Characterization of a flavoprotein oxidase from opium poppy catalyzing the final steps in sanguinarine and papaverine biosynthesis

Article abstract of DOI:10.1074/jbc.M112.420414

Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to ( S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K_m values of 201 and 146 μM for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.

Full text of DOI:10.1074/jbc.M112.420414

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 51, pp. 42972–42983, December 14, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Characterization of a Flavoprotein Oxidase from Opium
Poppy Catalyzing the Final Steps in Sanguinarine and
□
S
Papaverine Biosynthesis*
Received for publication, September 17, 2012, and in revised form, October 26, 2012 Published, JBC Papers in Press, November 1, 2012, DOI 10.1074/jbc.M112.420414
Jillian M. Hagel‡, Guillaume A. W. Beaudoin‡1, Elena Fossati§, Andrew Ekins§, Vincent J. J. Martin§2,
and Peter J. Facchini‡3
From the ‡Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada and the §Department of
Biology, Concordia University, Montréal, Québec H4B 1R6, Canada
Background: Oxidized forms of benzylisoquinoline alkaloids occur in plants.
Results: In vitro and in vivo characterization of flavoprotein oxidases led to the isolation of a novel alkaloid biosynthetic enzyme
in opium poppy.
Conclusion: The final conversions in sanguinarine and papaverine biosynthesis are catalyzed by a flavoprotein oxidase.
Significance: We have extended the importance of flavoprotein oxidases in benzylisoquinoline alkaloid metabolism.
Benzylisoquinoline alkaloids are a diverse class of plant port of DBOX as a multifunctional oxidative enzyme in BIA
specialized metabolites that includes the analgesic morphine, metabolism.
the antimicrobials sanguinarine and berberine, and the vaso-
dilator papaverine. The two-electron oxidation of dihydro-
Benzylisoquinoline alkaloids (BIAs)4 are a large and diverse
group of ϳ2500 plant specialized metabolites, many of which
possess potent pharmacological properties (1), including the
narcotic analgesics morphine and codeine, the cough suppres-
sant and potential anticancer drug noscapine (2), the antimi-
crobial agents sanguinarine and berberine, and the vasodilator
papaverine (Fig. 1). BIA biosynthesis begins with the condensa-
tion of two tyrosine derivatives, dopamine and 4-hydroxyphe-
nylacetaldehyde, yielding 1-benzylisoquinoline (S)-norcoclau-
rine as the central precursor to BIA biosynthesis (Fig. 1).
Internal carbon-carbon coupling of the (S)-norcoclaurine
derivative and branch point intermediate (S)-reticuline results
in the formation of various BIA structural subgroups, including
morphinan (e.g. morphine), protoberberine (e.g. berberine),
and benzophenanthridine (e.g. sanguinarine) alkaloids. Further
carbon-oxygen coupling and functional group modifications
within each branch pathway yield a multitude of structurally
related compounds. Several of the enzymes responsible for the
formation of novel backbone structures, hydroxylation of aro-
matic rings, and the modification of functional groups are oxi-
doreductases, including cytochromes P450, 2-oxoglutarate/
Fe2ϩ-dependent dioxygenases, and flavoproteins (1, 3).
sanguinarine catalyzed by dihydrobenzophenanthridine oxi-
dase (DBOX) is the final step in sanguinarine biosynthesis.
The formation of the fully conjugated ring system in sanguin-
arine is similar to the four-electron oxidations of (S)-cana-
dine to berberine and (S)-tetrahydropapaverine to papaver-
ine. We report the isolation and functional characterization
of an opium poppy (Papaver somniferum) cDNA encoding
DBOX, a flavoprotein oxidase with homology to (S)-tetrahy-
droprotoberberine oxidase and the berberine bridge enzyme.
A query of translated opium poppy stem transcriptome data-
bases using berberine bridge enzyme yielded several candi-
date genes, including an (S)-tetrahydroprotoberberine oxi-
dase-like sequence selected for heterologous expression in
Pichia pastoris. The recombinant enzyme preferentially cat-
alyzed the oxidation of dihydrosanguinarine to sanguinarine
but also converted (RS)-tetrahydropapaverine to papaverine
and several protoberberine alkaloids to oxidized forms,
including (RS)-canadine to berberine. The Km values of 201
and 146 ␮M for dihydrosanguinarine and the protoberberine
alkaloid (S)-scoulerine, respectively, suggested high concen-
trations of these substrates in the plant. Virus-induced gene
silencing to reduce DBOX transcript levels resulted in a cor-
responding reduction in sanguinarine, dihydrosanguinarine,
and papaverine accumulation in opium poppy roots in sup-
Berberine bridge enzyme (BBE) is a well characterized flavo-
protein oxidase that catalyzes the stereo-specific conversion of
the central intermediate (S)-reticuline to (S)-scoulerine (4) (Fig.
1). The formation of (S)-scoulerine is the first step in the bio-
synthesis of the benzophenanthridine and protoberberine alka-
loids sanguinarine and berberine, respectively. (S)-Tetrahydro-
* This work was supported by Genome Canada, Genome Alberta, Genome
Québec, the Government of Alberta, and the Canada Foundation for Inno-
vation-Leaders Opportunity Fund.
□
S
This article contains supplemental Table 1 and Figs. S1–S16.
1 Recipient of scholarships from the Natural Sciences and Engineering
Research Council of Canada and Fonds Québécois de la Recherche sur la 4 The abbreviations used are: BIA, benzylisoquinoline alkaloid; BBE, berberine
Nature et les Technologies.
bridgeenzyme;CID, collision-induceddissociation;DBOXandDHBO, dihy-
drobenzophenanthridine oxidase; DRS, 1,2-dehydroreticuline synthase;
FADOX, FAD-dependent oxidoreductases; STOX, (S)-tetrahydroprotober-
berine oxidase; VIGS, virus-induced gene silencing; FPKM, fragments per
kilobase of exon model per million mapped reads; contig, group of over-
lapping clones; RT-qPCR, real-time quantitative PCR.
2 Holder of the Canada Research Chair in Microbial Genomics and
Engineering.
3 Holder of the Canada Research Chair in Plant Metabolic Processes Biotech-
nology. To whom correspondence should be addressed. Tel.: 403-220-
7651; E-mail: pfacchin@ucalgary.ca.
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Flavoprotein Oxidase from Opium Poppy
protoberberine oxidase (STOX) is another flavoprotein that involved in a diverse array of biological processes (17). Many
catalyzes the four-electron oxidation of (S)-canadine to berber- flavoprotein oxidases act on amine substrates and can have
ine. Isolated STOX from Berberis wilsonae cell cultures either a broad or a highly restricted substrate range (18). Mem-
accepted a variety of tetrahydroprotoberberine alkaloid sub- bers of the vanillyl-alcohol oxidase flavoprotein family favor the
strates, including (S)-scoulerine, (S)-tetrahydrocolumbamine, covalent binding of FAD, a feature that increases the redox
and (S)-tetrahydropalmatine, along with certain 1-benzyliso- potential of the cofactor (19). BBE, STOX, and related vanillyl-
quinoline alkaloids, including (S)-norreticuline (5). Recently, alcohol oxidase flavoproteins, such as the marijuana (Cannabis
BBE from California poppy (Eschscholzia californica) was also sativa) enzymes tetrahydrocannabinolic acid synthase (20) and
shown to oxidize (S)-scoulerine in a manner similar to STOX, cannabidiolic acid synthase (21), and the sunflower (Helianthus
highlighting the catalytic similarities between the two enzymes annua) carbohydrate oxidase (22) are of interest due to their
(6).
unique bivalent 8␣-histidyl-6-S-cysteinyl mode of FAD attach-
The conversion of (S)-scoulerine to sanguinarine involves ment. Our work describes the isolation and functional char-
the closure of two methylenedioxy bridges by cytochromes acterization of an opium poppy cDNA encoding DBOX, a
P450 (7) followed by N-methylation (8), yielding the interme- flavoprotein oxidase catalyzing the terminal oxidations in
diate (S)-cis-N-methylstylopine. Two subsequent cytochrome sanguinarine and papaverine biosynthesis.
P450-dependent oxidations coupled with spontaneous intra-
EXPERIMENTAL PROCEDURES
molecular rearrangement convert the protoberberine (S)-cis-
N-methylstylopine through a protopine backbone to the ben-
Plants and Chemicals—Opium poppy (P. somniferum)
zophenanthridine dihydrosanguinarine (9). The two-electron plants were cultivated as described previously (16). Plant mate-
oxidation of dihydrosanguinarine to sanguinarine is catalyzed rials for gene expression and alkaloid analyses were harvested 1
by dihydrobenzophenanthridine oxidase (DBOX) (Fig. 1). Par- day before anthesis (60–80 days after germination) and stored
tially purified and characterized DBOX from E. californica cell at Ϫ80 °C. (RS)-Norlaudanosoline was purchased from Sigma-
cultures shares certain features with BBE, such as molecular Aldrich. (S)-Norreticuline was purchased from Toronto
weight (56000 for DBOX versus 57000 for BBE) and the nature Research Chemicals Inc. Dihydrosanguinarine and dihydro-
of the catalyzed reaction. However, whether or not E. califor- chelerythrine were prepared by NaBH4 reduction of sanguina-
nica DBOX is a flavoprotein oxidase has not been determined. rine and chelerythrine, respectively (23). (S)-Scoulerine was
A distinct dihydrobenzophenanthridine oxidase, designated prepared by the enzymatic oxidation of (S)-reticuline (100 mg)
DHBO to distinguish it from DBOX, was also isolated from using recombinant opium poppy BBE secreted from Pichia pas-
bloodroot (Sanguinaria canadensis) cell cultures (10). In con- toris cells and purified by two rounds of thin layer chromatog-
trast to DBOX, DHBO was a copper-dependent protein with a raphy. All other chemicals were synthesized or purchased as
molecular weight of 77000 (11). Corresponding genes for nei- described previously (3, 8, 16, 24).
ther DBOX nor DHBO have been isolated.
Selection of FADOX Candidates and Phylogenic Analysis—
Both sanguinarine and berberine are quaternary ammonium Using the translated opium poppy BBE sequence (GenBankTM
cations, which also occur at other points in BIA metabolism, accession number AAC61839) as a query, local sequence data-
including the oxidation of (S)-reticuline to 1,2-dehydroreticu- bases for opium poppy stem, root, and cell cultures were
line (Fig. 1). The introduction of a double bond into (S)-reticu- searched to identify homologous sequences encoding puta-
line, catalyzed by 1,2-dehydroreticuline synthase (DRS), is pro- tive FAD-dependent oxidoreductase (FADOX) candidates.
posed to initiate the formation of (R)-reticuline required for Available resources include individual Roche 454-based
morphine biosynthesis due to the stereospecificity of the sub- pyrosequencing databases for sanguinarine-producing elici-
sequent cytochrome P450, yielding the promorphinan inter- tor-treated cell cultures and eight plant chemotypes (25, 26),
mediate salutaridine (12). DRS was purified 5-fold from opium in addition to Sanger sequencing databases for stem from a high
poppy (Papaver somniferum) seedlings and was suggested to morphine variety (3) and elicitor-treated cell cultures (27).
require a covalently linked cofactor, such as FAD (13). How- RNA-seq databases for root and stem of the opium poppy vari-
ever, unlike the final oxidations in the formation of sanguina- ety Bea’s Choice were generated using Illumina HiSeq and
rine, berberine, and papaverine, the conversion of (S)-reticuline Genome Analyzer Ilx technologies, respectively. Following
ϩ
to 1,2-dehydroreticuline does introduce an additional aromatic RNA extraction (26), poly(A) RNA purification, cDNA library
ring system (Fig. 1). The major metabolic route to papaverine preparation, emulsion-based PCR and short-read sequencing
does not proceed through (S)-reticuline, but rather involves a were performed by the Genome Québec at the McGill Univer-
series of N-desmethylated intermediates (14–16). In a final sity Innovation Center. Quality control, de novo transcript
step, (S)-tetrahydropapaverine is dehydrogenated to papaver- assembly, annotation, and gene expression analysis were per-
ine (Fig. 1). Although a dedicated (S)-tetrahydropapaverine oxi- formed at the University of Calgary Visual Genomics Center.
dase has not been isolated from a papaverine-producing spe- Initial quality assessment of Illumina data were conducted
cies, such as opium poppy, STOX from Berberis wilsonae, a based on FastQC statistics (Brabraham Bioinformatics). Cut-
plant not known to produce papaverine, was reported to accept adapt (28) was employed for adapter/primer trimming, and in-
(S)-tetrahydropapaverine as a substrate (5).
house scripts were written to perform quality score conversion,
Flavoprotein oxidases catalyze the enantio- and regio-spe- trimming of reads based on a minimum quality score cut-off of
cific oxidation of a substrate with a concomitant reduction 25, and removal of read pairs where at least one member is
of molecular oxygen, yielding hydrogen peroxide, and are shorter than 35 base pairs. Short-read data were assembled
DECEMBER 14, 2012•VOLUME 287•NUMBER 51
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Flavoprotein Oxidase from Opium Poppy
FIGURE 1. Established and putative reactions catalyzed by flavoprotein oxidases in BIA metabolism. BIA biosynthesis begins with the formation of
(S)-norcoclaurine, a precursor to the central intermediate (S)-reticuline. (S)-Reticuline can be oxidized to (S)-scoulerine by BBE, a pivotal step toward the
formation of either benzophenanthridine (e.g. sanguinarine) or protoberberine (e.g. berberine) alkaloids. The final steps in these pathways are catalyzed by
DBOX and STOX, respectively. Alternatively, (S)-reticuline can be oxidized by DRS, leading to formation of (R)-reticuline and the morphinan alkaloid pathway.
(S)-Coclaurine is a key intermediate for papaverine biosynthesis, with tetrahydropapaverine oxidase (TPOX) catalyzing the final step. Corresponding genes
have been reported for BBE and STOX.
using Trinity (29), and contigs were annotated in MAGPIE. from P. pastoris cells into the culture medium, circumventing
Relative expression levels in RNA-seq databases were calcu- cell lysis prior to enzyme analysis and yielding a fraction highly
lated as fragments per kilobase of exon model per million enriched in recombinant protein. PCR amplicons were inserted
mapped reads (FPKM). Database queries yielded six unique, between the StuI and KpnI sites of pPink␣-HC. The same
full-length FADOX sequences and eight partial sequences. approach was used to assemble constructs for the expression of
Phylogeny (Fig. 2) and amino acid alignments (supplemental His6-tagged BBE1, BBE2, and FADOX5, except that different
Fig. S1) were performed using ClustalX (30), and phylogenetic primers were used for ORF amplification (supplemental Table
data were displayed using TREEVIEW (31).
S1). For these constructs, a flanking region encoding six histi-
Expression Vector Construction—The PichiaPink Expression dines followed by a stop codon was added to the reverse primer.
System (Invitrogen) was used for recombinant protein produc- Plasmid propagation was performed in Escherichia coli strain
tion in P. pastoris. Open reading frames (ORFs) encoding XL1-Blue, and expression constructs were transformed into
FADOX and BBE1 proteins were amplified using Phusion PichiaPink Strain 4, a double knock-out strain for endoge-
High-Fidelity DNA polymerase (Thermo Scientific) and sense nously secreted proteases prb1 and pep4. For recombinant pro-
and antisense primers with flanking StuI and KpnI restriction tein expression in S. cerevisiae, primers used to assemble
sites, respectively (supplemental Table S1), from cDNA pre- expression construct components are listed in supplemental
pared from opium poppy stem tissues. Sense primers were Table S1. The partial Kozak site AAAACA was introduced
designed to exclude known (32) or predicted signal peptides upstream of all ORFs, which were cloned into pYES2 (Invitro-
targeting the endoplasmic reticulum and to create fusion prod- gen). pYES2 was amplified using primers pYES2 HA Tag F and
ucts with the plasmid-encoded Saccharomyces cerevisiae pYES2 HA Tag R to eliminate a putative partial Kozak site and
␣-mating factor presequence. Fusion with the ␣-mating factor introduce an HA tag. The DBOX ORF was amplified using
presequence permitted the secretion of targeted gene products primers DBOX F and DBOX HA Tag R, introducing regions of
42974 JOURNAL OF BIOLOGICAL CHEMISTRY
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Flavoprotein Oxidase from Opium Poppy
homology to the modified pYES2 vector. pYES2 and DBOX
Enzyme Assays—To examine substrate range, triplicate
PCR amplicons were transformed into the yeast strain enzyme assays were performed in 100 mM Tris-HCl, pH 8.8,
CEN.PK113–13D (MAT␣ ura3-52, MAL2-8C, SUC2) resulting using 500 ␮M alkaloid substrate and desalted, concentrated
in the assembly of the vector pGC487 (pYES-2␮- ura3PGAL1
-
recombinant enzyme in a total volume of 80 ␮l. Assays were
FADOX5-HAtag-TCYC1). A further modified pYES2 vector, incubated for 2 h at 37 °C, stopped by adding 1 ml of quenching
whereby the URA3 auxotrophic marker was replaced with solution (20% (v/v) 10 mM ammonium acetate, pH 5.5, in etha-
LEU2, was used for the expression of 6OMT and 4ЈOMT2. This nol), and stored at Ϫ20 °C until analyzed. Immediately prior to
pYES2 vector was amplified using primers pYES F and pYES R, LC-MS/MS analysis, assays were centrifuged at room temper-
whereas 6OMT and 4ЈOMT2 codon-optimized synthetic genes ature for 5 min to remove insoluble debris. For Km and relative
(DNA2.0) were amplified using primers 6OMT F-6OMT R and
Vmax determinations, identical assay conditions were used with
4OMT F-4OMT R, respectively. Yeast promoters and termina- the exception that alkaloid substrate concentrations were var-
tors were isolated from CEN.PK genomic DNA using primers ied (supplemental Fig. S2). Saturation curves and kinetic con-
PMA1 F-PMA1 R for PPMA1, TDH3 F-TDH3 R for PTDH3
,
,
stants were calculated based on Michaelis-Menten kinetics
using GraphPad Prism 5 (GraphPad Software). To account for
CYC1 F-CYC1 R for Tcyc1, and ADH1 F-ADH1 R for TADH1
which added 25-bp linker sequences at their 5Ј and 3Ј termini. the spontaneous oxidation of the substrates dihydrosanguina-
Promoters and terminators with linker sequences were used as rine and dihydrochelerythrine, results from control enzyme
templates to amplify components for homologous recombina- assays using culture medium proteins from P. pastoris cells har-
tion using primers pYES:C1 F and PMA1 R2 for PPMA1, CYC1 boring the pPink␣-HC vector were subtracted from corre-
F2 and C6:H1 R for TCYC1, C1:H1 F and TDH3 R2 for PTDH3
,
sponding assays performed with recombinant enzyme extracts.
and ADH1 F2 and pYES:C6 R for TADH1. The seven PCR prod- Empty vector control data were also subtracted from recombi-
ucts were co-transformed into yeast strain CEN.PK113-16B nant enzyme assays using (S)-norreticuline due to the conver-
(MAT␣ leu2-3_112, MAL2–8C, SUC2), resulting in assembly sion of this substrate by endogenous P. pastoris proteins.
of vector pGC634 (pYES2–2␮-leu2 PPMA1-6OMT-TCYC1
-
Virus-induced Gene Silencing—Gene-specific silencing con-
P
TDH3-4ЈOMT2- TADH1). Constructs pGC487 and pGC634 structs were designed for in planta functional analysis of
were co-transformed into the haploid CEN.PK113 17A (MAT␣ FADOX1, FADOX3, FADOX5, and FADOX8 using unique
ura3-52, leu2-3_112, MAL2–8C, SUC2). CEN.PK113 13D 3Ј-UTR and/or 3Ј-ORF sequences. Due to a high sequence
(MAT␣ ura3-52, MAL2-8C, SUC2) was transformed separately identity (94%) between BBE1 and BBE2 (FADOX4), a single
with pGC487 for DBOX expression or the empty plasmid VIGS construct was designed based on the BBE1 sequence,
pYES2 for use as a negative control.
which contained numerous conserved stretches of Ͼ24 nucle-
Heterologous Expression in Yeast—For P. pastoris, recombi- otides capable of silencing both BBE1 and BBE2 (supplemental
nant proteins were generated according to the manufacturer’s Fig. S3). Full-length cDNAs were used as templates to amplify
instructions (PichiaPink Expression System, Invitrogen). gene-specific fragments (supplemental Fig. S3). Forward and
Briefly, freshly transformed cells were cultured in 500 ml of reverse PCR primers were designed with flanking BamHI and
growth medium to an OD between 2 and 6 and then transferred XhoI restriction sites, respectively (supplemental Table S1).
to 100 ml of induction medium containing 0.5% (v/v) methanol. Amplicons generated using Phusion High-Fidelity DNA
Following a 100-h induction period at 28 °C, the medium frac- polymerase (Thermo Scientific) were ligated into the BamHI
tion containing secreted, recombinant protein was centrifuged and XhoI sites of pTRV2. Constructs in pTRV2 along with
to remove cells and applied in batches to Amicon Ultra-15 cen- pTRV1 were independently mobilized in Agrobacterium tume-
trifugal filter units (Millipore) to concentrate the protein (final faciens strain GV3101 (33). Infiltration of 2-week-old seedlings
volume of 1 ml/100 ml of induction medium) and exchange to was performed as described previously (3). Plants were har-
appropriate buffering conditions (100 mM Tris-HCl, pH 8.8). vested just prior to anthesis and screened by PCR to identify
Desalted, concentrated fractions were stored at 4 °C for no lon- individuals harboring viral coat protein transcripts (supple-
ger than 48 h prior to enzyme analysis. For Saccharomyces mental Table S1; supplemental Fig. S4). Infected plants were
cerevisiae, strains were grown overnight in YNB supplemented analyzed by real-time quantitative PCR (RT-qPCR) to deter-
with synthetic dropout medium lacking uracil, histidine, tryp- mine relative target gene transcript levels. For BBE- and DBOX-
tophan, and leucine but containing 0.2% (w/v) glucose and 1.8% silenced plants, roots were ground to a fine powder under liquid
(w/v) galactose. After 24 h, yeast cultures were diluted 20-fold N2 and extracted three times with 4 ml of methanol, and pooled
in 10 ml of fresh medium containing 2% (w/v) galactose. After extracts were reduced to dryness under reduced pressure. Res-
6 h at 30 °C and 200 rpm, cells were collected by centrifugation idue was reconstituted in 1500 ␮l of methanol and stored at
at 2000 ϫ g for 5 min and resuspended in 200 ␮l of medium Ϫ80 °C until LC-MS/MS analysis. Latex alkaloids of FADOX1,
containing 2 mM norlaudanosoline. Cells were incubated at FADOX3, and FADOX8-silenced plants were analyzed by
30 °C and 200 rpm for 44 h and harvested by centrifugation. HPLC. Briefly, latex samples were suspended in 30 ␮l of meth-
Alkaloid content of cell extracts and culture medium was deter- anol, and 15 ␮l was further diluted with 235 ␮l of methanol,
mined using liquid chromatography-tandem mass spectrome- vortexed, and centrifuged to remove insoluble debris. 100 ␮l of
try (LC-MS/MS) analysis. Immunoblot analysis was performed the supernatant was analyzed as described previously (34).
on SDS-PAGE-fractionated cell lysate protein (50 ␮g) using Major alkaloids were identified based on retention times and
anti-HA DyLight antibodies (Rockland). Immunoblots were UV spectra compared with those of authentic standards. Statis-
visualized using a Typhoon imager (GE Healthcare).
tical analysis of transcript and metabolite data were performed
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Flavoprotein Oxidase from Opium Poppy
using GraphPad InStat 3.1a (GraphPad Software). For three- Power SYBR Green PCR Master Mix (Applied Biosystems).
way comparisons, Tukey-Kramer multiple comparison tests Reactions were subjected to 40 cycles of template denaturation,
were used to determine statistical significance at p values of primer annealing, and primer extension. To evaluate RT-qPCR
Ͻ0.05, Ͻ0.01, and Ͻ0.001. For two-way comparisons, paired t specificity, the amplicons of all primer pairs were subjected to
tests were performed using the same confidence intervals.
melt-curve analysis using the dissociation method as suggested
Ϫ⌬⌬
LC-MS/MS—Routine enzyme assay analyses were per- by the manufacturer. The 2
Ct method was used to deter-
formed using a 6410 Triple Quadropole LC-MS/MS system mine relative gene expression levels (35). The gene encoding
(Agilent Technologies, Santa Clara, CA). Two microliters of ubiquitin was used as the internal control (supplemental Table
quenched reactions were subjected to liquid chromatography S1), and the plant line showing the highest expression level
using a Zorbax Eclipse Plus C18 column (2.1 mm ϫ 50 mm, served as the calibrator for each target gene. The same method
1.8-␮m particle size; Agilent Technologies) at a flow rate of 0.2 was used to analyze VIGS experiments, except that nine plants
ml/min. For protoberberine alkaloids, the column was equili- per plant line were used to calculate mean transcript abun-
brated with solvent A (95:5 ratio of 10 mM ammonium acetate, dances, and alkaloid profiles were determined for the same
pH 5.5, to acetonitrile), and elution and washing were per- plants.
formed using the following gradient: 0–5 min, 0–30% solvent B
RESULTS
(100% acetonitrile); 5–10 min, 30–100% solvent B; 10–15 min,
100% solvent B; 15–15.1 min, 100–0% solvent B; 15.1–16 min,
Isolation of FADOX Gene Candidates—Opium poppy genes
0% solvent B. For benzophenanthridine alkaloids, gradient con- encoding FADOXs were identified from stem and elicitor-
ditions were as follows: 0–8 min, 0–100% solvent B; 8–12 min, treated cell culture transcriptome databases queried using
100% solvent B; 12–12.1 min, 100–0% solvent B; 12.1–16 min, opium poppy BBE. 16 non-redundant contigs were identified,
0% solvent B. For 1-benzylisoquinoline alkaloids, the flow was including eight full-length or nearly full-length sequences (sup-
adjusted to 0.4 ml/min, and the gradient conditions were as plemental Fig. S6). FADOX9 and FADOX10 were partial clones
follows: 0–1 min, 0% solvent B; 1–10 min, 0–35% solvent B; found only in a cell culture database generated by Sanger
10–11 min, 35–100% solvent B; 11–13 min, 100% solvent B; sequencing (27). FADOX clones 11–16 all consisted of partial
13–13.1 min, 100–0% solvent B; 13.1–16 min, 0% solvent B. sequences represented by few or single reads from individual
Injection into the mass analyzer was performed using an elec- 454 pyrosequencing databases (25, 26). Except for FADOX2
trospray ionization probe inlet. Ions were generated and and FADOX6, which contain stop codons and a deletion in the
focused using an electrospray ionization voltage of 4000 V, 10 ORF, respectively, amino acid sequences of other FADOX can-
liter/min gas flow, 50 p.s.i. nebulizing pressure, and gas temper- didates were aligned to opium poppy BBE1 (supplemental Fig.
ature of 350 °C. MS data acquisition was performed in positive S1). Opium poppy BBE1 contains an N-terminal ER-targeting
ion mode over 100–700 m/z. Product identification was based peptide (32), and online software predicted signal peptides in all
on comparisons of retention times and collision-induced disso- FADOX candidates. Both BBE1 and FADOX4 (BBE2) contain
ciation (CID) mass spectra of authentic standards or published Glu-421 as an equivalent of Glu-417, a key catalytic residue in
data (supplemental Table S2). Collision energies used for CID berberine bridge formation, found in E. californica BBE (4). All
spectra were either Ϫ30 or Ϫ35 eV, as noted in supplemental other FADOXs were substituted with other residues at this
Ϫ3
Fig. S5, with the argon collision gas at a pressure of 1.8 ϫ 10
position (supplemental Fig. S1). A phylogenetic tree based on
torr. For the analysis of VIGS experiments, 10 ␮l of root extract an alignment of protein sequences corresponding to function-
were subjected to liquid chromatography at a flow rate of 0.5 ally characterized plant flavoproteins shows high bootstrap
ml/min with the following gradient: 0–10 min, 0–50% solvent support for a monophyletic clade containing BBE homologues
B; 10–12 min, 50–99% solvent B; 12–13 min, 99% solvent B; from E. californica (36), barberry (B. wilsoniae) (37), and opium
13–13.1 min, 99–0% solvent B; 13.1–17.1 min, 0% solvent B. poppy (38) (Fig. 2). The nearest neighbor to FADOX5 (DBOX)
Ion source and MS parameters were identical to those used for is an enzyme from Mexican prickly poppy (Argemone mexi-
enzyme assays except that CID spectra were generated with cana), a member of the Papaveraceae along with opium poppy,
collision energies listed in supplemental Table S2.
that was recently shown to exhibit STOX activity (39). An addi-
Real-time Quantitative PCR Analysis—For gene expression tional clade was formed by partially characterized STOX
analysis in different opium poppy organs, tissues (ϳ0.1 g) from enzymes from B. wilsoniae and Japanese goldthread (Coptis
three plants were collected just prior to anthesis, and total RNA japonica), which are members of the Berberidaceae and Ranun-
was isolated with TRIzol (Invitrogen). Reverse transcription culaceae, respectively. Monophylogeny between the remaining
was performed at 42 °C for 60 min using 2.5 mM anchored oli- FADOXs, carbohydrate oxidase, tetrahydrocannabinolic acid
go(dT) primer (dT20VN), 0.5 mM dNTP, 10–40 ng/␮l RNA, synthase and either the BBE or STOX clade was not supported.
and 5 microunits/␮l reverse transcriptase (Fermentas, Burling- The relative transcript levels of full-length FADOX candidates
ton, Canada) after denaturing of the RNA/primer mix at 70 °C were compared in nine 454-pyrosequencing databases for
for 5 min. Real-time quantitative PCR using SYBR Green detec- opium poppy cell culture and stems from different chemotypes
tion was performed using a 7300 real-time PCR system (supplemental Fig. S7). All poppy chemotypes produce mor-
(Applied Biosystems). Each 10-␮l PCR included 1 ␮l of cDNA phine except for T, which accumulates the morphinan pathway
(taken directly from the RT reaction in the case of stem or intermediate thebaine (3), and Przemko, which is a nearly alka-
diluted 50% (v/v) with water for bud, leaf, and root), 300 nM loid-free variety (34). In addition to morphine, varieties Rox-
forward and reverse primers (supplemental Table S1), and 1ϫ anne and Veronica accumulate papaverine and the phthalide-
42976 JOURNAL OF BIOLOGICAL CHEMISTRY
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Flavoprotein Oxidase from Opium Poppy
ity using available substrates. For the purposes of detecting
BBE1, BBE2, and FADOX5 by immunoblot analysis and pursu-
ing affinity chromatography-based protein purification, we
attempted to generate His6-tagged proteins in P. pastoris. How-
ever, recombinant proteins were not detected by either SDS-
PAGE or immunoblot analysis, and no enzyme activities were
observed.
As a positive control for assay conditions, recombinant BBE1
and BBE2 (FADOX4) were tested for enzyme activity using 23
BIAs as potential substrates (Fig. 3 and supplemental Fig. S5).
As expected, both BBE1 and BBE2 catalyzed the efficient trans-
formation of the N-methyl group of (S)-reticuline into the C-8
berberine bridge carbon of (S)-scoulerine (supplemental Fig.
S9). In addition, columbamine and berberine were produced at
low levels from (RS)-tetrahydrocolumbamine and (RS)-cana-
dine, respectively, which represents a four-electron oxidation
characteristic of STOX activity. No other alkaloids were
accepted as substrates (Fig. 3 and supplemental Fig. S5).
Although reported for E. californica BBE (40), the four-electron
oxidation of (S)-scoulerine to dehydroscoulerine was not
detected with opium poppy BBE1 or BBE2.
FIGURE2. Unrootedneighbor-joiningphylogenetictreeforopiumpoppy
FADOXs and related flavoproteins. Bootstrap frequencies for each clade
are percentages of 1000 iterations. Species and associated GenBankTM acces-
sionnumbersforphylogenetictreeconstructionareasfollows:P. somniferum
(Ps) BBE1, AAC61839; B. stolonifera (Bs) BBE, AAD17487; E. californica (Ec) BBE,
AAC39358; C. sativa (Cs) ⌬1-tetrahydrocannabinolic acid synthase (THCAS),
AB057805; H. annua (Ha) carbohydrate oxidase (CHOX), AAL77103; A. mexi-
cana (Am) (S)-tetrahydroprotoberberine oxidase (STOX), ADY15027; B. wil-
soniae (Bw) STOX, ADY15026; C. japonica (Cj) STOX, BAJ40864.
DBOX (FADOX5) was capable of both two- and four-elec-
tron oxidations. For example, STOX activity was apparent with
most protoberberine substrates, with (RS)-canadine showing
the highest turnover rate under standard assay conditions (Figs.
isoquinoline alkaloid noscapine, whereas Marianne, Natasha, 3 and 4). Conversely, DBOX did not fully aromatize the C-ring
and Deborah accumulate only noscapine or the related com- of (S)-scoulerine but instead catalyzed a two-electron oxidation
pound narcotoline. Elicited cell cultures of opium poppy are to yield m/z 326 (Fig. 4). Extracted ion chromatographs
devoid of morphinan alkaloids but accumulate sanguinarine revealed two products at m/z 326 with essentially identical CID
(27). Only BBE1 and FADOX4 (BBE2) transcripts were rela- spectra, which were characterized as dihydroscoulerine iso-
tively abundant in all databases, including cell cultures. In con- mers (supplemental Fig. S10). The two-electron oxidation of
trast, FADOX5 and FADOX7 transcripts were absent in stem, (S)-scoulerine is consistent with a previous characterization of
occurring exclusively in cell cultures. Transcript levels for the STOX from A. mexicana (39). The formation of both dihydro-
remaining FADOXs were sporadic, with FADOX1, for exam- papaverine and papaverine was observed upon incubation of
ple, represented in all databases except for T and FADOX8 in DBOX with (RS)-tetrahydropapaverine, although other 1-ben-
five of eight chemotypes. Read counts for FADOX3 were also zylisoquinoline alkaloids, notably (S)-norreticuline, were not
variable and notably absent in cell cultures.
accepted as substrates (Fig. 3). CID analysis of dihydropapaver-
In Vitro Characterization—Flavoproteins containing a cova- ine revealed low abundance, approximately equal intensity ions
lently linked FAD cofactor require eukaryotic expression sys- of m/z 190 and 204, which represent signature fragment masses
tems to generate active proteins. Of the several expression sys- of 1,2-dihydropapaverine and 3,4-dihydropapaverine, respec-
tems previously employed for the production of E. californica tively (41). DBOX exhibited the highest relative activity with
BBE, the methylotrophic yeast P. pastoris has proven most suc- dihydrosanguinarine, producing the fully conjugated product
cessful (4, 6, 40). The secretory pathway of P. pastoris was used sanguinarine (Figs. 3 and 4). Dihydrochelerythrine was simi-
to produce opium poppy BBE and FADOX proteins. Replacing larly oxidized with lower efficiency. CID spectra for all products
the native endoplasmic reticulum secretion signal sequence are provided (supplemental Fig. S5). DBOX did not accept any
with S. cerevisiae ␣-mating factor has been shown to yield of the tested aporphine, morphinan, phthalideisoquinoline,
higher expression levels of E. californica BBE (6). For this rea- pavine, or bisbenzylisoquinoline alkaloids (supplemental Fig.
son, existing or predicted signal peptides were removed from S11).
opium poppy homologues prior to expression. Proteins with
Initial characterization of purified, native STOX from B. wil-
predicted molecular weight (57200 for BBE1 and BBE2 soniae suggested that (S)-norreticuline was a preferred 1-ben-
(FADOX4); 57500 for FADOX5) were routinely observed in zylisoquinoline substrate (5). Assays conducted using opium
concentrated and desalted medium fractions harvested 100 h poppy BBE1, DBOX, and empty expression vector controls all
postinduction (supplemental Fig. S8). These proteins were showed the conversion of (S)-norreticuline to two unidentified
never observed in empty vector controls, and enzyme activity compounds of m/z 328 that eluted separately during
was only associated with samples containing these proteins. LC-MS/MS analysis. These products were occasionally associ-
Recombinant FADOX1, FADOX3, and FADOX8 proteins were ated with two unidentified compounds of m/z 326. No turnover
not detected by SDS-PAGE, and enzyme assays performed on of (S)-norreticuline occurred if P. pastoris protein extracts were
concentrated and desalted medium fractions showed no activ- boiled for 10 min prior to performing assays, suggesting an
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Flavoprotein Oxidase from Opium Poppy
FIGURE 3. Relative conversion of selected substrates by BBE and DBOX (FADOX5) under standard assay conditions. Values represent mean Ϯ S.D. of
three independent experiments. The nature of each reaction is indicated below the activity values. The activity value for (RS)-tetrahydropapaverine includes
both dihydropapaverine and papaverine products. nd, not detected.
apparent consumption of (S)-norreticuline by native yeast methyltransferase and (RS)-3Ј-hydroxy-N-methylcoclaurine
enzymes. For this reason, DBOX activity with (S)-norreticuline 4Ј-O-methyltransferase coupled with the feeding of (RS)-nor-
as a substrate was re-evaluated using three different strains of laudanosoline (42). One strain also co-expressed a gene encod-
S. cerevisiae (supplemental Fig. S12). In two of these strains, an ing DBOX, which presumably could oxidize the endogenous
endogenous supply of (S)-norreticuline was generated by the (S)-norreticuline (m/z 316) to expected products of m/z 314 or
co-expression of genes encoding (RS)-norcoclaurine 6-O- 312. However, no masses corresponding to (S)-norreticuline
42978 JOURNAL OF BIOLOGICAL CHEMISTRY
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Flavoprotein Oxidase from Opium Poppy
FIGURE 5. Relative abundance of transcripts encoding BBE (A) and DBOX
(B) in opium poppy plant organs. RT-qPCR analysis was performed using
cDNA synthesized with RNA isolated from three individual plants. PCR signal
normalization was achieved using ubiquitin as the internal control, and the
plant organ exhibiting the highest gene expression served as the calibrator
for BBE or DBOX. Due to their extensive sequence identity, BBE1 and BBE2
could not be distinguished, and results reflect the cumulative abundance of
both transcripts. Error bars, S.D.
reduction products or to byproducts produced by endogenous
P. pastoris enzymes (i.e. m/z 328 or 326) were detected (supple-
mental Fig. S12), supporting the conclusion that (S)-norreticu-
line is not DBOX substrate.
K
m values of 146 Ϯ 9 and 201 Ϯ 67 ␮M were determined for
DBOX using the protoberberine and benzophenanthridine
substrates (S)-scoulerine and dihydrosanguinarine, respec-
tively (supplemental Fig. S2). All substrates were assayed using
the same enzyme preparations, which enabled the calculation
of relative Vmax values of 6.754 and 0.5308 pmol minϪ1, and
Ϫ1
Ϫ8
Ϫ9
relative Vmax Km ratios of 4.63 ϫ 10 and 2.64 ϫ 10
minϪ1, for (S)-scoulerine and dihydrosanguinarine, respec-
tively. The insolubility in aqueous solutions at saturating con-
centrations precluded Km analysis for (RS)-canadine (43).
Moreover, the formation of two products, dihydropapaverine
and papaverine, precluded a reliable Km determination for (RS)-
tetrahydropapaverine due to the possible occurrence of com-
petitive inhibition.
Gene Expression Analysis—Gene expression patterns of
DBOX and BBE in different organs of opium poppy were exam-
ined using RT-qPCR. Due to the extensive sequence identity
between BBE1 and BBE2, RT-qPCR analysis was unable to dis-
tinguish individual expression profiles (Fig. 5). However, rela-
tive transcript levels were for BBE1 and BBE2 were determined
using the FPKM values from Illumina-based stem and root
transcriptome databases (supplemental Fig. S13). RT-qPCR
results showed the highest DBOX transcript levels in roots
compared with other plant organs (Fig. 5). Although direct
FIGURE 4. Extracted ion chromatograms showing the substrates and prod-
ucts of DBOX (FADOX5) enzyme assays. In A–H, the tested substrate is indi-
cated in the top left or right corner, the bottom extracted ion chromatogram cor-
responds to an assay conducted with the empty pPINK␣-HC vector control, and
the top extracted ion chromatogram shows an assay performed with recombi- strate and product peaks. The latter were subjected to collision-induced dissoci-
nant DBOX. Parent ion mass-to-charge (m/z) values are indicated beside the sub- ation analysis for identification (supplemental Fig. S5).
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Flavoprotein Oxidase from Opium Poppy
comparisons of FPKM values are not possible using these data-
bases, DBOX transcripts were not detected in the stem data-
base but were relatively abundant in roots (supplemental Fig.
S13). RT-qPCR indicated abundant BBE transcript levels in
roots compared with aerial organs (Fig. 5), and FPKM data
showed that BBE1 transcripts were restricted to stems, whereas
BBE2 mRNAs were only in roots (supplemental Fig. S13).
VIGS Analysis—The organ-specific occurrence of DBOX
transcripts prompted a focus on the metabolic consequences of
DBOX gene silencing in roots. Our initial approach attempted
to quantify perturbations in the levels of alkaloids representing
in vitro substrates or products for DBOX and BBE in silenced
versus control plants. However, the low abundance of many
compounds, especially protoberberine alkaloids, in opium
poppy roots precluded such analysis. Both full ion scanning and
multiple reaction monitoring were used, together with CID
analysis to confirm compound identities. However, sanguina-
rine, dihydrosanguinarine, papavarine, and reticuline were
readily detected by full ion scanning analysis. Also included in
the full ion scanning analyses were thebaine and noscapine,
representing end products of the morphinan and pthalideiso-
quinoline branch pathways, respectively, N-methylcoclaurine,
an upstream precursor to (S)-reticuline, and protopine, which
serves as a sanguinarine pathway intermediate (supplemental
Fig. S14). Sanguinarine and dihydrosanguinarine were signifi-
cantly reduced in both DBOX- and BBE-silenced plants (Fig. 6).
However, spontaneous oxidation of dihydrosanguinarine dur-
ing the analysis might have contributed to a low dihydrosangui-
narine to sanguinarine ratio compared with levels in the plant.
In contrast, a reduction in papaverine and noscapine levels was
observed only in association with DBOX silencing. Tetrahydro-
papaverine and dihydropapaverine were not detected in either
control or silenced roots. BBE-silenced plants showed an accu-
mulation of N-methylcoclaurine and reticuline compared with
empty vector control and DBOX-silenced plants. Protopine and
thebaine levels were unaffected by the silencing of either gene.
Multiple reaction monitoring analysis yielded data for the rel-
ative abundance of the substrate/product pair stylopine/cop-
tisine, which remained unchanged in DBOX-silenced plants
(supplemental Fig. S15). The application of VIGS to silence
genes encoding FADOX1, FADOX3, and FADOX8 produced
no detectable alterations in major latex alkaloid levels com-
pared with empty vector controls (supplemental Fig. S16).
FIGURE 6. Effect of VIGS on root alkaloid levels. A, relative DBOX and BBE
transcript levels in roots as determined by RT-qPCR following VIGS treatment.
B, relative abundance of alkaloids in DBOX- or BBE-silenced roots, as deter-
mined by LC-MS/MS. Bars, mean Ϯ S.D. (error bars) of nine individual plants,
whichwereusedforbothgeneexpressionandalkaloidprofileanalyses. Aster-
isks denote a statistically significant difference relative to empty vector
(pTRV2) at p Ͻ 0.05 (*), p Ͻ 0.01 (**), or p Ͻ 0.001 (***) determined by the
Tukey-Kramer multiple comparisons test. pTRV2-BBE targeted both BBE1 and
BBE2 transcripts.
DISCUSSION
The isolation and characterization of DBOX as a novel flavo-
protein oxidase is a key step toward an understanding of several
key reactions within BIA metabolism. Our work demonstrates a
functional role for opium poppy FADOX5 as DBOX, an
enzyme previously uncharacterized at the genetic level. Results
also support a role for FADOX5 in papaverine biosynthesis,
although in vitro data (Figs. 3 and 4) combined with the exclu-
sive presence of the transcript in root (Fig. 5), which is the roots and elicited cell cultures, a feature shared with other
primary site of benzophenanthridine alkaloid accumulation, members of the Papaveraceae. Certain opium poppy cultivars
favored naming the enzyme DBOX. Most genes encoding also accumulate papaverine in roots and in the latex of stems,
enzymes in sanguinarine biosynthesis have been isolated (1), leaves, and seed capsules (16). In contrast, (S)-tetrahydroproto-
with DBOX catalyzing the terminal oxidation (Fig. 1). Opium berberine alkaloids are not major alkaloids in opium poppy. In
poppy accumulates relatively large quantities of sanguinarine in vitro characterization of recombinant DBOX revealed a versa-
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Flavoprotein Oxidase from Opium Poppy
tile enzyme capable of accepting a variety of benzophenanthri- in favor of dihydrosanguinarine. For example, in the Marianne
dine, 1-benzylisoquinoline, and (S)-tetrahydroprotoberberine chemotype, 80% of the benzophenanthridine alkaloid content
alkaloids (Figs. 3 and 4). Opium poppy DBOX was able to cat- of roots consists of dihydrosanguinarine and 10-hydroxydihy-
alyze both two- or four-electron oxidation reactions, depend- drosanguinarine, whereas sanguinarine and 10-hydroxysangui-
ing on the substrate, resulting in the introduction of either one narine account for only 20% (46). A high Km value for dihydro-
or two double bonds, respectively. The functional versatility of sanguinarine would contribute to the accumulation of
DBOX was corroborated by VIGS analysis, which highlighted dihydrosanguinarine.
changes in the levels of sanguinarine and papaverine in
Opium poppy DBOX yielded fully aromatized alkaloids from
response to DBOX silencing. Surprisingly, the levels of noscap- most tetrahydroprotoberberine substrates (Fig. 3). Purified
ine, which does not appear to be biosynthetically linked to STOX from B. wilsoniae showed a strict preference for (S)- over
DBOX, were also suppressed (Fig. 6).
(R)-enantiomers. The available racemic substrates (i.e. (RS)-ca-
Sequence analysis of opium poppy DBOX supported mono- nadine, (RS)-tetrahydrocolumbamine, and (RS)-tetrahydropal-
phylogeny with STOX from A. mexicana (Fig. 2). Opium poppy matine) might have reduced the apparent turnover rate of (S)-
and A. mexicana are members of the Papaveraceae, which gen- enantiomers. (S)-Scoulerine was the preferred protoberberine
erally accumulate sanguinarine and other benzophenanthri- substrate for opium poppy DBOX, which is in agreement with
dine alkaloids in roots, latex, or elicited cell cultures (44). Pre- the reported activity of B. wilsoniae STOX (5). Mechanistic
vious studies of STOX from A. mexicana and Berberis species investigation of B. wilsoniae STOX oxidation using (S)-scoul-
did not test benzophenanthridine alkaloids as substrates (5, 39). erine showed the formation of a single dihydroprotoberberine
Opium poppy BBE1 and BBE2 (FADOX4) formed a monophyl- with a double bond between N7 and C14. The lack of further
etic clade with BBE from E. californica (4) and Berberis stolo- oxidation to a fully aromatized dehydroprotoberberine was
nifera (Fig. 2). No relationship with either the STOX or BBE interpreted as an indication that the initial two-electron oxida-
clades was apparent for other FADOXs, which prompted us to tion to an iminium ion is enzymatic, whereas a second two-
focus on the functional characterization of FADOX5, with electron oxidation yielding dehydroprotoberberine products,
BBE1 and BBE2 serving as controls.
including berberine, columbamine, palmatine, and coptisine
Compounds representing nine BIA structural categories (Figs. 3 and 4), occurs spontaneously as the result of iminium
were tested, and DBOX accepted certain benzophenanthridine, ion instability. In contrast, mechanistic studies on E. californica
protoberberine, and 1-benzylisoquinoline alkaloids as sub- BBE, which exhibits STOX-like activity with (S)-scoulerine,
strates (Figs. 3 and 4 and supplemental Fig. S11). Dihydrosan- suggested an initial oxidation between N7 and C8 and a four-
guinarine showed the highest turnover rate compared with electron oxidation of (S)-scoulerine to dehydroscoulerine (40).
other substrates and underwent a two-electron oxidation to In this case, the second oxidation step did not appear to occur
complete the C-ring aromatization found in the sanguinarine spontaneously but was either enzymatic or the result of imi-
backbone structure (Fig. 3). DBOX also accepted dihydrochel- nium ion disproportionation. Opium poppy BBE1 and BBE2
erythrine at 29% of the turnover rate compared with dihydro- did not accept (S)-scoulerine but did exhibit STOX-like activity
sanguinarine, which was in contrast with partially purified with (RS)-tetrahydrocolumbamine and (RS)-canadine, yielding
DBOX from E. californica that did not convert dihydrochel- fully aromatized products (Fig. 3). Opium poppy DBOX cata-
erythrine (23). Interestingly, purified DHBO from S. canadensis lyzed only a two-electron oxidation of (S)-scoulerine, yielding
accepted dihydrochelerythrine as a substrate (10). The sub- two products with nearly identical CID spectra (supplemental
strate preference of opium poppy DBOX, combined with the Fig. S10, A–C). We propose that DBOX initially forms a single
metabolic effects of silencing DBOX in planta, strongly sup- iminium ion product, which in turn experiences a rearrange-
ports the physiological role of the enzyme in sanguinarine bio- ment of electrons around the nitrogen to form an isomer (sup-
synthesis. The molecular mass of opium poppy DBOX (58 kDa) plemental Fig. S10D). Whether DBOX-catalyzed oxidation
(supplemental Fig. S8) was similar to that of E. california DBOX occurs between N7 and C14 or between N7 and C8 is not
(56 kDa) compared with that of DHBO (77 kDa) (11). E. califor- known. Recently, it was shown that feeding (S)-scoulerine or its
nica DBOX was suggested as a flavin-dependent enzyme with a structural isomer (S)-coreximine to insect cell cultures express-
covalently linked cofactor based on shared features with STOX ing A. mexicana or B. wilsoniae STOX yielded two-electron
from B. wilsoniae, such as inhibition by morin and dicumarol, oxidation products with a possible double bond at N7ϭC8 (39).
and the lack of a dissociable cofactor (23, 45). In contrast with However, evidence supporting the introduction of an N7ϭC8
opium poppy DBOX, DHBO is not flavinylated and depends (versus an N7ϭC14) double bond was not provided.
instead on copper as a cofactor.
In addition to benzophenanthridine and protoberberine sub-
Although the opium poppy and E. californica enzymes are strates, opium poppy DBOX also accepted the 1-benzyliso-
probably sequence orthologues, the Km of opium poppy DBOX quinoline alkaloid (RS)-tetrahydropapaverine (Figs. 3 and 4),
for dihydrosanguinarine was substantially higher (201 ␮M) than yielding two-electron (dihydropapaverine) and four-electron
that reported for E. californica DBOX (16 ␮M) and DHBO (6 (papaverine) oxidation products. The proposed stepwise mech-
␮M) (supplemental Fig. S2). A high Km for dihydrosanguinarine anism for papaverine formation involves the initial loss of a
is not surprising, considering the benzophenanthridine alka- C3-hydrogen to yield an N2ϭC3 double bond, followed by
loid levels in the plant. Depending on the opium poppy chemo- deprotonation at C4 to create the enamine 1,2-dihydropapav-
type, the relative proportion of dihydrosanguinarine and san- erine (47). The imine-to-enamine isomerization is followed by
guinarine in roots can vary from approximately equal to largely the introduction of a double bond at C1ϭN2 to form papaver-
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Flavoprotein Oxidase from Opium Poppy
ine. Radioactive tracer experiments with opium poppy seed- tion, suggesting the possible direct or indirect involvement of
lings revealed the presence of 1,2-dihydropapaverine upon DBOX in phthalideisoquinoline alkaloid biosynthesis. How-
feeding (RS)-tetrahydropapaverine or (RS)-laudanosine in sup- ever, an off-target shift in BIA metabolism triggered by the
port of this mechanism (41). Whether DBOX-generated dihy- knockdown of DBOX transcript is equally plausible.
dropapaverine represents the 1,2-dihydro or 3,4-dihydro iso-
An initial expectation was that an enzyme exhibiting STOX
mer is not known because the purported diagnostic fragment activity would be responsible for the oxidation of (S)-reticuline
ions following CID analysis (m/z 190 and 204, respectively) (41) to 1,2-dehydroreticuline as the first step toward the generation
were detected at equally low abundance (supplemental Fig. of (R)-reticuline required for morphinan alkaloid biosynthesis
S5A). Tetrahydropapaverine and dihydropapaverine occur nat- (Fig. 1). Although the nature of the DRS reaction is similar to
urally in opium poppy at much lower levels than papaverine others catalyzed by DBOX, (S)-reticuline was not accepted in
(16), suggesting that physiological conditions strongly favor vitro, and silencing DBOX had no apparent effect on reticuline
papaverine formation. The inability of DBOX to convert (S)- or thebaine levels (supplemental Fig. S14). The lack of altera-
norreticuline (Fig. 3 and supplemental Fig. S12) was unex- tions in the major alkaloid content levels of FADOX1-,
pected because native STOX was reported to introduce a dou- FADOX3-, and FADOX8-silenced poppy plants excluded the
ble bond at C1ϭN2, yielding 1,2-dehydronorreticuline (5). The possibility that another FADOX enzyme functioned as DRS
acceptance of (RS)-tetrahydropapaverine, but not (S)-norreti- (supplemental Fig. S16). Although the possibility cannot be
culine or (RS)-norlaudanosoline (Fig. 3) suggests a requirement ruled out that an additional FADOX homologue could possess
for fully O-methylated 1-benzylisoquinoline substrates.
DRS activity, it is equally possible that an entirely different
The in vitro capacity of DBOX to convert different BIA struc- enzyme catalyzes this key reaction.
tural subgroups suggests that the enzyme participates in more
Acknowledgments—We thank Ye Zhang and Christoph Sensen (Vis-
than one biosynthetic pathway. The use of VIGS as a functional
genomics tool to investigate the physiological role of BIA bio-
synthetic genes is well established in opium poppy (3, 16, 48,
49). The restriction of DBOX transcripts to roots (Fig. 5 and
supplemental Fig. S13) was in contrast to the occurrence of BBE
transcripts, which were most abundant in roots but also present
in stems (supplemental Fig. S13). The significant reduction of
both sanguinarine and dihydrosanguinarine levels in BBE-si-
lenced opium poppy plants (Fig. 6) was consistent with the
action of BBE at an entry point to the benzophenanthridine
alkaloid branch pathway (supplemental Fig. S14). The lower
sanguinarine content of DBOX-silenced plants was also con-
sistent with the in vitro function of the enzyme (Fig. 6). How-
ever, the concomitant reduction of dihydrosanguinarine levels
in DBOX-silenced plants suggests additional mechanisms of
metabolic control. For example, lower sanguinarine levels or
initially higher dihydrosanguinarine levels could trigger regula-
tory alterations in the activity of other relevant BIA biosyn-
thetic enzymes. Interestingly, levels of upstream pathway
intermediates, such as protopine (Fig. 6) and stylopine (supple-
mental Fig. S15) were unaffected by DBOX silencing.
ual Genomics Centre, University of Calgary) for bioinformatics
support.
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DECEMBER 14, 2012•VOLUME 287•NUMBER 51
JOURNAL OF BIOLOGICAL CHEMISTRY 42983

Plant Biology:
Characterization of a Flavoprotein Oxidase
from Opium Poppy Catalyzing the Final
Steps in Sanguinarine and Papaverine
Biosynthesis
Jillian M. Hagel, Guillaume A. W. Beaudoin,
Elena Fossati, Andrew Ekins, Vincent J. J.
Martin and Peter J. Facchini
J. Biol. Chem. 2012, 287:42972-42983.
doi: 10.1074/jbc.M112.420414 originally published online November 1, 2012
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