1ꢀMϪ1) was 10-fold
higher than that for N-deacetylisoipecoside due to the 10-fold
Ϫ3
increase of the kcat value (3.8 ϫ 10
s
Ϫ1) with a similar Km
value (450 ␮M). IpeOMT2 catalyzed the 2-O-methylation of the
synthetic N-deacetylisoipecoside aglycon mimic, redipecam-
ine, with greater reaction efficiency (66 sϪ1ꢀMϪ1) than those for
N-deacetylisoipecoside and demethylalangiside. The apparent
reaction efficiency of IpeOMT2 for 7Ј-O-demethylcephaeline
(32,600 sϪ1ꢀMϪ1) was the highest among the substrates tested,
which is attributable to notably small Km (1.0 ␮M) and high kcat
(32.6 ϫ 10Ϫ3 sϪ1) values.
The apparent Km value (64 ␮M) of IpeOMT3 for 3-O-meth-
ylredipecamine, the synthetic 6-O-methyl-N-deacetylisoipeco-
side aglycon mimic, was 2-fold larger, and the apparent kcat
Ϫ3
value (10.5 ϫ 10
s
Ϫ1) was 5-fold higher than those of
IpeOMT2 for redipecamine.
Kinetic parameters of IpeOMT1, IpeOMT2, and IpeOMT3
for the methyl donor AdoMet were also determined using
N-deacetylisoipecoside (for IpeOMT1 and IpeOMT2) and (S)-
reticuline (for IpeOMT3) as methyl acceptors. The apparent
FIGURE 5. Subcellular localization of IpeOMT1-GFP, IpeOMT2-GFP,
IpeOMT3-GFP, and IpeGlu1-GFP fusion proteins. Onion epidermis was
bombarded with DNA-coated gold particles. GFP/YFP fluorescence (left
panel) and GFP/YFP transmission light overlay (right panel) are shown. Non-
targeted GFP is the bombardment with empty GFP vector (pBA103). Cytosol-
targeted YFP, endoplasmic reticulum-targeted GFP, and vacuole-targeted
YFP were also bombarded as controls. Fluorescence signals of IpeOMT-GFPs,
IpeGlu1-GFP, nontargeted GFP, and cytosol-targeted YFP in the nucleus are
attributable to free diffusion of the protein (41).
K
m values of IpeOMT1, IpeOMT2, and IpeOMT3 were com-
parable with each other (42, 20, and 34 ␮M, respectively).
Although the apparent kcat values of IpeOMT1 and IpeOMT3
were similar (7.1 ϫ 10Ϫ3 and 8.9 ϫ 10Ϫ3 sϪ1, respectively), that
Ϫ3
of IpeOMT2 (0.26 ϫ 10
s
Ϫ1) was remarkably lower than
those of IpeOMT1 and IpeOMT3.
Subcellular Localization of IpeOMT and IpeGlu1 Enzymes—
To examine the subcellular localization of IpeOMT1,
IpeOMT2, and IpeOMT3 enzymes, C-terminal GFP-fused
enzymes (IpeOMT1-GFP, IpeOMT2-GFP, and IpeOMT3-
GFP) were transiently expressed in onion epidermal cells. In
addition, subcellular localization of the ipecac alkaloid ␤-glu-
cosidase (IpeGlu1) (15) was also examined by expressing the
IpeGlu1-GFP fusion protein to see whether or not the IpeGlu1
enzyme is compartmentalized separately from the IpeOMT
enzymes. Three-dimensional images were reconstructed from
the confocal images, and the localization of GFP/YFP signals
from the transformed onion cells. Fluorescence profiles of
onion cells expressing each of the IpeOMT-GFPs and IpeGlu1-
GFP were the same as those expressing nontargeted GFP and
cytosol-targeted YFP but were totally different from those
expressing endoplasmic reticulum-targeted GFP and vacuole-
targeted YFP. These results showed the cytosolic localization of
IpeOMT and IpeGlu1 enzymes. This was supported by the
absence of signal peptides in IpeOMT and IpeGlu1 sequences
as predicted by the computer programs iPSORT, SignalP, and
was analyzed. Fig. 5 shows the representative sections obtained Target P.
MARCH 5, 2010•VOLUME 285•NUMBER 10
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Ipecac Alkaloid O-Methyltransferases
6,7-O,O-Dimethyl-N-deacetylisoipecoside was found to be
synthesized via 7-O-methyl-N-deacetylisoipecoside, because
none of the IpeOMTs catalyzed 7-O-methylation of 6-O-meth-
yl-N-deacetylisoipecoside but IpeOMT1 catalyzed the 6-O-
methylation of 7-O-methyl-N-deacetylisoipecoside. These
enzymatic properties seem to be the mechanism to effec-
tively prepare 6-O-methyl-N-deacetylisoipecoside to be
deglucosylated by IpeGlu1 for emetine biosynthesis. In fact,
6-O-methyl-N-deacetylisoipecoside is one of the best sub-
strates for IpeGlu1 among the glucosidal ipecac alkaloids
having an S-configuration, although its activity was
extremely poor toward 7-O-methyl-N-deacetylisoipecoside
and 6,7-O,O-dimethyl-N-deacetylisoipecoside (15).
Deglucosylation of 6-O-methyl-N-deacetylisoipecoside by
IpeGlu1 gives multiple products due to nonenzymatic intramo-
lecular reactions, of which the iminium cation formed by dehy-
dration of the immediate IpeGlu1 product was supposed to
FIGURE 6. Time course analysis of the transcript level of IpeOMT1,
IpeOMT2, IpeOMT3, and Ipeglu1 genes by quantitative real time RT-PCR
in P. ipecacuanha root culture. Precultured roots were transferred into new serve as intermediate for emetine biosynthesis (15). To examine
media, and the roots were harvested after each time period from 1 day to 8
the involvement of IpeOMTs in the other O-methylation reac-
weeks. Data are expressed as the mean of triplicate experiments with stand-
ard deviation.
tion (i.e. methylation of the 7-hydroxy group with respect to
isoquinoline skeleton) prior to protoemetine formation, we
tested synthetic 3-O-methylredipecamine (see supplemental
Fig. S1) as a mimic of endogenous 6-O-methyl-N-deacetyli-
soipecoside aglycon. That resulted in the detection of 2-O-
methylation activity of IpeOMT3 toward 3-O-methylredipe-
camine, which strongly supports the hypothesis that the
methylation of the 7-hydroxy group of the isoquinoline skele-
ton occurs after deglucosylation of 6-O-methyl-N-deacetyli-
soipecoside at a certain biosynthetic step leading to protoem-
etine (Fig. 7).
Transcript Profiles of IpeOMT and IpeGlu1 Genes in P. ipe-
cacuanha in Vitro Roots—Transcript profiles of the IpeOMT1,
IpeOMT2, and IpeOMT3 genes, as well as the Ipeglu1 gene,
were analyzed in P. ipecacuanha root culture to see whether the
three IpeOMT genes are coordinately transcribed with Ipeglu1.
Time course changes in the gene transcription in root cultures
were measured by quantitative RT-PCR analysis (Fig. 6).
Throughout the time course, transcript patterns of the three
IpeOMTs were well correlated with that of Ipeglu1, where tran-
script levels started to increase after subculture, reached max-
ima in 1–2 weeks, and then decreased to lower levels. The tran-
script level of IpeOMT3 was lower than the other two IpeOMTs
and Ipeglu1.
These results suggest that methylation of the 6-hydroxy
group before the 7-hydroxy group of the isoquinoline skeleton
is a common feature in the biosynthesis of isoquinoline alka-
loids. In the biosynthesis of the benzylisoquinoline alkaloids
papaverine, laudanine, and palmatine, 6-hydroxy group of iso-
quinoline skeleton is methylated in the early step by norcoclau-
DISCUSSION
Involvement of IpeOMTs in the Biosynthesis of Ipecac Alka- rine 6-OMT (16, 18, 22), and then the 7-hydroxy group is meth-
loids Derived from (1S)-N-Deacetylisoipecoside—Identification ylated in a later step by norreticuline 7-OMT (19), reticuline
of the three ipecac alkaloid O-methyltransferases, IpeOMT1, 7-OMT (16), and columbamine 2-OMT (21), respectively. In P.
IpeOMT2, and IpeOMT3, in this study, as well as the ipecac ipecacuanha, however, two sequential O-methylation steps
alkaloid ␤-glucosidase, IpeGlu1 (15), enabled us to elucidate a leading from 7Ј-O-demethylcephaeline, the condensation
major portion of the biosynthetic pathways to ipecac alkaloids. product of protoemetine and the second dopamine, to emetine
Although the EST data base of Psychotria root culture that was were performed in the opposite manner, where the 7Ј-hydroxy
constructed consisted of a relatively small number of ESTs group of 7Ј-O-demethylcephaeline was methylated first by the
(1050 members) (15), the results indicate that it is enriched in highly specific (Km ϭ 1.0 ␮M) IpeOMT2 to form cephaeline,
ipecac alkaloid biosynthetic genes.
followed by 6Ј-O-methylation of cephaeline to form the end
The elucidated pathway and the contribution of each of those product emetine by IpeOMT1 (Fig. 7). The catalytic efficiency
enzymes are shown in Figs. 7 and 8 for S- and R-forms of ipecac of IpeOMT1 toward cephaeline (3.5 sϪ1ꢀMϪ1) was much lower
alkaloids, respectively. Both IpeOMT1 and IpeOMT2 enzymes than that of IpeOMT2 toward 7Ј-O-demethylcephaeline
catalyzed the 6-O-methylation of N-deacetylisoipecoside, an (32,600 sϪ1ꢀMϪ1). These enzymatic properties may be the rea-
intermediate in emetine biosynthesis. Kinetic analysis showed son for accumulation of both cephaeline and emetine as major
that the reaction catalyzed by IpeOMT1 is 130 times as efficient ipecac alkaloids in P. ipecacuanha (1) despite the expression of
as that catalyzed by IpeOMT2 due to smaller Km and higher kcat IpeOMT1 and IpeOMT2 at the comparable levels (Fig. 6).
values, indicating that IpeOMT1 plays a major role in the for-
In addition to this central biosynthetic pathway of ipecac
mation of 6-O-methyl-N-deacetylisoipecoside. 7-O-Methyla- alkaloids, branch pathways were also elucidated. Because none
tion of N-deacetylisoipecoside was catalyzed by IpeOMT2, of the IpeOMT enzymes accepted the lactam species demeth-
although the reaction efficiency was much lower than the 6-O- ylisoalangiside, isoalangiside, and 3-O-demethyl-2-O-methyli-
methylation reaction catalyzed by IpeOMT1 and IpeOMT2. soalangiside as substrates, isoalangiside, 3-O-demethyl-2-O-
7734 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 10•MARCH 5, 2010

Ipecac Alkaloid O-Methyltransferases
FIGURE 7. Biosynthetic pathway of ipecac alkaloids having an S-configuration that derive from N-deacetylisoipecoside. The biosynthetic reactions identified
in this study (IpeOMTs) and IpeGlu1 (15) are shown. Bold arrows indicate emetine biosynthetic pathway. The detailed pathway leading to protoemetine from the
aglycon formed by IpeGlu1 has not yet been elucidated (shown by dashed line). Reactions not catalyzed by the IpeOMT enzymes are shown by ϫ.
methylisoalangiside, and methylisoalangiside were presumed way shown in Fig. 7. It is plausible that a certain amount of
to be synthesized by spontaneous lactamization of correspond- N-deacetylisoipecoside is deglucosylated by IpeGlu1 prior to
ing methylated N-deacetylisoipecoside as shown in Fig. 7. 9-O- 6-O-methylation by IpeOMT1 and IpeOMT2. Likewise, 10-O-
Demethylcephaeline and 10-O-demethylcephaeline have been demethylprotoemetine can be formed from 6-O-methyl-N-
isolated from Alangium (2), and 10-O-demethylcephaeline has deacetylisoipecoside aglycon. 9-O-Demethylprotoemetine and
also been found in Psychotria (30, Cephaelis in the literature). 10-O-demethylprotoemetine are probably condensed with do-
They are considered to be synthesized from 9-O-demethylpro- pamine, and the products are further methylated by IpeOMT2
toemetine and 10-O-demethylprotoemetine (Fig. 7). Consider- to form 9-O-demethylcephaeline and 10-O-demethylcepha-
ing that IpeGlu1 deglucosylates N-deacetylisoipecoside as effi- eline (Fig. 7).
ciently as 6-O-methyl-N-deacetylisoipecoside (15) and that
Involvement of IpeOMTs in the Biosynthesis of Ipecac Alka-
IpeOMT2 catalyzed 2-O-methylation of redipecamine, the syn- loids Derived from (1R)-N-Deacetylipecoside—Ipecac alkaloids
thetic mimic of the endogenous N-deacetylisoipecoside agly- derived from (1R)-N-deacetylipecoside is known to be accumu-
con, 9-O-demethylprotoemetine, is formed through the path- lated as alkaloidal glucosides such as ipecoside and alangiside
MARCH 5, 2010•VOLUME 285•NUMBER 10
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Ipecac Alkaloid O-Methyltransferases
7736 JOURNAL OF BIOLOGICAL CHEMISTRY
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Ipecac Alkaloid O-Methyltransferases
(5, 6). Their O-methyl derivatives (Fig. 8) have also been iden- is synthesized and acetylated in the vacuole. This is supported
tified (2). It was found herein that O-methylation reactions of by the fact that nonglucosidal ipecac alkaloid with an R-config-
the alkaloidal glucosides having an R-configuration are cata- uration has not been found so far in P. ipecacuanha. In P. ipe-
lyzed only by IpeOMT2 (Table 1), the catalytic property of cacuanha, however, 6-O-methylipecoside and 7-O-methylipe-
which revealed the biosynthetic pathway to the R-form ipecac coside are also accumulated (2). Because IpeOMT2 accepted
alkaloids (Fig. 8). IpeOMT2 activity for 6-O-methylation and N-deacetylipecoside but not ipecoside as substrate, 6-O-meth-
7-O-methylation of N-deacetylipecoside and the absence of ylipecoside and 7-O-methylipecoside are presumed to be
activity catalyzing 6-O-methylation and 7-O-methylation of formed via N-acetylation of 6-O-methyl-N-deacetylipecoside
ipecoside demonstrated that 6-O-methylipecoside and 7-O-meth- and 7-O-methyl-N-deacetylipecoside, respectively. For this to
ylipecosidearesynthesizedfrom6-O-methyl-N-deacetylipecoside occur, N-deacetylipecoside has to be transported into the
and 7-O-methyl-N-deacetylipecoside, respectively. Because none cytosol to react with IpeOMT2. N-Deacetylipecoside, however,
of the IpeOMTs catalyzed 3-O-methylation of demethylalangi- can then react with the cytosolic IpeGlu1, which exhibits a high
side, alangiside is considered to be formed by spontaneous lac- catalytic activity toward it. Moreover, the 6-O-methyl-N-
tamization of 6-O-methyl-N-deacetylipecoside. Meanwhile, 3-O- deacetylipecoside and 7-O-methyl-N-deacetylipecoside pro-
demethyl-2-O-methylalangiside is synthesized not only by the duced need to be transported into the vacuole again based on
lactamization of 7-O-methyl-N-deacetylipecoside but also by the the above-mentioned hypothesis that N-acetylation occurs in
enzymatic 2-O-methylation of demethylalangiside by IpeOMT2. the vacuole. Discrepancies in subcellular localization between
6,7-O,O-Dimethyl-N-deacetylipecoside and its lactam methyl- substrate and the corresponding enzyme in natural product
alangiside have not been found so far in P. ipecacuanha. This biosynthesis have also been reported for 2-O-␤-D-glucopyrano-
would be justified by the fact that none of the IpeOMTs catalyzed syl-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc)-metaboliz-
their formation (Fig. 8). However, we cannot exclude the possibil- ing enzymes in maize, where the substrate DIBOA-Glc was
ity that other OMTs that were not discovered in this study are considered to be sequestered in the vacuole, but two enzymes
involved in their formation, because methylalangiside has been (2-oxoglutarate-dependent dioxygenase and O-methyltrans-
isolated in Alangium (31, 32). Deep transcriptome sequencing ferase) metabolizing DIBOA-Glc were localized in cytosol (35).
would enable the complete identification of the remaining ipecac As exemplified by anthocyanin transport into the vacuole by a
alkaloid biosynthetic genes, including such OMT(s), as well as multidrug resistance-associated protein-type ATP-binding
those involved in the unidentified pathway leading to protoem- cassette transporter in maize (36, 37), the mechanism of vacu-
etine from 6-O-methyl-N-deacetylisoipecoside aglycon.
olar transport of plant secondary metabolites is becoming
Subcellular Network of Ipecac Alkaloid Biosynthetic Enzymes— unveiled (38). To reveal the subcellular network of ipecac alka-
It has been demonstrated that the terpenoid-indole alkaloid, loid biosynthetic enzymes, it is essential to identify such trans-
strictosidine, which is formed by condensation of tryptamine porters, as well as to determine subcellular localization of all
and secologanin, is synthesized and stored in the vacuole (33, biosynthetic enzymes.
34). Likewise, P. ipecacuanha N-deacetylisoipecoside and
Moreover, the cell-specific expression of the biosynthetic
N-deacetylipecoside that are formed by the condensation of genes must be taken into consideration. Immunofluorescence
dopamine and secologanin should be synthesized in the vacu- analysis of the morphine biosynthetic enzymes in opium poppy
ole. We demonstrated that IpeOMTs and IpeGlu1, which work P. somniferum demonstrated that in capsule and stem, 3Ј-hy-
toward N-deacetylisoipecoside and N-deacetylipecoside, are droxy-N-methylcoclaurine 4Ј-OMT, reticuline 7-OMT, and
localized in cytosol (Fig. 5). This means that N-deacetylisoipe- salutaridinol 7-O-acetyltransferase were found predominantly
coside and N-deacetylipecoside need to be transported outside in parenchyma cells within the vascular bundle, whereas
the vacuole to react with the subsequent enzymes. For emetine codeinone reductase is localized to laticifers (39). Accordingly,
biosynthesis, N-deacetylisoipecoside transported into the we can hypothesize that ipecac alkaloids having the S- and
cytosol immediately has to be captured by IpeOMT1 for 6-O- R-configuration would be synthesized in distinct types of cells;
methylation prior to deglucosylation by IpeGlu1. Despite co- the former cells need to co-express IpeOMTs and IpeGlu1 in an
localization of IpeOMT1 and IpeGlu1 in the cytosol, this may identical cell to complete the emetine biosynthesis, but the lat-
be possible because IpeOMT1 showed a considerably lower Km ter might express IpeOMT2 and an enzyme for N-acetylation
value (107 ␮M) for N-deacetylisoipecoside than did IpeGlu1 but not IpeGlu1 to avoid deglucosylation of N-deacetylipeco-
(5.8 mM) (15).
side and its IpeOMT2 products, as well as their acetylated
The biosynthetic process of glucosidal ipecac alkaloids hav- products.
ing an R-configuration appears to be more complicated. Con-
Functional Differentiation of IpeOMTs in the Evolution of
sidering that ipecoside is accumulated in P. ipecacunaha P. ipecacuanha—Because IpeOMTs catalyzed O-methylation
regardless of the presence of IpeGlu1 that exhibits the highest reactions of 6- and/or 7-hydroxy groups of the isoquinoline
catalytic activity toward ipecoside (15), ipecoside and IpeGlu1 skeleton of ipecac alkaloids, we expected that each of the three
must be localized in distinct subcellular compartments. Thus, IpeOMTs shows the closest relationship according to its regio-
we hypothesized that the R-diastereomer N-deacetylipecoside specificity to the known 6- and 7-OMTs for benzylisoquinoline
FIGURE 8. Biosynthetic pathway of ipecac alkaloids having an R-configuration that derive from N-deacetylipecoside. 6,7-O,O-Dimethyl-N-deacetylipe-
coside and the lactam methylalangiside not yet detected in Psychotria are shown in gray. Note that methylalangiside has been isolated in Alangium (31, 32).
Reactions not catalyzed by the IpeOMT enzymes are shown by ϫ.
MARCH 5, 2010•VOLUME 285•NUMBER 10
JOURNAL OF BIOLOGICAL CHEMISTRY 7737

Ipecac Alkaloid O-Methyltransferases
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and the clade is closer to the flavonoid OMTs and 16-hy-
droxytabersonine OMT than to those benzylisoquinoline alka-
loid OMTs (Fig. 2). Similarly, norreticuline 7-OMT showed a
closer relationship to norcoclaurine 6-OMT rather than to reti-
culine 7-OMT in P. somniferum (19). Pienkny et al. (19)
hypothesized that it would be because 7-OMT had been gener-
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Acknowledgments—We greatly thank Dr. Peter Spiteller (Technische
Universita¨t Mu¨nchen, Germany) for measuring NMR spectra. We are
grateful to Drs. Monica Schmidt and Howard R. Berg (Donald Dan-
forth Plant Science Center) for instrumental and technical support in
transient expression experiments and to Dr. Sona Pandey (Donald
Danforth Plant Science Center) for technical instruction in quantita-
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lor and Brian Kelly (Donald Danforth Plant Science Center) for the
gifts of reporter plasmids. We are grateful to Dr. Meinhart H. Zenk
(Donald Danforth Plant Science Center) for providing ipecoside,
cephaeline, kaempferol, myricetin, and benzylisoquinoline alkaloids.
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(Donald Danforth Plant Science Center) and of 7Ј-O-demethylcepha-
eline from Dr. Takao Tanahashi (Kobe Pharmaceutical University,
Japan). We also thank the Tissue Culture Facility of Donald Danforth
Plant Science Center for maintaining the root cultures of P.
ipecacuanha.
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7738 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 10•MARCH 5, 2010

Plant Biology:
Three New O-Methyltransferases Are
Sufficient for All O-Methylation Reactions
of Ipecac Alkaloid Biosynthesis in Root
Culture of Psychotria ipecacuanha
Taiji Nomura and Toni M. Kutchan
J. Biol. Chem. 2010, 285:7722-7738.
doi: 10.1074/jbc.M109.086157 originally published online January 8, 2010
Access the most updated version of this article at doi: 10.1074/jbc.M109.086157
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