G.A.W. Beaudoin, P.J. Facchini / Biochemical and Biophysical Research Communications 431 (2013) 597–603
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(CYP82N3; protopine 6-hydroxylase), KC154002; P. somniferum
PsCFS (CYP719A25; cheilanthifoline synthase), GU325749; P. som-
niferum PsSPS (CYP719A20; stylopine synthase), GU325750; P.
somniferum PsNMCH (CYP80B3; 30-hydroxy-N-methylcoclaurine
hydroxylase), AF191772; E. californica EcCYP719A5 (cheilanthifo-
line synthase), AB434654; E. californica EcCYP719A3 (stylopine
synthase), AB126256; E. californica EcCYP719A2 (stylopine syn-
thase), AB126257; E. californica EcCYP82N2v2 (protopine 6-
hydroxylase), AB598834; E. californica EcCYP82B1, AF014802; E.
californica EcCYP80B1 (30-hydroxy-N-methylcoclaurine hydroxy-
lase), AF014801; P. somniferum PsCYP719B1 (salutaridine syn-
thase), EF451150; Coptis japonica CjCYP719A1 (canadine
synthase), AB026122; Mentha x piperita MmCYP71A32 (menthofu-
ran synthase), AF346833; Ammi majus AmCYP71AJ1 (psoralen syn-
thase), AY532370; Catharanthus roseus CrCYP72A1 (secologanin
synthase), L10081; Helianthus tuberosus HtCYP76B1 (7-ethoxy-
coumarin O-deethylase), Y09920; Arabidopsis thaliana AtCYP79B2
(camalexin biosynthesis), NM_120158; Berberis stolonifera BsCY-
P80A1 (berbamunine synthase), U09610; Hyoscyamus niger
HnCYP80F1 (littorine mutase), DQ387048; C. japonica CjCYP80G2
(corytuberine synthase), AB288053; Sesamum indicum SiCYP81Q1
(methylenedioxy bridge formation), AB194714; A. thaliana At-
CYP82C2, O49394; Nicotiana tabacum NtCYP82E4v1 (nicotine N-
demethylase), DQ131886; A. thaliana AtCYP82G1, NM113423;
Zea mays ZmCYP92A1, AY072297; Glycyrrhiza echinata GeCYP93C2
(2-hydroxyisoflavanone synthase), AB023636; Lithospermum eryth-
rorhizon LeCYP98A6 (4-coumaroyl-40-hydroxyphenyllactic acid 3-
hydroxylase), AB017418; Cucurbita maxima CmCYP701A1 (ent-
kaurine oxidase), AF212990; A. thaliana AtCYP705A5 (flavonoid
30-monooxygenase), NM_124173; A. thaliana AtCYP71A13 (cama-
lexin biosynthesis), NM_128630; C. roseus CrCYP71BJ1 (taberso-
nine/lochnericine 19-hydroxylase), HQ901597; P. somniferum
PsCYP82X1, JQ659002; P. somniferum PsCYP82X2, JQ659004; P.
somniferum PsCYP82Y1, JQ659005.
and 20
l
g of microsomal protein in a total volume of 50
l
L. Assays
were incubated at 30 °C for 90 min and were stopped by adding
100 L of MeOH. The precipitate was discarded and the superna-
tant evaporated to dryness in a Speed-Vac concentrator (Savant;
l
l
L
of buffer A (95:5, 10 mM ammonium acetate, pH 5.5:acetonitrile).
All enzyme assays were performed in triplicate.
2.8. Liquid chromatography-tandem mass spectrometry
Analysis of enzyme assays was performed using a 1200 liquid
chromatograph and a 6410 triple quadropole mass spectrometer
(Agilent Technologies). Ten microliters of the reaction mixtures
were injected onto
(2.1 mm ꢀ 50 mm, 2.7
a
Poroshell 120 SB C18 column
lm particle size, Agilent Technologies) and
eluted at a flow rate of 0.7 mL/min. Liquid chromatography was
initiated at 100% solvent A, ramped to 60% solvent B (acetonitrile)
using a linear gradient over 6 min, further ramped to 99% solvent B
using a linear gradient over 1 min, held constant at 99% solvent B
for 1 min and returned to original conditions over 0.1 min for a
3.9 min equilibration period. Eluate was applied to the mass ana-
lyzer using an electrospray ionization probe operating in positive
mode with the following conditions: capillary voltage, 4000 V;
fragmentor voltage, 100 V; source temperature, 350 °C; nebulizer
pressure, 50 PSI; gas flow, 10 L/min. For full-scan analysis, quadru-
pole 1 and 2 were set to RF only, whereas the third quadrupole
scanned from 200–700 m/z. For collision-induced dissociation
(CID) analysis, the precursor m/z was selected in quadrupole 1
and collision energy of 25 eV was applied in quadrupole 2. The
resulting fragments were resolved by quadrupole 3 scanning from
40 m/z to 2 m/z greater than the precursor m/z and compared with
previously published spectra [28].
3. Results
2.5. S. cerevisiae expression vectors
3.1. Identification of a cDNA candidate encoding MSH from opium
poppy
A synthetic PsCPR gene [25] codon-optimized for expression in
amplified (forward primer: 50-GGATCCAACAATGGGGTCAAA-
CAACC-30; reverse primer: 50-CTCGAGCCATACATCTCTCAAGTATC-
30), and the amplicon was inserted in-frame with the c-Myc tag
in pESC-leu2d using BamHI and SalI restriction sites introduced
by PCR, yielding pESC-leu2d::PsCPR. PsMSH was amplified (for-
ward primer: 50-AAAAATGCGAACCGAATCAATC-30) and reverse pri-
mer: 50-TCTAGAAATATCTCGAGTCGAGGTTTGA-30) from cDNA
synthesized using total RNA isolated from elicitor-induced opium
poppy cell suspension cultures, and the amplicon inserted into
pESC-leu2d::PsCPR in-frame with the FLAG tag using NotI and SpeI,
yielding the dual expression plasmid pESC-leu2d::PsMSH/CPR.
The search for candidate cDNAs encoding MSH was performed
using uncharacterized sequences annotated as P450s from a previ-
ous microarray analysis that demonstrated the coordinated induc-
tion of all known sanguinarine biosynthetic genes [19] and
corresponding proteins [29] in elicitor-treated opium poppy cell
suspension cultures. Genes showing induced transcript levels that
correlated with the accumulation of sanguinarine included the
PsMSH candidate (Genbank accession number KC154003), in addi-
tion to PsNMCH, PsCFS, PsSPS and PsP6H (KC154003). The predicted
526 amino acid sequence of opium poppy MSH contained con-
served motifs found in many P450s (Supplementary Fig. S2). Phy-
logenic analysis comparing P450s involved in BIA metabolism
and other plant specialized metabolic pathways shows that opium
poppy MSH belongs to the CYP82 family (Fig. 2). Interestingly,
opium poppy MSH shows strong similarity to recently reported
P450s (CYP82X1, CYP82X2 and CYP82Y1) putatively involved in
the biosynthesis of noscapine in opium poppy [30] and Californica
poppy P6H (CYP82N2v2) [17] (Fig. 2). MSH shares 49%, 48%, 53%
and 54% amino acid sequence identity to CYP82X1, CYP82X2,
CYP82Y1 and P6H, respectively.
2.6. Heterologous expression in S. cerevisiae
The pESC-leu2d::PsMSH/CPR vector was introduced into S. cere-
visiae strain YPH499 (Agilent Technologies; Santa Clara, CA; http://
formed as described previously [26] except that the induction cul-
ture also contained 1% (w/v) raffinose. The microsomal fraction
was prepared by glass bead disruption [27].
2.7. Enzyme assays
3.2. Expression of PsMSH and PsCPR in S. cerevisiae
In vitro enzyme assays were performed using native and boiled
microsomal fractions in 50 mM HEPES, pH 7.5, 2.5 mM NADPH,
Heterologous co-expression of PsMSH and PsCPR was confirmed
by immunoblot analysis of microsomal fractions prepared from S.
cerevisiae transformed with the pESC-leu2d::PsMSH/CPR vector
using c-Myc and FLAG antibodies to detect tagged MSH and CPR
100
lM alkaloid substrate except for N-methylstylopine and N-
methylcanadine, which were used at a concentration of 10
lM