Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells

Article abstract of DOI:10.1016/j.phytochem.2012.02.013

Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common intermediate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.), stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine 6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia californica cells, based on an integrated analysis of metabolites and transcript expression profiles of transgenic cells with Coptis japonica scoulerine-9-O- methyltransferase. Using the full-length candidate cDNA for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characterization. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated that 6-hydroxylation played a role in the reaction.

Full text of DOI:10.1016/j.phytochem.2012.02.013

Phytochemistry xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Molecular cloning and characterization of a cytochrome P450 in sanguinarine
biosynthesis from Eschscholzia californica cells q,qq
Tomoya Takemura a, Nobuhiro Ikezawa a,1, Kinuko Iwasa b, Fumihiko Sato a,
⇑
a Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan
b Kobe Pharmaceutical University, Kobe, Hyogo 658-8558, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common interme-
diate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in
the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.),
stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine
6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia
californica cells, based on an integrated analysis of metabolites and transcript expression profiles of trans-
genic cells with Coptis japonica scoulerine-9-O-methyltransferase. Using the full-length candidate cDNA
for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characteriza-
tion. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference
spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and
allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated
that 6-hydroxylation played a role in the reaction.
Keywords:
Eschscholzia californica
Papaveraceae
Benzylisoquinoline alkaloids
Cytochrome P450
Protopine 6-hydroxylase
Sanguinarine biosynthesis
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
metabolic diversification (Takemura et al., 2010). In this, the most
critical step would be oxidative steps catalyzed by cytochrome
Isoquinoline alkaloids are a large group of alkaloids that include
many pharmacologically useful compounds, e.g., the analgesic
morphine, the antitussive codeine, and the antimicrobial agents
berberine and sanguinarine. These various types of isoquinoline
alkaloids (morphinans, protoberberines, and benzophenanthri-
dines) are biosynthesized from a central precursor, (S)-reticuline
(1) (Fig. 1) (Preininger, 1986; Kutchan, 1998; Sato et al., 2007; Zie-
gler and Facchini, 2008). Although the molecular process of this
chemical diversity has not yet been clarified, recent studies have
shown that addition of a branch pathway and relatively broad sub-
strate-specificity of endogenous enzymes may be involved in the
P450 (P450). P450s have been shown to play essential roles in
plant secondary metabolism (Chapple, 1998; Werck-Reichhart
et al., 2002; Mizutani and Sato, 2010).
California poppy (Eschscholzia californica), a Papaveraceae plant,
is a traditional medicinal plant of Native Americans and has been
intensively investigated because of the variety and pharmacologi-
cal effects of its alkaloids. While E. californica is known to produce
aporphine-, pavine-, protoberberine-, protopine-, and benzophe-
nanthridine-type alkaloids (Kutchan, 1998; Fabre et al., 2000),
the biosyntheses of benzophenanthridine-type alkaloids has been
most intensively studied at the enzyme level (Zenk, 1994; Kutchan,
1998). Biosynthesis of the major metabolite, sanguinarine (8), re-
quires seven reaction steps from (S)-reticuline (1), including the
four P450 reaction steps of two methylenedioxy bridge-forming
(cheilanthifoline synthase (CYP719A5) [EC 1.14.21.2.] and stylo-
pine synthase (CYP719A2/A3) [EC 1.14.21.1.]), N-methylstylopine
hydroxylase (MSH), and protopine 6-hydroxylase (P6H; EC
1.14.13.55.), respectively (Fig. 1).
Abbreviations: CjSMT, Coptis japonica (S)-scoulerine-9-O-methyltransferase;
CYP719A2/A3, stylopine synthase; CYP719A5, cheilanthifoline synthase; EST,
expressed sequence tag; LC–MS, liquid chromatography–mass spectrometry; P6H,
protopine 6-hydroxylase; P450, cytochrome P450; (S)-THB, (S)-tetrahydroberber-
ine; (S)-THC, (S)-tetrahydrocolumbamine.
q
This manuscript is dedicated to the late Prof. Meinhart Zenk for his great
contributions on isoquinoline alkaloid biosynthesis studies.
qq
In benzophenanthridine alkaloid biosynthesis, protopine 6-
hydroxylase (P6H) converts protopine (5) to dihydrosanguinarine
(7) (Tanahashi and Zenk, 1990). Tanahashi and Zenk (1990) charac-
terized P6H using [6-3H] protopine (5) and microsomal fractions of
elicitor-treated cultured E. californica cells, and measured the re-
Nucleotide sequence of full-length cDNA of EcCYP82N2v2 (AB598834) was
deposited in the DDBJ/Genbank/EMBL database.
⇑
Corresponding author. Tel.: +81 75 753 6381; fax: +81 75 753 6398.
E-mail address: fsato@lif.kyoto-u.ac.jp (F. Sato).
Present address: Department of Biotechnology, Graduate School of Engineering,
1
Osaka University, Suita, Osaka 565-0871, Japan.
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2012.02.013
Please cite this article in press as: Takemura, T., et al. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Esc-
hscholzia californica cells. Phytochemistry (2012), doi:10.1016/j.phytochem.2012.02.013

2
T. Takemura et al. / Phytochemistry xxx (2012) xxx–xxx
MeO
NMe
HO
HO
lease of 3H from the substrate through formation of dihydrosan-
H
guinarine (7). It was estimated therein that the 6-hydroxylation
of protopine (5) and further non-enzymatic intramolecular rear-
rangement produced dihydrosanguinarine (7) from 6-hydroxypro-
topine (6), since no stable intermediate was detected.
MeO
(S)-reticuline
In this report, the isolation of cDNA of a P450 is described, which
converts protopine (5) to dihydrosanguinarine (7), from cultured E.
californica cells based on an integrated analysis of metabolites and
transcript expression profiles (Takemura et al., 2010). The candi-
date P450 (CYP82N2v2) was heterologously expressed in Saccharo-
myces cerevisiae and a recombinant microsomal protein was used
for the P6H assay. Further characterization of the substrate-speci-
ficity supports a unique reaction characteristic of CYP82N2v2. The
potential role of the broad substrate-specificity of CYP82N2v2 is
discussed from the perspective of metabolic diversification.
BBE
MeO
HO
N
H
OH
OMe
(S)-scoulerine
CjSMT
CYP719A5
MeO
HO
MeO
HO
N
N
H
H
2. Results
OMe
OMe
O
O
2.1. Isolation of P6H cDNA candidate
(S)-cheilanthifoline
CYP719A2/A3
(S)-THC
In a previous study (Takemura et al., 2010), the relationship be-
tween the accumulation of metabolite and biosynthetic gene
expression in transgenic E. californica cells was reported with ecto-
pic expression of scoulerine-9-O-methyltransferase of Coptis japon-
ica. Enhanced variation of gene expression of biosynthetic enzymes
provided a considerable variation of metabolites, and a good corre-
lation was found between the gene expression of biosynthetic en-
zymes and metabolites (Takemura et al., 2010). An EST clone
(EcCYP-A in Takemura et al., 2010) identified from four indepen-
dent putative P450 sequences from an EST library of California
poppy (http://pgn.cornell.edu/index.pl) showed a good negative
correlation with the accumulation of allocryptopine (11), which
is a potential substrate for protopine 6-hydroxylase in transgenic
E. californica cells with the overexpression of CjSMT (Fig. 1). This
means that EcCYP-A might be protopine 6-hydroxylase, the inhibi-
tion of which induces the accumulation of protopine (5) in biosyn-
thesis. Since EcCYP-A only contained the N-terminal sequence of
P450, a full-length cDNA was isolated from transgenic cultured
California poppy cells using 30 RACE.
O
O
O
O
N
H
N
H
OMe
OMe
O
O
(S)-stylopine
(S)-THB
TNMT
MSH
O
O
O
O
Me
N
Me
N
O
O
OMe
OMe
O
O
protopine
allocryptopine
CYP82N2v2
(P6H)
OH
O
OH
Me
O
Me
N
N
O
O
O
O
OMe
O
Isolated cDNA clone of EcCYP-A contained 1572 nucleotides
with an open reading frame of 514 amino acids (DDBJ/Gen-
Bank™/EMBL Accession No. AB598834). The predicted amino acid
sequence had conserved eukaryotic P450 domains: a helix K re-
gion, an aromatic region, and a heme-binding region. The P450
6-hydroxy- O
protopine
6-hydroxy- OMe
allocryptopine
O
O
nomenclature
committee
(http://drnelson.uthsc.edu/Cyto-
O
O
Me
N
Me
N
chromeP450.html) named it CYP82N2v2, since CYP82N2v1 with
only eight amino acid sequence difference had been registered in
P450 nomenclature database. CYP82N2v2 also had a hydrophobic
endoplasmic reticulum sorting signal at the N-terminal region.
CYP82N2v2 was compared with other P450s, especially those in
isoquinoline alkaloid biosynthesis (BsCYP80A1, EcCYP80B1,
CjCYP80G2, EcCYP719A2/A3, EcCYP719A9, and PsCYP719B1)
(Kraus and Kutchan, 1995; Pauli and Kutchan, 1998; Ikezawa
et al., 2007, 2008, 2009; Gesell et al., 2009). CYP82N2v2 was dis-
tant from the CYP719 and CYP80 families on the phylogenetic tree.
CYP82N2v2 had the highest sequence similarity to AtCYP82C2
(45% identity), followed by AtCYP82G1 (42% identity) and NtCY-
P82E4v1 (39% identity) among the functionally-characterized
P450s (Fig. 2). Among P450s that are known to play a role in iso-
quinoline alkaloid biosynthesis, CYP82N2v2 showed the highest
sequence similarity (43% identity) to CYP80B1 (N-methylcoclau-
rine-30-hydroxylase). CYP82N2v2 also had highly conserved Gly/
Ala residues in the helix I region, whereas the CYP719 family, un-
ique to isoquinoline biosynthesis, has less-conserved residues in
this region (Mizutani and Sato, 2010; Fig. 3).
OMe
OMe
O
O
dihydro-
sanguinarine
dihydro-
chelerythrine
DHBO
O
O
O
O
Me
N
Me
N
+
+
OMe
OMe
O
O
sanguinarine
chelerythrine
Fig. 1. Biosynthetic pathways for isoquinoline alkaloids. Both native and metabol-
ically modified pathways in transgenic E. californica cells with CjSMT are shown.
Down-regulation of P6H (CYP-A/CYP82N2v2) induced the accumulation of allo-
cryptopine in transgenic E. californica cells (Takemura et al., 2010). BBE: berberine
bridge enzyme, CYP719A5: cheilanthifoline synthase, SMT: scoulerine-9-O-meth-
yltransferase, CYP719A2 and CYP719A3: stylopine synthase, TNMT: tetrahydropro-
toberberine-N-methyl transferase, MSH: methylstylopine hydroxylase, P6H:
protopine-6-hydroxylase, DHBO; dihydro-benzophenanthriddine oxidase.
Please cite this article in press as: Takemura, T., et al. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Esc-
hscholzia californica cells. Phytochemistry (2012), doi:10.1016/j.phytochem.2012.02.013

T. Takemura et al. / Phytochemistry xxx (2012) xxx–xxx
3
P80B1
Y
2
G
EcC
0
P8
CY
Cj
9B2
7
CYP
At
95
90
45
93
39
96
56
58
50
96
99
99
100
35
90
100
92
100
49
Cr
C
YP72
A1
0.2
Fig. 2. Phylogenetic tree of plant cytochrome P450 families. Plant cytochrome P450 amino acid sequences obtained from GenBank or SwissProt were used for tree building.
Accession Nos. are: NM101040, Arabidopsis thaliana AtCYP51G1 (sterol 14-demethylase); AF346833, Mentha x piperita MmCYP71A32 (menthofuran synthase); AY532370,
Ammi majus AmCYP71AJ1 (psoralen synthase); L10081, Catharanthus roseus CrCYP72A1 (secologanin synthase); Y09920, Helianthus tuberosus HtCYP76B1 (7-ethoxycoumarin
O-deethylase); AF069495, Arabidopsis thaliana AtCYP79B2 (conversion of amino acid to aldoxime); U09610, Berberis stolonifera BsCYP80A1 (berbamunine synthase);
DQ387048, Hyoscyamus niger HnCYP80F1 (littorine mutase/monooxygenase); AB288053, Coptis japonica CjCYP80G2 (corytuberine synthase); AB194714, Sesamum indicum
SiCYP81Q1 (two methylenedioxy bridge formation); O49394, Arabidopsis thaliana AtCYP82C2 (monooxygenase); DQ131886, Nicotiana tabacum NtCYP82E4v1 (nicotine N-
demethylase); NM113423 Arabidopsis thaliana AtCYP82G1 (DMNT/TMTT homoterpene synthase); DQ374443, Lycopersicon esculentum LeCYP85A1 (6-deoxocastasterone
oxidase); AF212991, Cucurbita maxima CmCYP88A2 (ent-kaurenoic acid oxidase); AY072297 Zea mays ZmCYP92A1 (monooxygenase); AB023636, Glycyrrhiza echinata
GeCYP93C2 (2-hydroxyisoflavanone synthase); AB017418, Lithospermum erythrorhizon LeCYP98A6 (4-coumaroyl-40-hydroxyphenyllactic acid 3-hydroxylase); AF212990,
Cucurbita maxima CmCYP701A1 (ent-kaurene oxidase); BAB11063, Arabidopsis thaliana AtCYP705A5 (flavonoid 30-monooxygenase); NM129002, Arabidopsis thaliana
AtCYP710A1 (sterol C-22 desaturase); EF451150, Papaver somniferum PsCYP719B1 (salutaridine synthase); AF014801, Eschscholzia californica EcCYP80B1 ((S)-N-
methylcoclaurine 30-hydroxylase); AB026122 Coptis japonica CjCYP719A1 (canadine synthase); AB126257 Eschscholzia californica EcCYP719A2 (stylopine synthase);
AB126256 Eschscholzia californica EcCYP719A3 (stylopine synthase); AB434655 Eschscholzia californica EcCYP719A9 (involved in alkaloid biosynthesis). The branch length is
proportional to the estimated divergence distance of each protein. The scale bar (0.2) corresponds to a 20% change. The percentage of replicate trees in which the associated
taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches.
2.2. P450 nature of CYP82N2v2
showed a reduced CO-difference that was characteristic of a
P450, which had a peak at 450 nm (Fig. 4). The CYP82N2v2 con-
tent in the microsomal fraction was estimated to be 80 pmol
P450 per mg microsomal protein.
To determine the P450 nature of CYP82N2v2, its expression
vector was constructed and introduced into S. cerevisiae. The
microsomal fraction prepared from transgenic S. cerevisiae cells
Please cite this article in press as: Takemura, T., et al. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Esc-
hscholzia californica cells. Phytochemistry (2012), doi:10.1016/j.phytochem.2012.02.013

4
T. Takemura et al. / Phytochemistry xxx (2012) xxx–xxx
EcCYP82N2v2 - - - MD S L M L A Y L F P I S V A S I I A F V F L YN L F S SR T - L KN K K I R T A PMA TG AWP V L GH L H L F 56
AtCYP82G1
- - - M T F L F S T L Q L S L F S L A L V I FG Y I F L R KQ L SR - C E VD S S T I P - E P L G A L P L FGH L H L L 55
NtCYP82E4v1 - - - M V F P I E A I VG - - - - - - L V T F T F L F F F L WT K K - SQ K P S K P L P P K I PGGWP V I GH L FH F 50
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
- - - - - - - - - M E V V T V A L I A V I I S S I L Y L L FG S SG - H KN - - - - - L P PG P K PWP I VGN L L Q L 45
- - - - - - - - - MD Y I - VG F V S I S L V A L L Y F L L F K P K - H TN - - - - - L P P S P P AWP I VGH L PD L 44
- - - - - - - - - MD L Q - I A L F S L I P V I L V F I L L L K P K - Y KN - - - - - L P PG PH PWP L I GN L P I L 44
M E EM K I L MMN N PW I L T A T A T T L L I S I F L F F TR K - - - - - S S KM VWP AG P K T L P I I GNMH L L 55
M A P I N I EG - N D F WM I AC T V I I V F A L V K FMF S K I S F YQ S AN T T EWP AG P K T L P I I GN L HQ L 59
EcCYP82N2v2 G SG - - - E L PH KM L A AM AD K YG S A FRMK FG - KH T T L V V SD TR I V K EC F T TND T L F SNR P S T 112
AtCYP82G1 R G K - - - K L L C K K L A AM SQ KHG P I F S L K L G - F YR L V V A SD P K T V KDC F T TND L A T A TR PN I 111
NtCYP82E4v1 NDDGDDR P L AR K L GD L AD K YG P V F T FR L G - L P L V L V V S S Y E A V KDC F S TND A I F SNR P A F 109
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
G E K P - - - - - H AQ F A E L AQ T YGD I F T L KMG - T E T V V V A S T S S A A S E I L K THDR I L S AR Y V F 99
I S KN S - P P F L D YM SN I AQ K YG P L I H L K FG - L H S S I F A S T K E A AM E V L Q TND K V L SGRQ P L 102
F T N T E - V P L H I T L ANM AR THG P I M I L WL G - TQ P T VMA S T A E A AM E I L K THDR I F S ARH I R 102
GG - - - - T A L Q V V L HN L A K VHG S VMT I W I G SWR P V I V V SD I ER AWE V L VN K S SD Y S ARDM P 111
GGG - - - V P L Q V A L AN L A K V YGG A F T I W I G SWV PM I V I SD I DN AR E V L VN K S AD Y S ARD V P 116
EcCYP82N2v 2 K A F Q L M T YDN E S V A F T P YG S YWR E I R K I S T L K L L SNHR L Q A I KD VR A S E VN VC F K T L YDQ 172
AtCYP82G1 A F GR Y VG YNN A S L T L A P YGD YWR E L R K I V T VH L F SNH S I EML GH I R S S E VN T L I KH L Y K - 170
NtCYP82E4v 1 L YGD Y L G YNN AM L F L AN YG P YWR KNR K L V I Q E V L S A SR L E K F KH VR F AR I Q A S I KN L Y TR 169
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
Q S F R V KGH V EN S I VWSDC T E TWKN L R K VCR T E L F TQ KM I E SQ AH VR E K KC E EM V E Y L MK K 159
PC F R I K PH I D Y S I L WSD SN S YWK KGR K I L H T E I F SQ KM L Q AQ E KNR ER V AGN L VN F I M T K 162
M S F R L KHH I K Y S L VWSDC TD YWK L L R K I VR T E I F S P KM L Q AQ SH VR EQ K V A E L I D F L R S K 162
D I T K I I S ADWK T I S T SD SG PHWTN L R KG L QN V A L S PHN L A AQ FQ FQ E KDM T KM I Q T L E E E 171
D I L K I I T ANG KN I ADCD SG P FWHN L K KG L Q - SC I N P SN VM S L SR L Q E KDMQN L I K SMQ ER 175
EcCYP82N2v2 C KN P SG S A P I L I DM K KWF E E V SNN V VMR V I VGRQN FG S K I VQG E E E A I H Y K K VMD E L L R L 232
AtCYP82G1 GN GG T S - - - - I V K I DM L F E F L T FN I I L R KM VG KR - - - I G FG E VN SD EWR Y K E A L KHC E Y L 223
NtCYP82E4v 1 I D GN S S - - - - T I N L TDWL E E L N FG L I V KM I AG KN - - - Y E SG KGD EQ V ER F K K A F KD FM I L 222
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
QG E - - - - - - - E V K I V E V I FG T L VN I FGN L I F SQN - - I F E L G X PN SG S S E F K E Y L WRM L E L 210
VGD - - - - - - - V V E L R SWL FGC A L N V L GH V V F S KD - - V F E Y S - DQ SD E VGMD K L I HGM L M T 212
EGQ - - - - - - - V V K I SQ F V FG T L L N I L GN V V F S KD - - V F V YG - D E TD KGG I QN L I R EM L M I 212
AR N N N G - - - - I V K P L DHM K K A T L R L I SR L V FGQD - - - - - - FNND K Y VDDMH L A I E E L I R V 221
A SQH N G - - - -
I I K P L DH A K E A SMR L L SR V I FGHD - - - - - - F SN ED L V I G V KD A L D EM VR I 225
EcCYP82N2v 2 A S L SM F SD - - F A P L L G F VD I FQGN L S AM KRN A K K VD A I L ENWL E EHR K K KN S V A E SQQ - - 288
AtCYP82G1 A V I PM I GD - - V I PWL GWL D - F A KN SQ - M KR L F K E L D S VN T KWL H EH L K KR SRN E KDQ E - - 277
NtCYP82E4v 1 SM E F V L WD A F P I P L F KWVD - FQGH V K AM KR T F KD I D S V FQNWL E EH I N KR E KM E VN A EGN 281
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
GN S T N P AD - - Y F PM L G K FD - - - - L FGQR K E V A EC L KG I Y A I WG AM L Q ER K L A K K VDG YQ S 264
GGD F D V A S - - Y F P V L AR FD - - - - L HG L KR KMD EQ F K L L I K I WEG E V L ARR ANRN P E - - - - 262
G A E PN V A E - - F Y P S L E E L D - - - - L QG L K K KCD ER F I R VMKMWEG T V K ER K ANRN E E - - - - 262
SG Y AR L A E - - A F Y Y A K Y L P SH K K A VR E V E E AQRR VQN L V S P F L S L N P P TN - - - - - - - - - - 269
SG L A S L AD - - A F K I A K Y L P SQR KN I RDM Y A TRDR V YN L I Q PH I V PN L P AN - - - - - - - - - - 273
EcCYP82N2v 2 - - D F MD VM L S I V - E E S K L SGHD AD A V I K A TC L AM I MGG TD T T A V S L TW I I S L L MNNRH A L 345
AtCYP82G1 - R T I MD L L L D I L P ED I V I SGH VRD V I V K A T I L A L T L TG SD S T S I T L TWA V S L L L NN P A A L 336
NtCYP82E4v 1 EQD F I D V V L S KM SN E Y L G EG Y SRD T V I K A T V F S L V L D A AD T V A L H I NWGM A L L I NNQ K A L 341
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
KN D F VD VC L D SG L ND YQ I N - - - - - - - - - A L L M E L FG AG T E T S A S T I EWAM T E L T KN P K I T 315
P KD M L D V L I AND FN EHQ I N - - - - - - - - - AM FM E T FG PG SD TN SN I I EWA L AQ L I KN PD K L 313
S KD M L D V L L AND FND AQ I N - - - - - - - - - A L F L E T FG PG S E T S S A T I EWV I A E L I K S P K EM 313
- - T Y L H F L R SQ K YDD E V I
I - - - - - - - - - F A I F E A Y L L G VD S T S L T T AWA L A F L I R E PN VQ 318
- - S F L Y F L T SQD Y SD E I I Y - - - - - - - - - SM V L E I FG L G VD S T A A T A VWA L S F L VG EQ E I Q 322
EcCYP82N2v 2 K K AR E E L D - - A L VG KDRQ V ED SD L KN L V YMN A I V K E TMRM Y P L G T L L - ER E T K EDC E I DG 402
AtCYP82G1 E A AQ E E I D - - N S VG KGRW I E E SD I QN L K Y L Q A I V K E THR L Y P P A P L TG I R E AR EDC F VGG 394
NtCYP82E4v 1 T K AQ E E I D - - T K VG KDRWV E E SD I KD L V Y L Q A I V K E V L R L Y P PG P L L V PH EN V EDC V V SG 399
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
A K L R S E L Q - - T V VG - ER S V K E SD F PN L P Y L E A T V K E T L R L H P P T P L L L PRR A L E TC T I L N 372
A K L R E E L D - - R V VGR S S T V K E SH F S E L P Y L Q AC V K E TMR L Y P P I S I M I PHRCME TCQ VMG 371
A K VR K E L N - - E V VG - T S T I K E SD L PQ L P Y L Q AC I K E AMR L H P A A P F L L PRR A A E TC E VMG 370
E K L YQ E L E S F A S KNDRR I L K V ED I N K L Q Y L Q A V I K E TMRM K P I A P L A I PH K ACRD T S L MG 378
E K L YR E I N - - NR TGGQR P V K V VD L K E L P Y L Q A VM K E T L RM K P I A P L A V PH V A A KD T T F KG 380
EcCYP82N2v2 FH V KGG TR L L VN VWK L QRD PN VWVD P T E FR P ER F L T EN AD I D VGG - - - - QH F E L L P FG AG 458
AtCYP82G1 YR V E KG TR L L VN I WK L HRD P K I WPD P K T F K P ER FM ED - - K SQC E K - - - - SN F E Y I P FG SG 448
NtCYP82E4v 1 YH I P KG TR L F AN VMK L QRD P K L WSD PD T FD P ER F I A T - - D I D FRG - - - - Q Y Y K Y I P FG SG 453
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
Y T I P KDCQ I M VN AWG I GRD P K TW I D P L T F S P ER F L N S - - S VD FRG - - - - ND F S L I P FG AG 426
Y T I P KGMD VH VN AH A I GRD P KDWKD P L K FQ P ER F L D S - - D I E YNG - - - - KQ FQ F I P FG SG 425
Y T I P KN SQ V L VN A Y A I GRD P K SWKD P S T FWP ER F L E S - - D VD FHG - - - - AH YQ F I P FG SG 424
K K I D KG TR VM VN I F A L HHN KN V FND P F K FM P ER FM K - VD SQD ANG - - K AM EQ S L L P F S AG 435
R R I V KG T K VM VN L Y A I HHD PN V F P A P Y K FM P ER F L KD VN SDGR FGD I N TM E S S L I P FG AG 440
EcCYP82N2v2 RR VC PG V X F A L Q FMH L V L AR L I HG YD L N T L N E EN VD L T E S P EGH VNH K A S P L D L I L T PR L 518
AtCYP82G1 RR SC PG VN L G L R V VH F V L AR L L QG F E L H K V SD E P L DM A EG P - G L A L P K I N P V E V V VM PR L 507
NtCYP82E4v 1 RR SC PGM T Y A L Q V EH L TM AH L I QG FN YR T PND E P L DM K EG A - G I T I R K VN P V E L I I A PR L 512
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
RR I C PG L P I ANQ F I A L L V A T F VQN L DWC L PN GM S VDH L I V E E K FG L T L Q K E P P L F I V P K S 486
RR I C PGR P L A VR I I P L V L A S L VH A FGWE L PDG V PN E K L DM E E L F T L S L CM A K P L R V I P K V 485
RR TC VGM P L A TR T I P L I VG S L VHN YD FG L PGGNR P ED L KMN EM L S L T L A I D P S L C V V P K A 484
MR I C AGM E L G K L Q F S F A L AN L A Y A F KWSC V ADG V L PDM SDQ L G F V L L MK T P L E AR I NRRN 495
MR I CGG V E L A KQM V A F A L A SM VN E F KWD C V S EG K L PD L S E A I S F I L YMKN P L E A K I T PR T 500
EcCYP82N2v 2 H Y K L Y E -
AtCYP82G1 D P K L Y S L L 515
NtCYP82E4v 1 A P E L Y - - 517
R V - - - - - - 488
-
524
-
EcCYP80B1
BsCYP80A1
Cj CYP80G2
EcCYP719A2
PsCYP719B1
R I
-
- - - -
-
-
487
486
R A -
-
- - -
- - - - - - - - 495
K P F R Q - - 505
-
Fig. 3. Amino acid sequence alignment of CYP82N2v2 and related P450s. Boxes indicate the conserved regions of eukaryotic P450; i.e., the helix K region, aromatic region, and
heme-binding region. Underline indicates consensus sequence in the Helix I region. Asterisk indicates conserved Gly/Ala, open circle indicates conserved Thr, which are
conserved in monooxygenase-type P450. EcCYP80B1, BsCYP80A1, CjCYP80G2, AtCYP82G1, NtCYP82E4v1, EcCYP719A2, and PsCYP719B1 are (S)-N-methylcoclaurine-30-
hydroxylase (Accession No. AF014801), berbamunine synthase (Accession No. P47195), corytuberine synthase (Accession No. BAF80448), homoterpene synthase
(NM113423), nicotine N-demethylase (DQ131886), stylopine synthase (Accession No. AB126257), and salutaridine synthase (Accession No. ABR14720), respectively. Highly
conserved amino acid residues are shown in white in black box-shading, while similar amino acid residues are shown in black in gray box-shading.
Please cite this article in press as: Takemura, T., et al. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Esc-
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T. Takemura et al. / Phytochemistry xxx (2012) xxx–xxx
5
2.5. Enzyme kinetics of CYP82N2v2
0.02
0.01
0
To understand the physiological role of CYP82N2v2 in California
poppy cells, the substrate affinities for protopine (5) and allocryp-
topine (11) were determined using recombinant microsomal frac-
tions. CYP82N2v2 was reacted with different concentrations of
substrates and product formation was quantified by HPLC. The
CYP82N2v2 reaction followed Michaelis–Menten-type reaction
kinetics and the apparent km values for protopine (5) and allocryp-
topine (11) were estimated to be 6.45 and 8.70
lM, with estimated
Vmax values of 24.5 and 23.0 pkat/ g P450, respectively (Supple-
l
mentary Fig. S3). The P450 content was estimated from the CO-dif-
ference spectrum. These data suggest that CYP82N2v2 can convert
both protopine (5) and allocryptopine (11) to the corresponding
benzophenanthridine-type alkaloids at comparable rates, as shown
in transgenic California poppy cells with CjSMT, which efficiently
metabolized the new product produced by CjSMT (see Fig. 1). In
comparison with other P450s in the biosynthetic pathway for iso-
quinoline alkaloids, CYP82N2v2 has a higher km value than that of
-0.01
400
420
440
460
480
500
wavelength (nm)
Fig. 4. Reduced CO-difference spectra of CYP82N2v2.
A microsomal fraction
containing CYP82N2v2 was prepared from transgenic S. cerevisiae and used for
reduced CO-difference spectrum measurement at 1.65 mg protein/ml. Spectra of
the microsomal fraction from S. cerevisiae transformed with empty vector (dotted
line) and CYP82N2v2-expressing S. cerevisiae (solid line) are shown.
CYP719A1
for
(S)-tetrahydrocolumbamine
((S)-THC)
(9)
(0.269 M), and a lower km value than that of CYP80B1 for (S)-N-
methylcoclaurine (15 lM).
l
2.3. Enzymological activity of CYP82N2v2
To determine the enzymological activity of CYP82N2v2, the
microsomal fraction was reacted with either protopine (5) or allo-
cryptopine (11) as a substrate under P450 assay conditions. Re-
combinant CYP82N2v2 clearly showed protopine (5) or
allocryptopine (11) conversion activity to produce dihydrosan-
guinarine (7) or dihydrochelerythrine (13), respectively (Fig. 5).
The identities of the products were confirmed by direct compari-
son with authentic standards (Fig. 5 and Supplementary Fig. S1).
During the experiments, an intermediate, such as 6-hydroxyproto-
pine (6) from protopine (5), was not detected even in the single ion
mode in LC–MS.
To understand the reaction properties of CYP82N2v2, the
dependence of the reaction on NADPH and oxygen was deter-
mined. As shown in Table 1, the absence of NADPH or removal of
O2 from the reaction mixture using a glucose/glucose oxidase/cat-
alase system (Bauer and Zenk, 1991) clearly inhibited the activity
of CYP82N2v2. Furthermore, ketoconazole, a known P450 inhibi-
tor, also inhibited its activity. These results clearly indicated that
CYP82N2v2 reacted as P450 to convert protopine (5) or allocrypto-
pine (11) to dihydrosanguinarine (7) or dihydrochelerythrine (13),
respectively.
3. Discussion
3.1. Isolation of cDNA of a key P450, CYP82N2v2, in
benzophenanthridine biosynthesis
The conversion of protopine (5) to dihydrosanguinarine (7) is
the crucial step in benzophenanthridine alkaloid biosynthesis. In
this study, it was determined that CYP82N2v2 catalyzed conversion
of protopine (5) to dihydrosanguinarine (7) by the P450 reaction.
CYP82N2v2 had conserved eukaryotic P450 regions i.e., helix K re-
gion, aromatic region and heme-binding region at the C-terminal
end (Fig. 3), and depended on NADPH/O2 (Table 1). Whereas some
P450s in isoquinoline alkaloid biosynthesis such as CYP719 and
CYP80G2 have unique substitutions at a conserved Gly/Ala residue
in the helix I region, EcCYP82N2v2 has conserved Gly, suggesting
that EcCYP82N2v2 would basically function as a monooxygenase-
type enzyme. Conserved Gly is also found in AtCYP82C2 (Accession
No. NM119348), which functions as methoxypsoralen hydroxylase
(Kruse et al., 2008), whereas AtCYP82G1 (Accession No.
NM113423), which functions as homoterpene synthase for oxida-
tive degradation of geranyllinalool/nerolidol (Lee et al., 2010),
has a Thr residue, and CYP82E4v1 (Accession No. DQ131886),
which functions as nornicotine synthase for demethylation (Simi-
nszky et al., 2005), has an Asp residue (Fig. 3). While several mem-
bers of the CYP80 and CYP719 families have been reported in
isoquinoline biosynthesis, CYP82N2v2 is the first gene in the
CYP82 family that has been shown to be involved in isoquinoline
alkaloid biosynthesis. It would be interesting to determine how
CYP82N2v2 evolved, since the CYP719 family is unique to isoquin-
oline alkaloid biosynthesis. Since some P450 genes are clustered in
the genome (Shimada et al., 2003), characterization of the genome
structure of isoquinoline alkaloid-producing plants would be
useful.
2.4. Substrate specificity of CYP82N2v2
To obtain more insight about the reaction mechanism of
CYP82N2v2, the substrate-specificity of CYP82N2v2 was examined
using several types of alkaloids at 10 lM (Supplementary Fig. S2).
Among protopine-type alkaloids, corycavine (15) and 13-oxoproto-
pine (17) (see Fig. 6 for structure) in addition to protopine (5) and
allocryptopine (11) were converted by CYP82N2v2 to give new
products.
The reaction product of corycavine (15) was determined to be
corynoloxine (16) by direct comparison with a standard compound
using LC–MS (Fig. 6A–C). On the other hand, the reaction product
of 13-oxoprotopine (17) by CYP82N2v2 showed a reduction of
2 m/z by LC–MS (Fig. 6D and E, Supplementary Fig. S4). Among pro-
topine-type substrates, protopine-N-oxide (18, Supplementary
Fig. S2) did not show any new product when reacted with
CYP82N2v2. Simple benzylisoquinolines such as reticuline (1),
and protoberberines such as scoulerine (2) and N-methyl stylopine
(19, Supplementary Fig. S2) also did not generate new product.
3.2. Reaction mechanism of CYP82N2v2
Recombinant CYP82N2v2 expressed in S. cerevisiae converted
protopine (5) to dihydrosanguinarine (7) without detectable for-
mation of any intermediate. Tanahashi and Zenk (1990) used
6-3H protopine (5) and detected the removal of HO3H from the
substrate by the microsomal fraction of E. californica cells during
formation of [11-3H] dihydrosanguinarine (7). It was concluded
Please cite this article in press as: Takemura, T., et al. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Esc-
hscholzia californica cells. Phytochemistry (2012), doi:10.1016/j.phytochem.2012.02.013

6
T. Takemura et al. / Phytochemistry xxx (2012) xxx–xxx
O
Me
N
(A)
O
O
O
Height
(x107)
O
protopine
(5)
TIC
334
354
retention time (min)
retention time (min)
reaction product
(B)
Height
(x107)
O
O
Me
N
dihydrosanguinarine
O
(C)
O
(7)
dihydro-
Height
sanguinarine
(x107)
retention time (min)
O
O
Me
N
O
(D)
OMe
OMe
Height
(x107)
allocryptopine (11)
reaction product
retention time (min)
retention time (min)
(E)
Height
(x107)
O
O
(F)
Me
N
dihydrochelerythrine
Height
OMe
(13)
OMe
(x107)
dihydro-
chelerythrine
retention time (min)
Fig. 5. CYP82N2v2 reaction with protopine (5) or allocryptopine (11) as a substrate. (A) Vector control reaction with protopine (5) (m/z 354, [M+H]+), (B) CYP82N2v2 reaction
with protopine (5), (C) authentic dihydrosanguinarine (7) (m/z 334, [M+H]+), (D) vector control reaction with allocryptopine (11) (m/z 370, [M+H]+), (E) CYP82N2v2 reaction
with allocryptopine (11), and (F) authentic dihydrochelerythrine (13) (m/z 350, [M+H]+). Reaction conditions were as shown in Section 5. Total ion chromatogram (TIC) and
single ion chromatogram for each m/z were monitored. Asterisks show the reaction products. Mass fragment patterns of reaction products are shown in comparison with
those of standard chemicals.
Please cite this article in press as: Takemura, T., et al. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Esc-
hscholzia californica cells. Phytochemistry (2012), doi:10.1016/j.phytochem.2012.02.013

T. Takemura et al. / Phytochemistry xxx (2012) xxx–xxx
7
Table 1
that hydroxylation of protopine (5) at the 6-position and succes-
sive spontaneous conversion produces dihydrosanguinarine (7).
The sequence information for CYP82N2v2 as a simple monooxy-
genase supports this speculation.
NADPH or oxygen dependency of CYP82N2v2 activity. The reaction was carried out
under standard assay conditions with protopine as the substrate, after the removal of
NADPH or oxygen with glucose/glucose oxidase, or the addition of ketoconazole. The
CYP82N2v2 activity of control assay was 6300 pmol/30 min/8.5 ng P450. n.d. means
that the activity was less than 2% of control assay.
Characterization of the substrate-specificity of CYP82N2v2 fur-
ther provided additional support for the 6-hydroxylation of proto-
pine (5): when CYP82N2v2 was reacted with corycavine (15), the
formation of corynoloxine (16) was detected (Fig. 6). Its formation
was speculated to occur through an enamino aldehyde intermedi-
ate in equilibrium with 6-hydroxy protopine-type alkaloids (Sup-
plementary Fig. S5, Iwasa et al., 1989). In fact, when CYP82N2v2
was reacted with 13-oxoprotopine (17) (Fig. 6), CYP82N2v2 pro-
duced a novel compound with an m/z of 366. This product had a
unique MS fragment pattern, which resembled that of the amino
alcohol-type intermediate and was distinct from the substrate
13-oxoprotopine (17) (Fig. S4). The limited reaction with 13-oxo-
protopine (17) also indicates that the hydrogen at the 13-position
plays a critical role in benzophenanthridine alkaloid biosynthesis.
Although the first 6-hydroxylation step is not detectable, the iden-
tification of CYP82N2v2 provides a useful tool for understanding
the molecular mechanism of this reaction.
Addition
Relative
activity
%
None
ꢀNADPH
100
n.d.
15
40 mM glucose + 5 units of glucose oxidase + 10 units of
catalase
40 mM glucose + boiled glucose oxidase + 10 units of
catalase
92
25
lM ketoconazole
n.d.
O
O
Me
(A)
Height
(x107)
N
O
Me
O
O
corycavine (15)
TIC
366
368
3.3. Physiological role of CYP82N2v2
The identification of CYP82N2v2 also provides useful informa-
tion on the regulation of benzophenanthridine alkaloid biosynthe-
sis. In fact, CYP82N2v2 can react with several protopine-type
alkaloids, such as protopine (5), allocryptopine (11), 13-oxoproto-
pine (17) and corycavine (15). Both protopine (5) and allocrypto-
pine (11) are particularly good substrates for CYP82N2v2, but
this broad reactivity is not common for P450s in alkaloid biosyn-
thesis, as shown for EcCYP719A2 or CjCYP719A1 (Ikezawa et al.,
2003, 2007). The results herein also suggest that a CYP82N2v2
homolog may be involved in the formation of corynoloxine (16)
from corycavine (15) found in Corydalis incisa cells (Supplementary
Fig. S5, Iwasa et al., 1989). CYP82N2v2 would be a good example of
the importance of endogenous enzymes for metabolic
diversification.
retention time (min)
(B)
Height
(x107)
TIC
366
368
*
retention time(min)
O
O
Me
N
(C)
O
O
O
Height
Me
H
(16)
corynoloxine
(x107)
TIC
366
368
4. Conclusions
retention time(min)
A cDNA of P450 (CYP82N2v2) in sanguinarine biosynthesis was
isolated from E. californica, based on an integrated analysis of
metabolites and the mRNA expression profile of transgenic cells
with C. japonica scoulerine-9-O-methyltransferase (Takemura
et al., 2010). Recombinant CYP82N2v2 produced in S. cerevisiae
clearly showed protopine (5) or allocryptopine (11) conversion to
produce dihydrosanguinarine (7) or dihydrochelerythrine (13),
respectively. Further characterization of the substrate-specificity
of CYP82N2v2 indicated that 6-hydroxylation played a role in the
reaction. Although the first 6-hydroxylation step is not detectable,
the identification of CYP82N2v2 provides a useful tool for under-
standing the molecular mechanism of this reaction.
O
(D)
Height
(x107)
Me
N
O
O
O
O
(17)
13-oxoprotopine
O
TIC
366
368
retention time(min)
(E)
Height
(x107)
TIC
366
368
5. Experimental
*
5.1. Plant material
retention time(min)
E. californica seeds were obtained from Takii Seed Co., Ltd. (Kyo-
to, Japan). Suspension cultures of E. californica with ectopic expres-
sion of C. japonica scoulerine-9-O-methyltransferase (CjSMT) cDNA
were established and maintained in Linsmaier–Skoog medium
Fig. 6. CYP82N2v2 reaction with corycavine (15) or 13-oxoprotopine (17) as the
substrate. Vector control reaction with corycavine (15) (A), CYP82N2v2 reaction
with corycavine (15) (m/z 368, [M+H]+) (B), authentic corynoloxine (16) (m/z 366,
[M+H]+) (C), vector control reaction with 13-oxoprotopine (17) as substrate (D), and
CYP82N2v2 reaction with 13-oxoprotopine (17) (E). Asterisks show the reaction
products.
(Linsmaier and Skoog, 1965) with 3% sucrose, 10
lM a-naphtha-
leneacetic acid, and 1 M 6-benzylaminopurine on a gyratory
l
Please cite this article in press as: Takemura, T., et al. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Esc-
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8
T. Takemura et al. / Phytochemistry xxx (2012) xxx–xxx
shaker (90 rpm) at 26 °C in the dark in a 21-day growth cycle, as
described previously (Takemura et al., 2010).
fraction (protein concentration of 1.65 mg/mL) in 50 mM Hepes/
NaOH (pH 7.6) was measured before and after the addition of so-
dium dithionite, with the bubbling of CO, to obtain the reduced
CO-bound spectrum of CYP82N2v2. The P450 hemoprotein content
in the microsomal fraction was then determined using a difference
of 91 mMꢀ1 cmꢀ1 between the extinction coefficients at 450 and
490 nm (Omura and Sato, 1964).
5.2. Chemicals
Protopine (5), allocryptopine (11) and sanguinarine (8) were
purchased from SIGMA–ALDRICH, Co., (St. Louis, MO, USA). 13-
Oxoprotopine (17), corycavine (15), protopine-N-oxide (18), cory-
noloxine (16), (S)-N-methylstylopine (19), dihydrosanguinarine
(7) and dihydrochelerythrine (13) were from the collection of K.
Iwasa. (S)-Reticuline (1) and (S)-scoulerine (2) were gifts from Mit-
sui Chemicals, Inc. (Tokyo, Japan).
5.8. P6H enzyme assay
P6H activity was determined in a reaction mixture that con-
sisted of 50 mM Hepes/NaOH (pH 7.6), 500 lM NADPH, 10 lM
substrate, and the enzyme preparation (8.5 ng P450). Protopine
(5) and allocryptopine (11) were used as standard substrates in
the enzymological characterization of CYP82N2v2, and corycavine
(15), 13-oxoprotopine (17), protopine N-oxide (18), reticuline (1),
scoulerine (2), and N-methyl stylopine (19) were used for the char-
acterization of substrate-specificity. The assay mixture was incu-
bated at 30 °C for 30 min, except for the determination of kinetic
parameters (incubation time; 5–10 min), and the reaction was
then terminated by the addition of Cl3CCO2H (final concentration
of 2%) and MeOH (final concentration of 40%). After protein precip-
itation, the reaction products were determined quantitatively by
reversed-phase HPLC with a Shimadzu LC-10 A system: column,
5.3. Preparation of cDNAs for P6H candidate gene
cDNA was prepared from transgenic cells at day 14 of culture.
Since cDNA candidate (EcCYP-A) clone for P6H contained only 50-
termini, 30-RACE (rapid amplification of cDNA ends) was per-
formed using a GeneRacer kit (Invitrogen) with a total RNA sample
(1 l
g), gene-specific primer, 30-GSP (50-ATCATTTCATGGGTGG-
GAAA-30) and the universal primer (50-GCTACGTAACGGCATGA-
CAGT-30) following the manufacture’s instructions. The resultant
PCR product of ꢁ1.5 kb was subcloned into pGEM T-easy vector
and the nucleotide sequence was determined.
COSMOSIL
pNAP column (4.6 ꢂ 250 mm; Nacalai Tesque); solvent
5.4. Alignment analysis
system, 0.05% CF3CO2H/CH3CN containing 0.05% CF3CO2H (1:1);
flow rate, 0.5 mL/min; detection, absorbance measurement at
280 nm with a SPD6A photodiode array detector. Product forma-
tion was confirmed by LC–MS (LCMS-2020, Shimadzu; ESI-MS at
70 eV, positive ion mode) with the same conditions as in the HPLC
analysis except for the solvent system: CH3CN/H2O/AcOH (99:99:2,
v/v/v) and flow rate 0.5 mL/min.
To determine the kinetic parameters of P6H, the amount of
dihydrosanguinarine (7) produced was estimated using a calibra-
tion curve of standard sample at 280 nm. Kinetic parameters were
obtained from three independent experiments and then averaged
to yield final estimates with a standard deviation.
The nucleotide sequence was deposited in DDBJ/GenBank™
(AB598834) and also in the P450 nomenclature committee, who
named it CYP82N2v2. CYP82N2v2 was aligned using Clustal W
(Thompson et al., 1994; Higgins et al., 1996) and BioEdit (http://
www.mbio.ncsu.edu/BioEdit/bioedit.html). GenBank™ accession
numbers for the sequences used are BsCYP80A1 (U09610), Ec-
CYP80B1 (AF014800), CjCYP80G2 (AB288053), EcCYP719A2
(AB126257), PsCYP719B1 (EF451150), NtCYP82E4v1 (DQ131886),
AtCYP82C2 (NM119348), and AtCYP82G1 (NM113423).
5.5. Construction of expression vector
Acknowledgments
To determine the P6H activity of CYP82N2v2, a yeast expression
vector was constructed in pGYR-SpeI with the S. cerevisiae NADPH-
P450 reductase gene and an SpeI cloning site (Ikezawa et al., 2008).
Full-length CYP82N2v2 cDNA was amplified by PCR with the fol-
lowing primers to introduce an XbaI site (underlined): forward pri-
mer (50-CTACAAAATCTAGAAATGGATTCCTTAATC-30) and reverse
primer (50-AACCACATTCTAGACTATTCGTACAACTTG-30). After sub-
cloning in pGEM T-easy vector and confirmation of the nucleotide
sequence, full-length cDNA of CYP82N2v2 was cut with XbaI, and
then ligated into the SpeI site of pGYR-SpeI to generate yeast
expression vector, pGS-CYP82N2v2.
We thank Drs. Y. Yabusaki and M. Mizutani for providing the
expression vector, pGYR, and for the reduced CO-difference spectra
measurement, respectively. We also thank Mitsui Chemicals, Inc.
for generous gifts of the alkaloids. This research was supported
by the Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan [Grant-in-Aid (Nos. 21248013 and 23108511 to F.S.)];
the Japan Society for the Promotion of Science [fellowship to T.T.].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2012.02.013.
5.6. Heterologous expression of CYP82N2v2 in S. cerevisiae
pGS-CYP82N2v2 was introduced into S. cerevisiae strain AH22 (a
leu2–3 leu2–112 his4–519 can1 (cir+)) (Oeda et al., 1985) by the
LiCl method (Ito et al., 1983), and transgenic S. cerevisiae cells were
cultivated in concentrated SD medium at 30 °C and 220 rpm (Sak-
aki et al., 1990). A microsomal fraction was prepared in the buffer
(100 mM Hepes/NaOH pH 7.6) as described previously (Ikezawa
et al., 2003), and used for the enzyme assay.
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