isolation and characterization of o-methyltransferases …...isolation and characterization...

Isolation and Characterization of O-methyltransferasesInvolved in the Biosynthesis of Glaucine inGlaucium flavum1

Limei Chang, Jillian M. Hagel, and Peter J. Facchini*

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterizedO-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionallytested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 49-OMT, reticuline7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy(G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C69 coupling and four O-methyl groups at C6, C7, C39, andC49 as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differentialsubstrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showeda preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 39 and 49 hydroxyl groups of certain alkaloids.GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 49OMT and catalyzed the 6-O-methylation ofnorlaudanosoline, but more efficiently 49-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and itsN-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 39-O-methylated both reticuline and norreticuline.GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 39- and 79-O-methylations of several1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine andtetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinationsof Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase fromCoptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with theO-methylation patterns of alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry.

Glaucine is a benzylisoquinoline alkaloid (BIA) ofthe aporphine subclass produced in members of thePapaveraceae, including yellowhorned poppy (Glauciumflavum; Lapa et al., 2004), Glaucium oxylobum (Morteza-Semnani et al., 2003), and Corydalis yanhusuo (Xu et al.,2004), and in some plants such as Croton lechleri of theEuphorbiaceae (Milanowski et al., 2002). Glaucine func-tions as a phosphodiesterase-4 inhibitor and calciumchannel blocker (Cortijo et al., 1999), displays broncho-dilator and antiinflammatory effects, and is used to treatcoughs and asthma in Iceland and several eastern Euro-pean countries (Dargan et al., 2008). Glaucine has alsobeen shown to decrease heart rate, lower blood pressure(Orallo et al., 1995), and relieve pain, although less ef-fectively than other analgesics (Zetler, 1988). Side effects

such as sedation, fatigue, and hallucinations have con-tributed to the increased recreational use of glaucine as apsychoactive drug (Dargan et al., 2008). G. flavum is na-tive to Europe, northern Africa, Macaronesia, westernAsia, and the Caucasus, growing exclusively on sea-shores, and the plant is an introduced species and nox-ious weed in some parts of North America.

Although the reaction sequence is not known, the bio-synthesis of glaucine begins with the central BIA inter-mediate (S)-norcoclaurine (Hagel and Facchini, 2013),which must undergo (1) 39-hydroxylation, (2) 6-, 7-, 39-,and 49-O-methylations, (3) N-methylation, and (4) oxida-tive C8-C69 coupling (Fig. 1A). Initial tracer experimentsin wild bleeding heart (Dicentra eximia; Papaveraceae)suggested that glaucine and other aporphine alkaloidswere derived from norprotosinomenine (Battersbyet al., 1971), which is norcoclaurine substituted via39-hydroxylation, and 7- and 49-methoxylation. However,later tracer studies inbollywood (Litsea glutinosa; Lauraceae)indicated that the key BIA branch-point intermediate(S)-reticuline (Hagel and Facchini, 2013) was preferentiallyconverted by C8-C69 oxidative coupling to (S)-isoboldine,which is subsequently O-methylated at C39 andC7 yielding glaucine (Bhakuni and Jain, 1988). Othersubstituted 1-benzylisoquinolines, including proto-sinomenine (N-methylated norprotosinomenine), ori-entaline (6- and 39-methoxy-substituted norcoclaurine),and laudanosine (6-, 7-, 39-, and 49-methoxy-substitutednorcoclaurine) were also incorporated at lower levels.

1 This work was supported by Genome Canada, Genome Alberta,the Government of Alberta, the Canada Foundation for Innovation,and the Natural Sciences and Engineering Research Council of Can-ada (to P.J.F.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Peter J. Facchini ([email protected]).

L.C. conducted most of the experiments and cowrote the article;J.M.H. performed the mass spectrometry analysis; P.J.F. conceivedand supervised the research and cowrote the article.

www.plantphysiol.org/cgi/doi/10.1104/pp.15.01240

Plant Physiology�, October 2015, Vol. 169, pp. 1127–1140, www.plantphysiol.org � 2015 American Society of Plant Biologists. All Rights Reserved. 1127

https://plantphysiol.orgDownloaded on April 27, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.15.01240

https://plantphysiol.org

Several S-adenosyl-L-Met (SAM)-dependent, and sub-strate- and regiospecific, O-methyltransferases (OMTs)involved in BIA biosynthesis have been isolated (Fig. 1B),including (1) 6OMT from Japanese goldthread (Coptisjaponica; Morishige et al., 2000) and opium poppy(Papaver somniferum; Ounaroon et al., 2003); (2) N7OMTfrom opium poppy (Pienkny et al., 2009); (3) 7OMT fromopium poppy (Ounaroon et al., 2003); (4) 49OMT from C.japonica (Morishige et al., 2000), opium poppy (Ziegleret al., 2005), and California poppy (Eschscholzia californica;Inui et al., 2007); (5) SOMT from C. japonica (Takeshitaet al., 1995) and opium poppy (Dang and Facchini, 2012);and (6) CoOMT from C. japonica (Morishige et al., 2002).In addition, a 6-O -methylnorlaudanosoline 39-OMT (Fig.1B) was detected in Argemone platyceras cell cultures(Rueffer et al., 1983). SOMT and CoOMT show a pre-ference for the protoberberine substrates (S)-scoulerineand (S)-tetrahydrocolumbamine, respectively (Fig. 1B),and are involved in the biosynthesis of diverse com-pounds such as berberine and noscapine (Hagel andFacchini, 2013).

The monophyletic origin of BIA metabolism in mem-bers of the order Ranunculales (Liscombe et al., 2005)provides a foundation for the application of biochemicalgenomics to the isolation of genes encoding enzymevariants with similar or unique catalytic functions. Wehave established deep transcriptome resources based on acombination of 454 and Illumina sequencing for 20 plantspecies representing four families in the Ranunculales,includingG. flavum (Xiao et al., 2013). The availability of atranscriptome database for yellowhorned poppy roots, inwhich glaucine accumulates to high levels, provided anopportunity to functionally characterize all expressedgenes encoding orthologs of known OMTs involved inBIA biosynthesis using the reported signature substratefor each enzyme. We hypothesized that variants of eachenzyme would be detected, and that the missing 39OMTwould be identified. In this article, we report the func-tional characterization of seven OMT orthologs fromG. flavum roots with respect to BIAmetabolism. Enzymescatalyzing efficientO-methylation at all positions on both1-benzylisoquinoline and protoberberine scaffolds wereidentified. However, most active enzymes were multi-functional, accepting several substrates and efficientlytargeting more than one position. The efficacy of thedetected catalytic activities was confirmed in vivo usingvarious combinations of Escherichia coli strains expressingindividual recombinant enzymes. Inclusion of a strainproducing recombinant coclaurine N-methyltransferase(CNMT) from C. japonica allowed certain strain combi-nations to convert norlaudanosoline (39-hydroxylatednorcoclaurine; Fig. 1) to laudanosine, which possessesthe same O-substitutions as glaucine, but lacks the

Figure 1. Substitutions involved in the conversion of (S)-norcoclaurineto (S)-glaucine (A), and known O-methyltransferase reactions inBIA biosynthesis (B). Substitutions are indicated in red. 6OMT,Norcoclaurine/norlaudanosoline 6-O-methyltransferase; 39OMT,6-O-methylnorlaudanosoline 39-O-methyltransferase; N7OMT,

norreticuline 7-O-methyltransferase; 7OMT, reticuline 7-O-methyl-transferase; 49OMT, 6-O-methyllaudanosoline 49-O-methyltransferase;SOMT, scoulerine 9-O-methyltransferase; CoOMT, tetrahydrocolumb-amine O-methyltransferase.

1128 Plant Physiol. Vol. 169, 2015

Chang et al.



C8-C69 coupling. The primary O-methylation reactionsequence(s) were identified.

RESULTS

Selection of G. flavum O-Methyltransferases

Seven orthologs of characterized OMTs involved inBIA biosynthesis were isolated from assembled 454 andIllumina Genome Analyzer databases (Xiao et al., 2013)of G. flavum (GFL). The candidate selection strategy wasbased on a cutoff of 40% amino acid sequence identitycompared with at least one functionally characterizedOMT involved in BIA metabolism (Supplemental TableS1). Phylogenetic analysis showed that GFLOMT1 toGFLOMT4 and GFLOMT6 formed separate clades withcharacterized OMTs (Fig. 2), whereas GFLOMT5 andGFLOMT7 formed a new clade. GFLOMT1 shares 77%and 71% amino acid sequence identities with Ps49OMT2from opium poppy and Cj49OMT from C. japonica, re-spectively. GFLOMT2 shows 80% and 70% sequenceidentities with Ps6OMT from opiumpoppy andCj6OMTfrom C. japonica, respectively. GFLOMT3 shares 63% se-quence identities with Ps7OMT from opium poppy, andGFLOMT4 shows 38% sequence identity with CjCoOMTfrom C. japonica. GFLOMT6 shares 60% and 63% se-quence identities with PsSOMT from opium poppy andCjSOMT from C. japonica, respectively. In contrast,GFLOMT5 and GFLOMT7 display only 42% and 44%sequence identity with the nearest neighbor CjSOMTfrom C. japonica, respectively. Phylogenetic relationshipsbased on overall sequence identity were used to formu-late an initial hypothesis that 49OMT, 6OMT, 7OMT,SOMT, and CoOMT functions were associated withGFLOMT1, GFLOMT2, GFLOMT3, GFLOMT4, andGFLOMT6 orthologs in G. flavum. Moreover, the un-known 39OMTactivity required for glaucine biosynthesiswas proposed to reside with GFLOMT5 or GFLOMT7,which do not clade with characterized enzymes.

Purification and Characterization of GFLOMTs

Full-length complementary DNAs (cDNAs) for theseven GFLOMT candidates were cloned into the pRSETAexpression vectorwith anN-terminal His-tag translationalfusion. Recombinant GFLOMTs were purified from totalprotein extracts using a cobalt-affinity resin. All purifiedrecombinant enzymes displayed Mr values corre-sponding to expected protein sizes, as determined bySDS-PAGE (Supplemental Fig. S1). Enzyme assayswere performed on each of the purified, His-taggedrecombinant proteins to screen for O-methylation ac-tivity using seven compounds that serve as the sig-nature substrates for characterized OMTs involved inBIA metabolism (Fig. 1B).In the presence of SAM, GFLOMT1 showed differen-

tial activity with all seven substrates (Table I; Fig. 3).Norlaudanosoline was the preferred substrate, dis-playing 96% conversion in the standard OMT assay;

however, 6-O-methylnorlaudanosoline (87%) and 6-O-methyllaudanosoline (68%) were also efficiently con-verted. Scoulerine (32%), tetrahydrocolumbamine (19%),and reticuline (6%) were also accepted, but with rela-tively lower conversion efficiencies. Norlaudanosolinewas also the best substrate for GFLOMT2 (100%),whereas 6-O-methylnorlaudanosoline (1%) and 6-O-methyllaudanosoline (14%) were not efficiently con-verted (Table I; Fig. 4). In further contrast to GFLOMT1,scoulerine (75%), tetrahydrocolumbamine (32%), andreticuline (22%) were accepted with relatively higherconversion efficiencies. Scoulerine was the preferredsubstrate for GFLOMT6 (100%), with reticuline (97%) andtetrahydrocolumbamine (90%) also efficiently converted,and norreticuline (36%) and 6-O-methyllaudanosoline(23%) accepted at moderate levels (Table I; Fig. 5).GFLOMT6 did not accept norlaudanosoline or 6-O-methylnorlaudanosoline. GFLOMT7 showed relativelylow activity with scoulerine (12%) and tetrahydro-columbamine (8%), but did not accept other BIAs (Table I;Fig. 5). GFLOMT3, GFLOMT4, and GFLOMT5 did notshow activity with any of the tested substrates (Table I).

Kinetic analyses with preferred substrates yieldingsingle reaction products showed that the three mostactive GFLOMTs followed the Michaelis-Mentenmodel (Supplemental Fig. S2). GFLOMT1 exhibited a Kmof 12 mM for 6-O-methylnorlaudanosoline, GFLOMT2showed a Km of 15 mM for norlaudanosoline, andGFLOMT6 displayed a Km of 22 mM for scoulerine

Figure 2. Unrooted neighbor-joining phylogenetic tree combiningfunctionally characterized OMTs involved in BIA metabolism andseven orthologs from yellow horned poppy. Bootstrap frequencies foreach cladewere based on 1,000 iterations. Abbreviations andGenBankaccessions numbers for each enzyme are listed in the “Materials andMethods.” The scale bar corresponds to 0.2 amino acid substitutions persite.

Plant Physiol. Vol. 169, 2015 1129

Benzylisoquinoline Alkaloid OMTs


http://www.plantphysiol.org/cgi/content/full/pp.15.01240/DC1





(Table II). Catalytic efficiencies (kcat/Km) were relativelyhigh for all three conversions.

Reaction Product Identification

Enzyme assays were subjected to positive-modeelectrospray ionization (ESI[+]) liquid chromatography(LC)-tandem mass spectrometry (MS/MS) for reactionproduct characterization, including collision-induceddissociation (CID) fragmentation analysis. ESI[+]-CIDof 1-benzylisoquinoline and protoberberine alkaloids atlow ionization energy yields isoquinoline and/or benzylmoieties as major ion fragments. Using the ESI[+]-CIDspectra of authentic standards (Supplemental Table S2),the identity of recombinant GFLOMT reaction productswas determined (Supplemental Table S3). Positions ofnew O-methyl groups could be inferred from the in-creased mass-to-charge ratio (m/z; in multiples of 14 D)of dissociated isoquinoline and benzyl ion fragmentseven in the absence of authentic standards, althoughmost were available.

Incubation of GFLOMT1 with norlaudanosoline (m/z288) yielded twomajor peaks, withm/z 302 at 2.62minand m/z 316 at 2.95 min (Fig. 3A), suggesting singleand double O-methylation events, respectively. Theparent ion with m/z 302 produced an ESI[+]-CIDspectrum corresponding to authentic 6-O-methyl-norlaudanosoline, whereas the parent ion withm/z 316yielded an ESI[+]-CID spectrum matching that ofnorreticuline. Assays containing GFLOMT1 and 6-O-methylnorlaudanosoline (m/z 302) generated major andminor products ofm/z 316 andm/z 330, with ESI[+]-CIDspectra corresponding to norreticuline and norcodamine,respectively (Fig. 3B). Although an authentic standardfor norcodamine was not available, compound iden-tity could be inferred. Compared with the ESI[+]-CIDspectrum of 6-O-methylnorlaudanosoline, which dis-plays the fragment ions m/z 178 (isoquinoline moiety)and m/z 123 (benzyl moiety), the m/z 330 reactionproduct yieldedmajor fragment ions ofm/z 178 andm/z 151 (increase of 28 D), the latter of which correspondsto a 39- and 49-O-methylated benzyl moiety. Incubation

of GFLOMT1 with 6-O-methyllaudanosoline (m/z 316)yielded major and minor products with m/z 330 at 2.99min andm/z 344 at 3.17min, corresponding to single anddouble O-methylation events, respectively (Fig. 3C). Them/z 330 parent ion produced an ESI[+]-CID spectrumcorresponding to authentic reticuline, whereas thedouble O-methylated m/z 344 parent ion yielded anESI[+]-CID spectrum matching that of codamine. Inassays containing GFLOMT1 and reticuline, a minorproduct of m/z 344 with an ESI[+]-CID spectrumcorresponding to codamine was also produced (Fig.3D). The major and minor products resulting fromthe incubation of GFLOMT1 with scoulerine showedparent ions of m/z 342 and m/z 356, with ESI[+]-CIDspectra corresponding to tetrahydropalmatrubine andtetrahydropalmatine (Fig. 3E). An authentic standard fortetrahydropalmatrubine was not available; however,product identification was inferred from the 14-D increasein the isoquinolinemoiety of scoulerine (m/z 178) tom/z192. In assays containing GFLOMT1 and tetrahy-drocolumbamine, aminor productwas generatedwith aparent mass of m/z 356 and an ESI[+]-CID spectrumcorresponding to tetrahydropalmatine (Fig. 3F).

GFLOMT2 efficiently converted norlaudanosoline(m/z 288) to a product with m/z 302, which yield-ed an ESI[+]-CID spectrum corresponding to 6-O-methylnorlaudanosoline (Fig. 4A). In contrast, GFLOMT2incubated with 6-O-methylnorlaudanosoline generated aminor product withm/z 316 (Fig. 4B), whichwas inferredas nororientaline based on the detection of major frag-ment ions of m/z 178 (isoquinoline moiety) and m/z 137(39-O-methylated benzyl moiety). The different retentiontime compared with norreticuline (i.e. 49-O-methylated6-O-methylnorlaudanosoline) confirmed 39- rather than49-O-methylation. Incubation of GFLOMT2 with 6-O-methyllaudanosoline (m/z 316) yielded three productswith m/z 330 at 2.85 min, m/z 330 at 2.98 min, and m/z344 at 3.15 min, indicating both single and double O-methylation events (Fig. 4C). The identity of the m/z 330parent ion at 2.85 min was inferred as orientalinebased on the detection of major fragment ions of m/z192 (isoquinoline moiety) and m/z 137 (39-O-methylated benzyl moiety). The different retention time

Table I. Substrate range of GFLOMTs

Values represent the percentage of each substrate converted to reaction product(s) in a standard assay. Specific enzyme activities are provided inthe footnote. nd, Not detected.

SubstrateEnzyme

GFLOMT1 GFLOMT2 GFLOMT3 GFLOMT4 GFLOMT5 GFLOMT6 GFLOMT7

(%)(R,S)-norlaudanosoline 100a 100b nd nd nd nd nd(R,S)-6-O-methylnorlaudanosoline 90 1 nd nd nd nd nd(R,S)-6-O-methyllaudanosoline 71 14 nd nd nd 23 nd(R,S)-norreticuline nd nd nd nd nd 36 nd(S)-reticuline 6 22 nd nd nd 97 nd(R,S)-scoulerine 3 75 nd nd nd 100c 100d

(R,S)-tetrahydrocolumbamine 20 32 nd nd nd 91 67

a2,560 nmol min21 mg21 protein. b2,667 nmol min21 mg21 protein. c2,667 nmol min21 mg21 protein. d320 nmol min21 mg21 protein.


Chang et al.





compared with reticuline (i.e. 49-O-methylated 6-O-methyllaudanosoline and the m/z 330 parent ion at2.98 min) confirmed 39- rather than 49-O-methylation.The doubleO-methylation productwithm/z 344 yieldedan ESI[+]-CID spectrum corresponding to codamine. Theminor GFLOMT2 reaction product of reticuline alsoshowed a parent mass of m/z 344 and an ESI[+]-CIDspectrum corresponding to codamine (Fig. 4D). Incuba-tion of GFLOMT2 with scoulerine (m/z 328) yieldedmajor and minor products with m/z 342 and m/z 356,identified as tetrahydropalmatrubine and tetrahy-dropalmatine, respectively (Fig. 4E). The reaction productof GFLOMT2 incubated with tetrahydrocolumbamine

showed a parent mass of m/z 356, which was also iden-tified as tetrahydropalmatine (Fig. 4F).

Incubation of GFLOMT6with 6-O-methyllaudanosoline(m/z 316) yielded a reaction product with m/z 330 (Fig.5A),whichwas inferred as 6,7-O,O-dimethyllaudanosolinebased on ESI[+]-CID spectrum and a unique retentiontime. Compared with the ESI[+]-CID spectrum of 6-O-methyllaudanosoline, which displayed major fragmentions ofm/z 192 andm/z 123, them/z 330 reaction productyielded fragment ions of m/z 206 (i.e. the 7-O-methylatedisoquinoline moiety) and m/z 123 (i.e. the unchangedbenzyl moiety). Incubation of GFLOMT6 with norreticu-line (m/z 316) yielded two major and one minor reaction

Figure 3. Total ion chromatograms showing theO-methylation activityof recombinant GFLOMT1 on various substrates. Numbers in squarebrackets correspond to identified reaction products based on retentiontimes and ESI[+]-CID spectra (Supplemental Table S3).

Figure 4. Total ion chromatograms showing theO-methylation activityof recombinant GFLOMT2 on various substrates. Numbers in squarebrackets correspond to identified reaction products based on retentiontimes and ESI[+]-CID spectra (Supplemental Table S3).







products with m/z 330 at 3.16 min, m/z 330 at 3.25 min,and m/z 344 at 3.43 min (Fig. 5B), corresponding to singleand double O-methylation events. Identity of the m/z 330parent ion at 3.16 min was inferred as norcodamine based

on the ESI[+]-CID spectrum and a unique retentiontime. The m/z 330 parent ion at 3.25 min produced anESI[+]-CID spectrum corresponding to norlaudanine. Theminor double O-methylation product with m/z 344 pro-duced an ESI[+]-CID spectrum matching that of tetrahy-dropapaverine. GFLOMT6 efficiently converted reticuline(m/z 330) to three products withm/z 344 at 3.17 min,m/z344 at 3.24 min, and m/z 358 at 3.43 min (Fig. 5C), indi-cating single and double O-methylation events. The m/z344 parent ions at 3.17 and 3.24 min yielded ESI[+]-CIDspectra corresponding to codamine and laudanine. Thedouble O-methylation product with a parent ion of m/z358 generated an ESI[+]-CID spectrum matching that oflaudanosine. GFLOMT6 efficiently converted scoulerine(m/z 328) to two major reaction products with m/z 342 at3.29 min andm/z 356 at 3.59 min (Fig. 5D), correspondingto single and double O-methylation events, respectively.Them/z 342 parent ion produced an ESI[+]-CID spectrumcorresponding to tetrahydrocolumbamine, whereas them/z 356 parent ion yielded an ESI[+]-CID spectrummatching that of tetrahydropalmatine. Incubation ofGFLOMT6 with tetrahydrocolumbamine (m/z 342) gen-erated a major product with m/z 356 and an ESI[+]-CIDspectrumcorresponding to tetrahydropalmatine (Fig. 5E).

Incubation of GFLOMT7 with scoulerine (m/z 328)yielded twominor reaction productswithm/z 342 at 3.33min and m/z 342 at 3.44 min (Fig. 5F), with ESI[+]-CIDspectra corresponding to tetrahydrocolumbamine (i.e.the methylated benzyl moiety of scoulerine) and tetra-hydropalmatrubine (i.e. the methylated isoquinolinemoiety of scoulerine), respectively. The reaction productof GFLOMT7 incubated with tetrahydrocolumbamine(m/z 342) generated a reaction product with m/z 356and an ESI[+]-CID spectrum corresponding to tetra-hydropalmatine (Fig. 5G).

Transformations of Norlaudanosoline in E. coli

(R,S)-norlaudanosoline was fed to mixed cultures ofE. coli harboring different combinations and permutationsof the expression constructs pGFLOMT1, pGFLOMT2,pGFLOMT6, and pCNMT to determine the in vivo effi-ciency of each OMT using both N-methylated andN-desmethyl 1-benzylisoquinolines (Fig. 6). Norlauda-nosolinewas not recovered in ethyl acetate extractions orwas not detected. The empty vector control showed thatE. coli was inherently incapable of transforming norl-audanosoline to other BIAs (Fig. 6A). Transformationproduct identifications (Supplemental Table S4) weredetermined using the ESI[+]-CID spectra of authenticstandards and inferences described above.

Incubation of an E. coli strain harboring pGFLOMT1with norlaudanosoline (m/z 288) yielded one com-pound identified as norcodamine (m/z 330; Fig. 6B).In contrast, incubation of an E. coli strain harboringpGFLOMT2 with norlaudanosoline generated threeproducts withm/z 316 at 2.85 min,m/z 330 at 3.15 min,and m/z 344 at 3.42 min (Fig. 6C), corresponding tonororientaline, norcodamine, and tetrahydropapaverine,

Figure 5. Total ion chromatograms showing the O-methylation activity ofrecombinantGFLOMT6 (A–E) andGFLOMT7 (FandG)onvarious substrates.Numbers in square brackets correspond to identified reaction products basedon retention times and ESI[+]-CID spectra (Supplemental Table S3).


Chang et al.





respectively. Incubation of mixed E. coli strains harboringpGFLOMT1 and pGFLOMT2 with norlaudanosolineyielded compounds with m/z 330 at 3.15 min and m/z344 at 3.42 min (Fig. 6D), corresponding to norcod-amine and tetrahydropapaverine, respectively. Addi-tion of an E. coli strain harboring pCNMT to this seriesaltered the profile of products formed in all pGFLOMTcombinations. Incubation of mixed E. coli strains har-boring pGFLOMT1 and pCNMT with norlaudanoso-line yielded products withm/z 330 at 2.99 min andm/z344 at 3.17 min, which were identified as reticuline andcodamine (Fig. 6E). Incubation of mixed E. coli strainsharboring pGFLOMT2 and pCNMT with norlaudano-soline resulted in the production of four compoundswith m/z 330 at 2.85 min, m/z 330 at 2.98 min, m/z 344at 3.16 min, and m/z 358 at 3.4 min, correspondingto orientaline, reticuline, codamine, and laudanosine(Fig. 6F). Incubation of mixed E. coli strains harboringpGFLOMT1, pGFLOMT2, and pCNMT with norlau-danosoline also produced orientaline, reticuline, cod-amine, and laudanosine, but with an apparently higheryield compared with incubations lacking one of thestrains (Fig. 6G).Incubation of mixed E. coli strains harboring pGFLO-

MT1 and pGFLOMT6 with norlaudanosoline yieldedtwo products with m/z 330 at 3.14 min and m/z 344 at3.43 min, corresponding to norcodamine and tetrahy-dropapaverine, respectively (Fig. 6H). Incubation ofmixedE. coli strains harboring pGFLOMT2 and pGFLOMT6with norlaudanosoline generated three compounds withm/z 316 at 2.87 min, m/z 330 at 3.14 min, and m/z 344 at3.43 min, identified as nororientaline, norcodamine, andtetrahydropapaverine, respectively (Fig. 6I). CombiningE. coli strains harboring pGFLOMT1, pGFLOMT2, andpGFLOMT6 and incubating with norlaudanosoline onlychanged the relative abundance of nororientaline, nor-codamine, and tetrahydropapaverine (Fig. 6J) comparedwith the absence of pGFLOMT1 (Fig. 6I). However, ad-dition of an E. coli strain harboring pCNMT to this seriesaltered the profile of products generated by all pGFLOMTcombinations. Incubation ofmixedE. coli strains harboringpGFLOMT1, pGFLOMT6, and pCNMT with norlauda-nosoline produced five compounds with m/z 330 at2.99 min,m/z 330 at 3.16 min,m/z 344 at 3.18 min,m/z344 at 3.24 min, and m/z 358 at 3.42 min, which wereidentified as reticuline, norcodamine, codamine, lau-danine, and laudanosine, respectively (Fig. 6K). MixedE. coli strains harboring pGFLOMT2, pGFLOMT6, and

pCNMT converted norlaudanosoline to compoundswithm/z 330 at 2.88 min, m/z 330 at 3 min, m/z 344at 3.16min,m/z 344 at 3.23min, andm/z 358 at 3.42min,corresponding to orientaline, reticuline, codamine, lau-danine, and laudanosine, respectively (Fig. 6L). Finally,incubation of mixed E. coli strains harboring pGFLOMT1,pGFLOMT2, pGFLOMT6, and pCNMT with norlauda-nosoline produced the same five compounds, but withan apparently higher yield than incubations lacking theE. coli strain harboring pGFLOMT1 (Fig. 6M).

GFLOMT Gene Expression and Alkaloid Accumulationin G. flavum

GFLOMT1, GFLOMT2, and GFLOMT6 showed gener-ally higher levels of expression in different G. flavum or-gans compared with genes encoding other OMTs (Fig.7A). Overall, transcripts encoding GFLOMT1, GFLOMT2,and GFLOMT6 were also relatively lower in stems com-pared with other organs. High-resolution FTMS per-formed in positive ion mode on corresponding G. flavumorgans yielded unbiased exact mass data sets, each con-taining.200 putativemetabolites (Supplemental Data SetS1). Comparison of the retention times and CID spectraof authentic standards (Farrow et al., 2012) facilitatedthe annotation of putative alkaloids, seven of whichcould be unambiguously identified (SupplementalTable S5). Glaucine was the predominant alkaloid inaerial organs, occurring at levels up to 100-fold higherthan the combined accumulation of all other identifiedBIAs (Fig. 7B). Conversely, the benzophenanthridinealkaloids sanguinarine and chelerythrine, and the pro-topine alkaloids, in particular protopine and allo-cryptopine, predominated in root (Fig. 7C). Levels ofthe aporphine alkaloids isocorydine and glaucine weresimilar in root, but isocorydine accumulation was rel-atively low in aerial organs.

DISCUSSION

Elucidation of BIA metabolic pathways in plants hasadvanced rapidly over the past decade with a nearcomplete catalog of biosynthetic genes and correspond-ing enzymes involved in the formation of major pro-ducts, including berberine, sanguinarine, morphine, andnoscapine (Hagel and Facchini, 2013; Dang et al., 2015).Among the BIA biosynthetic genes not yet isolated is

Table II. Kinetic data for GFLOMT1, GFLOMT2, and GFLOMT6

Enzyme Substrate Km Vmax kcat kcat/Km

(mM) (nmol min21 mg21 protein) (s21) (M21 s21)GFLOMT1 6-O-methylnorlaudanosoline 17.2 6 2.8 2,360 6 204 0.93 6 0.04 54,100

S-adenosyl-Met 24.3 6 4.5 2,442 6 230 0.96 6 0.05 39,500GFLOMT2 Norlaudanosoline 18.3 6 2.5 3,591 6 255 2.78 6 0.09 151,900GFLOMT2 Scoulerine 31.0 6 5.0 3,220 6 253 1.25 6 0.06 40,300

S-adenosyl-Met 27.1 6 7.1 4,110 6 529 3.18 6 0.22 117,300GFLOMT6 Scoulerine 26.4 6 4.1 4,343 6 332 3.68 6 0.15 139,200

S-Adenosyl-Met 27.4 6 4.6 4,079 6 337 3.46 6 0.16 126,300









one encoding an enzyme capable of the efficient 39-O-methylation of 1-benzylisoquinoline and/or aporphinesubstrates. Several major 1-benzylisoquinoline deriva-tives, such as glaucine in yellow horned poppy and pa-paverine in opium poppy, are tetra-O-methylated. Anumber of BIAOMTswith a wide range of substrate andregiospecificity have been isolated and characterizedfrom opium poppy although none have shown effici-ent 39-O-methylation activity with 1-benzylisoquinolinesubstrates (Ounaroon et al., 2003; Ziegler et al., 2005;Pienkny et al., 2009; Dang and Facchini, 2012). Theavailability of deep transcriptome resources for severalBIA-producing plant species (Xiao et al., 2013) facili-tated a more comprehensive approach to the character-ization of OMT orthologs involved in the biosynthesis ofunique compounds, such as glaucine, with the isolationof a 39OMT as a principal objective. The monophyleticorigin of BIA biosynthetic genes in members of theRanunculaceae suggests that orthologs in opium poppyand other plant species could then be readily identified(Liscombe et al., 2005).

Despite names suggesting strict functions, charac-terized BIA OMTs often display a range of substrateand regiospecificities. For example, among several1-benzylisoquinoline and protoberberine substratestested, opium poppy 6OMT specifically 6-O-methylated(R,S)-norcoclaurine, whereas (R,S)-reticuline, (R,S)-ori-entaline, and (R,S)-protosinomenine were 7-O-methylatedby opiumpoppy 7OMT (Ounaroon et al., 2003).However,both enzymes also 7-O-methylated (R,S)-isoorientaline.In contrast, opium poppy N7OMT only accepted (S)-norreticuline among a variety of tested N-methylatedandN-desmethyl 1-benzylisoquinolines, including (R,S)-reticuline (Pienkny et al., 2009). Opium poppy 49OMTdisplayed its highest activity with (R,S)-6-O-methyl-laudanosoline and (R,S)-6-O-laudanosoline, but also lessefficiently accepted (R,S)-6-O-methylnorlaudanosoline and(R,S)-norlaudanosoline (Ziegler et al., 2005). 49-O-methyl-ation of all substrates was assumed, but only 6- and 7-O-methylations could be ruled out. Opium poppy SOMTefficiently 9-O-methylated (S)-scoulerine and showed2-O-methylation of (S)-tetrahydrocolumbamine, but alsoweakly 7- and 39-O-methylated (S)-reticuline and (S)-norreticuline (Dang and Facchini, 2012). Reported ortho-logs from other species, especially Japanese goldthread(C. japonica; Ranunculaceae), generally showed functionalsimilarity to opium poppy OMTs in terms of signaturesubstrates, but also displayed variances in their range ofsubstrate and regiospecificities. 6OMT from C. japonicaefficiently 6-O-methylated (R,S)-norcoclaurine, (R,S)-nor-laudanosoline, and (R,S)-laudanosoline, but weaklyO-methylated (R,S)-6-O-methylnorlaudanosoline, (S)-scou-lerine, and (R,S)-coclaurine, clearly not at the alreadyO-methylated 6-position in each case (Sato et al.,1993; Morishige et al., 2000). 49OMT from C. japonica

Figure 6. Total ion chromatograms showing the products of variouscombinations of E. coli strains harboring individual pGFLOMT con-structs, plus or minus a strain harboring pCNMT from C. japonica, andfed 25 mM (R,S)-norlaudanosoline. Cultures were incubated overnightand alkaloid extracts subjected to LC-MS/MS analysis. Product

identifications were based on retention times and ESI[+]-CID spectra(Supplemental Table S4). The empty expression vector served as thenegative control (A).


Chang et al.




preferred (R,S)-6-O-laudanosoline and (R,S)-6-O-methyllaudanosoline, but also 49-O-methylated thecorresponding N-desmethyl compounds (R,S)-norlau-danosoline and (R,S)-6-O-methylnorlaudanosoline, andshowed weak activity with (S)-scoulerine (Sato et al.,1993; Morishige et al., 2000). Similar to opium poppySOMT, the C. japonica ortholog showed low activitywith1-benzylisoquinolines such as (R,S)-norreticuline (Satoet al., 1993; Morishige et al., 2000). CoOMT fromC. japonica showed exclusive activitywithprotoberberines,preferring columbamine over (R,S)-scoulerine (Morishigeet al., 2002). Only limited information is available forOMTs from other species. California poppy (E. californica)has been suggested to utilize a single OMT with both49- and 6-O-methylation activities (Inui et al., 2007).SOMT from barberry (Berberis wilsonae; Berberidaceae)

appeared specific for the 9-O-methylation of (S)-scoulerine and did not accept other tested proto-berberines or (R,S)-reticuline (Muemmler et al., 1985).Finally, Berberis koetineuna 49OMT effectively 49-O-methylated (S)-6-O-methyllaudanosoline, (R,S)-6-O-laudanosoline, and (R,S)-7-O-methylnorlaudanosoline,and showed weak activity with (S)-norlaudanosoline(Frenzel and Zenk, 1990).

Homology searches of combined 454- and Illumina-generated transcriptome databases for G. flavum resultedin the isolation of seven candidate cDNAs encoding pro-teins with .40% amino acid identity to at least one char-acterized OMT involved in BIA metabolism (Fig. 2;Supplemental Table S1). The characterizedOMT, or groupof functionally related enzymes, displaying the maximumamino acid sequence identity was the basis for predictingsubstrate- and regiospecific activity for each GFLOMT.Partially purified recombinant protein obtained for eachGFLOMT (Supplemental Fig. S1) was assayed with thesignature substrate for each of seven characterized OMTs(Fig. 1). In one case, 6-O-methylnorlaudanosoline wasthe only substrate accepted by an apparently regiospecificenzyme partially purified from A. platyceras cell cultures(Rueffer et al., 1983), although the corresponding genehas not been isolated. In general, the signature substrateof the nearest phylogenetic orthologwas accepted by eachactive G. flavum OMT. GFLOMT1 exhibited the highestamino acid identity with characterized 49OMTs and cat-alyzed the 49-O-methylation of 6-O-methyllaudanosolineto reticuline (Fig. 3; Table I). GFLOMT2 showed thehighest amino acid sequence identity with characterized6OMTs (and with opium poppy N7OMT) and catalyzedthe 6-O-methylation of norlaudanosoline yielding 6-O-methylnorlaudanosoline (Fig. 4; Table I). GFLOMT6was the closest SOMT ortholog and catalyzed the 9-O-methylation of scoulerine to tetrahydrocolumbamine,but also efficiently 2-O-methylated tetrahydrocolumb-amine yielding tetrahydropalmatine (Fig. 5; Table I).However, the functions of the remaining GFLOMTs didnot correspond to those of characterized enzymes.GFLOMT3 displayed substantial amino acid sequenceidentity with 7OMTs, but did not accept reticuline or anyother tested 1-benzylisoquinoline or protoberberine sub-strates (Table I). Similarly, GFLOMT4 was the closestortholog to an uncharacterized enzyme from opiumpoppy (SOMT2; Dang and Facchini, 2012; Winzer et al.,2012), and GFLOMT5 showed low amino acid sequenceidentity with characterized SOMTs. However, neitherenzyme was active with any tested substrates. GFLOMT7also showed low amino acid sequence identity withcharacterized SOMTs and displayed relatively weak ac-tivity with scoulerine (Table I). However, both the 9- and2-hydroxyl positionswere independently targeted (Fig. 5).Interestingly, despite the depth of transcriptome resourcesfrom the alkaloid-rich roots of G. flavum, no ortholog of C.japonica CoOMT was detected (Supplemental Table S1).

An initial hypothesis for the identification of a 39OMTwas that orthologs displaying relatively low amino acidsequence identity with functionally characterized OMTs,none of which display efficient 39-O-methylation activity,

Figure 7. Quantitative reverse transcription-PCR showing the relativeexpression of seven GFLOMT genes (A) and high-resolution, Fourier-transformmass spectrometry (FTMS) of BIAs (B andC) in different organs ofG. flavum. Bars represent the mean 6 SD of four biological replicates.








could represent a unique substrate and regiospecificcatalyst. The only indication that a 39OMT might behighly substrate specific was based on the characteriza-tion of a partially purified A. platyceras enzyme, whichaccepted only 6-O-methylnorlaudanosoline (Ruefferet al., 1983). However, GFLOMT3, GFLOMT4, GFL-OMT5, or GFLOMT7 failed to accept this substrate.Further examination of the range of products producedby the five tested 1-benzylisoquinoline substratesshowed that GFLOMT1, GFLOMT2, and GFLOMT6catalyzed additional regiospecific O-methylations withdifferential efficiency, in addition to the aforemen-tioned signature reactions. The least multifunctionalenzyme was GFLOMT2, which appears to serve as anefficient 6OMT in G. flavum. Nevertheless, GFLOMT2also performed 39- and 49-O-methylations on various1-benzylisoquinolines, and functioned as a regiospe-cific scoulerine 2-O-methyltransferase yielding tetra-hydropalmatrubine (Fig. 4).

GFLOMT1 and GFLOMT6 were more versatile en-zymes, but also displayed striking functional differences.With 1-benzylisoquinoline substrates, GFLOMT1 wasrelatively efficient at the 6- and 49-O-methylation ofnorlaudanosoline and 6-O-methylnorlaudanosoline, re-spectively (Fig. 3), whereas GFLOMT6 did not acceptthese substrates (Table I). With less apparent efficiency,GFLOMT1 also catalyzed 39-O-methylations on sub-strates yielding low levels of codamine (Fig. 3). Incontrast, GFLOMT6 catalyzed the efficient 7- and 39-O-

methylation of norreticuline and reticuline, althoughthere was a preference forN-methylated substrates with,at minimum, a 6-O-methyl and ideally a 49-O-methylgroup (Fig. 5). Taking an unbiased and comprehensiveapproach to the functional characterization of OMTcandidates from an unexplored BIA-producing plant ledto both (1) corroboration of previously reported activitiesfor several orthologs and (2) unique and important in-sights into the associations between amino acid sequenceidentity, and substrate and regiospecificity. In G. flavum,we conclude that only three OMTs are responsible forfour and two O-methylations on 1-benzylisoquinolineand protoberberine substrates, respectively.

To test whether GFLOMT1, GFLOMT2, and GFLO-MT6 were capable of the efficient tetra-O-methylationof the 1-benzylisoquinoline scaffold, individual E. colistrains producing each enzyme (Supplemental Fig. S1)were combined in various permutations and combina-tions, with or without a strain producing a functionalCNMT, and fed norlaudanosoline. Analysis was pri-marily qualitative, although equivalent scaling allowedan estimate of relative product accumulation. Resultsfrom in vivo feeding (Fig. 6) were generally consistentwith in vitro assays (Figs. 3–5). As expected fromthe range of substrate and regiospecificity shown byGFLOMT1, GFLOMT2, and GFLOMT6 in vitro, the in-clusion of strains producing all three enzymes togetherwith the introduced capacity for N-methylation was themost effective combination for the production of the

Figure 8. Proposedmajor andminorO-methylation routes involved in theO-methylation of (R,S)-norlaudanosoline based on thedetected functions of GFLOMT1, GFLOMT2, and GFLOMT6. Diagonal blue arrows represent N-methylation reactions. Majorsubstrates and reaction products are highlighted in yellow. Enzymes in bold catalyze relatively efficient reactions compared withother conversions.


Chang et al.




6,7,49-, 6,39,49-, and 6,7,39,49-O-methylated compoundslaudanine, codamine, and laudanosine, respectively.Fewer products were obtained at lower levels in theabsence of CNMT, demonstrating the importance ofN-methylation to complete the tetra-O-methylation pro-cess, at least in G. flavum. Differences in substrate prefer-ence would be expected in species, including opiumpoppy, that produce high levels of an N-desmethyl1-benzylisoquinoline, such as papaverine. Although ahitherto untested OMT might catalyze efficient 39-O-methylation of 1-benzylisoquinolines in opiumpoppy, thesearch for this functionality should not preclude previ-ously characterized enzymes (Hagel and Facchini, 2013).GFLOMT1, GFLOMT2, and GFLOMT6 showed rela-

tively low Km values and high catalytic efficiencies as de-termined using substrates yielding a single product, andthus allowing the calculation of reliable kinetic data (TableII). Based primarily on the apparent in vitro conversionefficiency of each enzyme, supported by in vivo feedingexperiments, themajor routes for the transformation of thetetra-hydroxylated and N-desmethylated norlaudanoso-line to the tetra-methoxylated and N-methylatedlaudanosine were mapped (Fig. 8). Among all possibleO-methylation sequences involving bothN-desmethylatedand N-methylated intermediates (Supplemental Fig. S3),only routes through norreticuline and reticuline appearactive, consistent with the proposed glaucine biosyntheticpathway based on tracer feeding experiments (Bhakuniand Jain, 1988). For the formation of (S)-glaucine, C8-C69oxidative coupling of (S)-reticuline to (S)-isoboldine isalso consistent with substrate specificity of corytuberinesynthase (CYP80G2), which leads to the aporphinemagnoflorine in C. japonica (Ikezawa et al., 2008). Thestructural similarity of the 1-benzylisoquinoline andaporphine scaffolds suggests that GFLOMTs also usecertain aporphine derivatives as glaucine pathway inter-mediates. Unfortunately, the key aporphine (S)-isoboldinewas not available to test this hypothesis.Alkaloid content in G. flavum has been reported to

show substantial variation with glaucine, in particular,occurring as a major alkaloid in some ecotypes, but ap-parently absent in others (Peled et al., 1988). Previouslydetected BIAs generally belong to the protopine, benzo-phenanthridine, or aporphine subgroups. HPLC-UVanalysis showed that glaucine predominates in aerial or-gans, whereas protopine occursmostly in roots (Bournineet al., 2013). Isocorydine, an aporphine alkaloid with onefree hydroxyl group, has also been reported in G. flavum(Daskalova et al., 1988; Peled et al., 1988; Kintsurashviliand Vachnadze 2000; Petitto et al., 2010; ). Our high-resolution, FTMS analysis provided unequivocal confir-mation of the alkaloid profile in G. flavum, with glaucinefound predominantly in aerial organs (Fig. 7, B and C).Gene expression analysis shows that GFLOMT1, GFL-OMT2, and GFLOMT6 are expressed in all plant organs(Fig. 7A) in support of their proposed roles in the bi-osynthesis of glaucine above ground and other alka-loids in the root.In conclusion, we have characterized all apparent

OMTs involved in BIA metabolism from the roots of

G. flavum, and shown that threemultifunctional enzymesare responsible for four substrate and regiospecificO-methylations. In addition to improving our under-standing of plant alkaloid metabolism, functional OMTvariants are important to the synthetic biology objectiveof reconstituting BIA biosynthetic pathways in microor-ganisms (Hawkins and Smolke, 2008; Nakagawa et al.,2011; Fossati et al., 2014). A comprehensive characteri-zation of enzyme functionality is clearly essential forthe rational engineering of metabolic pathways in anyorganism.

MATERIALS AND METHODS

Plants and Chemicals

Glauciumflavumplantswere obtained from the collection at the JardinbotaniquedeMontréal (espacepourlavie.ca/jardin-botanique). (R,S)-6-O-methylnorlaudanosolinewas purchased from Toronto Research Chemicals. (R,S)-6-O-methyllaudanosolinewas generated from (R,S)-6-O-methylnorlaudanosoline using purified recombi-nant CNMT Coptis japonica (Choi et al., 2002). Enzymatic conversion was per-formed at 37°Cwith 1.2 mM (R,S)-6-O-methylnorlaudanosoline, 200 mM SAM, and150 mg of purified CjCNMT. Reactions were extracted by ethyl acetate three times.The purity and identity of 6-O-methyllaudanosolinewas confirmed by LC-MS/MS.(R,S)-norreticuline was produced from (R,S)-6-O-methylnorlaudanosolineusing purified recombinant C. japonica 49OMT (Morishige et al., 2000).(S)-tetrahydrocolumbamine was prepared from (R,S)-scoulerine using purifiedrecombinant Papaver somniferum SOMT1 (Dang and Facchini, 2012). Other al-kaloids were obtained as described previously (Liscombe and Facchini, 2007;Hagel and Facchini, 2010; Dang and Facchini, 2012). SAM was purchased fromSigma-Aldrich. All other chemicals were purchased from Bioshop.

Phylogenetic Analysis

AminoacidalignmentswereperformedusingClustalW(Larkinetal., 2007), andaphylogenetic tree was built by the neighbor-joining method using the Geneious(Biomatters) software package. Abbreviations and GenBank accession numbers forsequencesused to construct thephylogenetic tree are as follows:Cj49OMT,C. japonicaSAM:39-hydroxy-N-methylcoclaurine 49-O-methyltransferase (BAB08005); Cj6OMT,C. japonica SAM:norcoclaurine 6-O-methyltransferase (BAB08004); CjCoOMT, C.japonica columbamine O-methyltransferase (BAC22084); CjSOMT, C. japonica SAM:scoulerine 9-O-methyltransferase (BAA06192); Ec49OMT, Eschscholzia californica 39-hydroxy-N-methylcoclaurine-49-O-methyltransferase (BAM37633); Ec6OMT, E. cal-ifornica O-methyltransferase (BAM37634); Ec7OMT, E. californica reticuline-7-O-methyltransferase (BAE79723); Ps49OMT1, P. somniferum SAM:39-hydroxy-N-meth-ylcoclaurine 49-O-methyltransferase 1 (AAP45313); Ps49OMT2, P. somniferumSAM:39-hydroxy-N-methylcoclaurine 4’-O-methyltransferase 2 (AAP45314);Ps6OMT, P. somniferum SAM:norcoclaurine 6-O-methyltransferase (AAP45315);Ps7OMT, P. somniferum reticuline 7-O-methyltransferase (AAQ01668); PsN7OMT,P. somniferum norreticuline-7-O-methyltransferase (ACN88562); PsSOMT, P. som-niferum scoulerine-9-O-methyltransferase (AFK73709); PsSOMT2,P. somniferum O-methyltransferase 2 (AFK73710); PsSOMT3, P. somniferumO-methyltransferase 3(AFK73711); Tf49OMT, Thalictrum flavum 39-hydroxy-N-methylcoclaurine 49-O-methyltransferase (AAU20768); Tf6OMT, T. flavum norcoclaurine 6-O-methyl-transferase (AAU20765); and TfSOMT, T. flavum scoulerine 9-O-methyltrans-ferase (AAU20770).

RNA Extraction and cDNA Synthesis

Total RNA was isolated from G. flavum tissues ground to a fine powderunder liquid nitrogen using a Tissue Lyser (Qiagen) and extracted using thecetyl-trimethyl-ammonium bromide method (Gambino et al., 2008). Reversetranscription was performed in a 20-mL reaction containing approximately 1.5 mgof total RNA using Moloney murine leukemia virus reverse transcriptase(Invitrogen) according to the manufacturer’s instructions.

Cloning and Expression of GFLOMT cDNAs

Full-length GFLOMT coding regions were amplified from cDNA derivedfrom total G. flavum RNA using Phusion High-Fidelity Taq DNA polymerase






(New England Biolabs) and the primer sets listed in Supplemental Table S6. Forheterologous expression of His-tagged GFLOMT proteins, PCR products wereligated into pRSETA (Invitrogen) and transformed in Escherichia coli strainRosetta (DE3) pLysS (EMD Millipore). For the production of recombinantGFLOMT proteins, bacteria were cultured in Luria-Bertani medium to an op-tical density at 600 nm of 0.6 and subsequently induced with 1 mM isopropylb-D-thiogalactopyranoside (IPTG). Cultures were incubated at 30°C on a gy-ratory shaker at 200 rpm. Cells were harvested and sonicated in binding buffer(50 mM potassium phosphate [pH 7.5], 100 mM NaCl, 10% [v/v] glycerol, and1 mM b-mercaptoethanol). Cleared lysates obtained after centrifugation at10,000g for 10 min were loaded onto Talon cobalt affinity resin (Clontech). Theresin was washed three times in binding buffer and eluted using aliquots ofbinding buffer containing increasing concentrations of imidazole to obtainpurified proteins. Purified and His-tagged recombinant GFLOMT proteinswere desalted on PD10 columns (GE Healthcare) in storage buffer (50 mM

potassium phosphate [pH 7.5], 10% [v/v] glycerol, and 1 mM b-mercaptoeth-anol), and protein concentrations were determined using the Bradford assay(Bio-Rad) using bovine serum albumin as the standard. The purity of recom-binant proteins was evaluated by SDS-PAGE.

Enzyme Assays and Characterization of GFLOMTs

The standard enzyme assay forOMTactivitywas performed at 37°C in 50mLof 50mM potassium phosphate (pH 7.0), 25 mM sodium ascorbate, 100mM SAM,100 mM potential alkaloid substrate, and 5 mg of the purified recombinant en-zyme. Reactions were incubated for 2 h and stopped by adding 500 mL ofmethanol. Proteins were precipitated by centrifugation at 20,000g for 20 min,and supernatants were subjected to LC-MS/MS. Controls were performedwithdenatured purifiedHis-tagged proteins prepared by boiling inwater for 20min.Conversion rates were calculated based on substrate loss. Kinetic parameterswere determined at 37°C for 30 min by (1) varying alkaloid substrate concen-trations from 1 to 300mM at a fixed concentration of 100mM SAMand (2) varyingSAM concentrations from 1 to 300 mM at a fixed concentration of 100mM for eachalkaloid substrate. Kinetic constants were determined by fitting initial velocityversus substrate concentration to theMichaelis-Menten equation usingGraphPadPrism 5 (www.graphpad.com).

LC-MS/MS Analysis of Enzyme Assays

Enzyme assays were analyzed using a 6410 Triple Quadrupole LC-MS/MS(Agilent Technologies) for the identification and quantification of alkaloids. LCwas carried out using a Poroshell 120 SB C18 column (2.1 3 50 mm, 2.7-mmparticle size; Agilent Technologies) at a flow rate of 0.7 mL min21. LC wasinitiated at 100% solvent A (1% [v/v] formic acid), ramped to 60% (v/v) solventB (acetonitrile) using a linear gradient over 6 min, further ramped to 99% (v/v)solvent B using a linear gradient over 1 min, held constant at 99% (v/v) solventB for 1 min, and returned to original conditions over 0.1 min for a 3.9-minequilibration period. Eluate was applied to the mass analyzer using an elec-trospray ionization probe operating in positive mode with the following con-ditions: capillary voltage, 4,000 V; fragmentor voltage, 100 V; source temperature,350°C; nebulizer pressure, 50 pounds per square inch; gas flow, 10 L min21. Forfull-scan analysis, quadrupole 1 and 2 were set to radio frequency only, whereasthe third quadrupole scanned from 200 to 700 m/z. ESI[+]-CID spectra were an-alyzed, the precursorm/zwas selected in quadrupole 1, and a collision energy of30 eV and an argon collision gas pressure of 1.8 3 1023 torr were applied inquadrupole 2. The resulting ion fragments were resolved in quadrupole 3 scan-ning from 40m/z to 2m/z greater than the precursor ionm/z. Compounds wereidentified based on retention times and ESI[+]-CID spectra compared with au-thentic standards or compared with previously published spectral data (Des-gagné-Penix et al., 2012; Winzer et al., 2012).

Bioconversions

IPTG-induced E. coli cultures harboring pGFLOMT1, pGFLOMT2,pGFLOMT6, and/or pCNMT were combined at equal ratios, and 25 mM (R,S)-norlaudanosoline was added to 1 mL of the mixture. The mixed cultures wereincubated overnight at 30°C and subsequently quenched with methanol. Celldebris was removed by centrifugation, and the supernatant was dried andextracted three times using ethyl acetate. Pooled extracts were dried, subse-quently dissolved in 50 mL of 1% (v/v) formic acid, and subjected to LC-MS/MS analysis. IPTG-induced cultures expressing empty vector pRSETA wereused as the negative control.

LTQ-Orbitrap Analysis of Plant Extracts

G. flavum organs from four individual plants were flash frozen and stored at280°C. Frozen tissues were ground to fine powder using a TissueLyser II(Qiagen), extracted with 5 mL of methanol:chloroform (50:50), and centrifugedat 8,000g for 10 min. Liquid phases (aqueous and organic) were aspirated frominsoluble debris and pooled. Insoluble material was extracted twice more withmethanol:chloroform (50:50), all liquid phases were pooled, and the remainingdebris was dried and weighed. Liquid phases were reduced to dryness undervacuum, resuspended in 1.5 mL of methanol:chloroform (50:50), and centri-fuged at 10,000g for 10 min to remove particulates. Dilutions of 1:100 and1:1,000 were prepared for (1) the acquisition of exact mass data and quantifi-cation of low-abundance alkaloids, and (2) the quantification of high-abundancealkaloids, respectively. Ten microliters of each sample was fractionated on aZorbax C18 column (2.1 3 50 mm, 1.8 mm; Agilent) using an Accela HPLCsystem (Thermo Scientific) at a flow rate of 0.5 mL min21 and a solvent (10 mM

ammonium acetate, pH 4.5) gradient of 100% to 80% over 5 min, 80% to 50%over 3 min, 50% to 0% over 3 min, isocratic at 0% for 2 min, 0% to 100% over 0.1min, and isocratic at 100% for 1.9 min. The second solvent was 100% acetoni-trile. Total run time was 15min, but data were collected for only 10min. HeatedESI source and interface conditions were operated in positive ion mode asfollows: vaporizer temperature, 400°C; source voltage, 3 kV; sheath gas, 60 au,auxiliary gas, 20 au; capillary temperature, 380°C; capillary voltage, 6 V; tubelens, 45 V. LTQ-Orbitrap-XL (Thermo Scientific) instrumentation was per-formed as three scan events in data-dependent, parallel detection mode. Thefirst scan consisted of high-resolution FTMS from 200 to 700 m/z with ion in-jection time of 500 ms and scan time of approximately 1.5 s. The second andthird scans (approximately 0.5 s each) collected CID spectra in the ion trap,where the parent ions represented the first- and second-most abundant alkaloidmasses, respectively, as determined by fast Fourier transform preview using aparent ion mass list corresponding to exact masses of known alkaloids.Dynamic-exclusion and reject-ion-mass-list features were enabled. External andinternal calibration procedures ensured ,2 ppm. FTMS data were processedvia R i386 v. 3.2.0 using the centWave, obiwarp, group, and fillPeaks features ofPackage XCMS v.1.44.0 (Tautenhahn et al., 2008). Exact mass, retention times,and CID spectra of authentic standards were used to identify alkaloids, andquantification was performed using standard curves.

Gene Expression Analysis

After denaturing the RNA-primer mix for 15 min at 70°C, cDNA synthesiswas performed at 37°C for 50 min using 2.5 mM anchored oligo(dT) primer(dT20VN), 0.5 mM deoxynucleotide triphosphate mix, 500 ng G. flavum RNA,and 5 microunits mL21 reverse transcriptase (Invitrogen). Quantitative reversetranscription-PCR was performed using SYBR Green detection on an AppliedBiosystems 7300 real-time PCR system. Each 10-mL reaction included 1 mL ofsynthesized cDNA, 300 nM forward and reverse primers (Supplemental TableS6), and 13 Power SYBR Green PCR Master Mix (Applied Biosystems). Ther-mal cycling conditions for relative quantification included 40 cycles of templatedenaturation, primer annealing, and primer extension. To evaluate PCR spec-ificity, the amplified products of all primer pairs were subjected to melt curveanalysis using the dissociation method suggested by Applied Biosystems. The22DDCT method was used for the analysis of relative gene expression (Livak andSchmittgen, 2001) with b-actin as the internal control. Values were calculatedbased on three biological replicates for each GFLOMT gene.

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers KP176693 to KP176699.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Purification of recombinant proteins from E. coliexpressing pGFLOMT constructs.

Supplemental Figure S2. Steady-state enzyme kinetics of affinity-purifiedrecombinant GFLOMT1, GFLOMT2, and GFLOMT6 for (R,S)-6-O-methylnorlaudanosoline, (R,S)-norlaudanosoline, (R,S)-scoulerine, and SAM.

Supplemental Figure S3. Possible permutations in the sequence of O- andN-methyl transfer reactions and the detected functions of GFLOMT1,


Chang et al.



http://www.graphpad.com







GFLOMT2, and GFLOMT6 in the conversion of (R,S)-norlaudanosolineto (R,S)-laudanosine.

Supplemental Table S1. Amino acid sequence identity matrix forGFLOMTs with functionally characterized OMTs involved in BIA bio-synthesis.

Supplemental Table S2. Chromatographic and mass spectral data for au-thentic BIA standards.

Supplemental Table S3. Chromatographic and mass spectral data for thereaction products of recombinant GFLOMTs assayed with various sub-strates.

Supplemental Table S4. Chromatographic and mass spectral data for com-pounds produced from various combinations of E. coli strains harboringindividual pGFLOMT constructs, plus or minus a strain producingpCNMT from C. japonica, and fed (R,S)-norlaudanosoline.

Supplemental Table S5. Primers used to amplify open reading frames ofGFLOMTs for insertion into pRSETA expression vector.

Supplemental Table S6. Primers used to amplify open reading frames ofGFLOMTs for insertion into pRSETA expression vector.

Supplemental Data Set S1. Exact mass data obtained by high-resolutionFourier-transform mass spectrometry performed in positive mode onextracts from different organs of G. flavum.

ACKNOWLEDGMENTS

We thank Scott Farrow for assistance with the preparation of norreticuline.

Received August 6, 2015; accepted August 20, 2015; published August 21, 2015.

LITERATURE CITED

Battersby AR, McHugh JL, Staunton J, Todd M (1971) Biosynthesis of theapparently “directly coupled” aporphine alkaloids. J Chem Soc ChemCommun 1971: 985–986

Bhakuni DS, Jain S (1988) The biosynthesis of glaucine in Litsea glutinosa.J Chem Soc Perkin Trans 1: 1447–1449

Bournine L, Bensalem S, Wauters JN, Iguer-Ouada M, Maiza-BenabdesselamF, Bedjou F, Castronovo V, Bellahcène A, Tits M, Frédérich M (2013)Identification and quantification of the main active anticancer alkaloids fromthe root of Glaucium flavum. Int J Mol Sci 14: 23533–23544

Choi KB, Morishige T, Shitan N, Yazaki K, Sato F (2002) Molecularcloning and characterization of coclaurine N-methyltransferase fromcultured cells of Coptis japonica. J Biol Chem 277: 830–835

Cortijo J, Villagrasa V, Pons R, Berto L, Martí-Cabrera M, Martinez-LosaM, Domenech T, Beleta J, Morcillo EJ (1999) Bronchodilator and anti-inflammatory activities of glaucine: In vitro studies in human airwaysmooth muscle and polymorphonuclear leukocytes. Br J Pharmacol 127:1641–1651

Dang TTT, Chen X, Facchini PJ (2015) Acetylation serves as a protectivegroup in noscapine biosynthesis in opium poppy. Nat Chem Biol 11:104–106

Dang TTT, Facchini PJ (2012) Characterization of three O-methyltrans-ferases involved in noscapine biosynthesis in opium poppy. PlantPhysiol 159: 618–631

Dargan PI, Button J, Hawkins L, Archer JR, Ovaska H, Lidder S, RamseyJ, Holt DW, Wood DM (2008) Detection of the pharmaceutical agentglaucine as a recreational drug. Eur J Clin Pharmacol 64: 553–554

Daskalova E, Iskrenova E, Kiryakov HG, Evstatieva L (1988) Minor al-kaloids of Glaucium flavum. Phytochemistry 27: 953–955

Desgagné-Penix I, Farrow SC, Cram D, Nowak J, Facchini PJ (2012) In-tegration of deep transcript and targeted metabolite profiles for eightcultivars of opium poppy. Plant Mol Biol 79: 295–313

Farrow SC, Hagel JM, Facchini PJ (2012) Transcript and metabolite pro-filing in cell cultures of 18 plant species that produce benzylisoquinolinealkaloids. Phytochemistry 77: 79–88

Fossati E, Ekins A, Narcross L, Zhu Y, Falgueyret JP, Beaudoin GA,Facchini PJ, Martin VJJ (2014) Reconstitution of a 10-gene pathway for

synthesis of the plant alkaloid dihydrosanguinarine in Saccharomycescerevisiae. Nat Commun 5: 3283

Frenzel T, Zenk MH (1990) S-adenosyl-L-methionine:39hydroxyl-N-methyl-(S)-coclaurine-49-O-methyltransferase, a regio- and stereoselec-tive enzyme of the (S)-reticuline pathway. Phytochemistry 29: 3505–3511

Gambino G, Perrone I, Gribaudo I (2008) A rapid and effective method forRNA extraction from different tissues of grapevine and other woodyplants. Phytochem Anal 19: 520–525

Hagel JM, Facchini PJ (2010) Dioxygenases catalyze the O-demethylationsteps of morphine biosynthesis in opium poppy. Nat Chem Biol 6: 273–275

Hagel JM, Facchini PJ (2013) Benzylisoquinoline alkaloid metabolism: acentury of discovery and a brave new world. Plant Cell Physiol 54: 647–672

Hawkins KM, Smolke CD (2008) Production of benzylisoquinoline alka-loids in Saccharomyces cerevisiae. Nat Chem Biol 4: 564–573

Ikezawa N, Iwasa K, Sato F (2008) Molecular cloning and characterizationof CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-Cphenol coupling of (S)-reticuline in magnoflorine biosynthesis, fromcultured Coptis japonica cells. J Biol Chem 283: 8810–8821

Inui T, Tamura K, Fujii N, Morishige T, Sato F (2007) Overexpression ofCoptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in benzylisoquinoline alkaloid biosynthesis in culturedEschscholzia californica. Plant Cell Physiol 48: 252–262

Kintsurashvili LG, Vachnadze VY (2000) Alkaloids of Glaucium cornicu-latum and G. flavum growing in Georgia. Chem Nat Compd 36: 225–226

Lapa GB, Sheichenko OP, Serezhechkin AG, Tolkachev ON (2004) HPLCdetermination of glaucine in yellow horn poppy grass (Glaucium flavumCrantz). Pharm Chem J 38: 441–442

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA,McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al (2007)Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948

Liscombe DK, Facchini PJ (2007) Molecular cloning and characterization oftetrahydroprotoberberine cis-N-methyltransferase, an enzyme involvedin alkaloid biosynthesis in opium poppy. J Biol Chem 282: 14741–14751

Liscombe DK, MacLeod BP, Loukanina N, Nandi OI, Facchini PJ (2005)Evidence for the monophyletic evolution of benzylisoquinoline alkaloidbiosynthesis in angiosperms. Phytochemistry 66: 2501–2520

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression datausing real-time quantitative PCR and the 22DDCT method. Methods 25:402–408

Milanowski DJ, Winter RE, Elvin-Lewis MP, Lewis WH (2002) Geo-graphic distribution of three alkaloid chemotypes of Croton lechleri. J NatProd 65: 814–819

Morishige T, Dubouzet E, Choi KB, Yazaki K, Sato F (2002) Molecularcloning of columbamine O-methyltransferase from cultured Coptis ja-ponica cells. Eur J Biochem 269: 5659–5667

Morishige T, Tsujita T, Yamada Y, Sato F (2000) Molecular characteriza-tion of the S-adenosyl-L-methionine:39-hydroxy-N-methylcoclaurine 49-O-methyltransferase involved in isoquinoline alkaloid biosynthesis inCoptis japonica. J Biol Chem 275: 23398–23405

Morteza-Semnani K, Amin G, Shidfar MR, Hadizadeh H, Shafiee A(2003) Antifungal activity of the methanolic extract and alkaloids ofGlaucium oxylobum. Fitoterapia 74: 493–496

Muemmler S, Rueffer M, Nagakura N, Zenk MH (1985) S-adenosyl-L-methionine: (S)-scoulerine 9-O-methyltransferase, a highly stereo- andregio-specific enzyme in tetrahydroprotoberberine biosynthesis. PlantCell Rep 4: 36–39

Nakagawa A, Minami H, Kim JS, Koyanagi T, Katayama T, Sato F, KumagaiH (2011) A bacterial platform for fermentative production of plant alkaloids.Nat Commun 2: 326

Orallo F, Fernández Alzueta A, Campos-Toimil M, Calleja JM (1995)Study of the in vivo and in vitro cardiovascular effects of (+)-glaucineand N-carbethoxysecoglaucine in rats. Br J Pharmacol 114: 1419–1427

Ounaroon A, Decker G, Schmidt J, Lottspeich F, Kutchan TM (2003) (R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferaseof Papaver somniferum - cDNA cloning and characterization of methyl transferenzymes of alkaloid biosynthesis in opium poppy. Plant J 36: 808–819

Peled B, Waisel Y, Carmeli S (1988) Alkaloid content in various chemo-ecotypes of Glaucium flavum from Israel. Phytochemistry 27: 1021–1024

Petitto V, Serafini M, Gallo FR, Multari G, Nicoletti M (2010) Alkaloidsfrom Glaucium flavum from Sardinia. Nat Prod Res 24: 1033–1035












Pienkny S, Brandt W, Schmidt J, Kramell R, Ziegler J (2009) Functionalcharacterization of a novel benzylisoquinoline O-methyltransferasesuggests its involvement in papaverine biosynthesis in opium poppy(Papaver somniferum L). Plant J 60: 56–67

Rueffer M, Nagakura N, Zenk MH (1983) A highly specific O-methyltransferasefor nororientaline synthesis isolated from Argemone platyceras cell cultures.Planta Med 49: 196–198

Sato F, Takeshita N, Fitchen JH, Fujiwara H, Yamada Y (1993) S-adenosyl-L-methionine:scoulerine-9-O-methyltransferase from cultured Coptis japonicacells. Phytochemistry 32: 659–664

Takeshita N, Fujiwara H, Mimura H, Fitchen JH, Yamada Y, Sato F (1995)Molecular cloning and characterization of S-adenosyl-L-methionine:scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica.Plant Cell Physiol 36: 29–36

Tautenhahn R, Böttcher C, Neumann S (2008) Highly sensitive featuredetection for high resolution LC/MS. BMC Bioinformatics 9: 504

Winzer T, Gazda V, He Z, Kaminski F, Kern M, Larson TR, Li Y, Meade F,Teodor R, Vaistij FE, et al (2012) A Papaver somniferum 10-gene cluster

for synthesis of the anticancer alkaloid noscapine. Science 336: 1704–1708

Xiao M, Zhang Y, Chen X, Lee EJ, Barber CJ, Chakrabarty R, Desgagné-Penix I, Haslam TM, Kim YB, Liu E, et al (2013) Transcriptome analysisbased on next-generation sequencing of non-model plants producingspecialized metabolites of biotechnological interest. J Biotechnol 166:122–134

Xu XH, Yu GD, Wang ZT (2004) Resource investigation and quality eval-uation on wild Corydalis yanhusuo. Zhongguo Zhong Yao Za Zhi 29: 399–401

Zetler G (1988) Neuroleptic-like, anticonvulsant and antinociceptive effectsof aporphine alkaloids: bulbocapnine, corytuberine, boldine and glau-cine. Arch Int Pharmacodyn Ther 296: 255–281

Ziegler J, Diaz-Chávez ML, Kramell R, Ammer C, Kutchan TM (2005)Comparative macroarray analysis of morphine containing Papaversomniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis. Planta222: 458–471


Chang et al.



isolation and characterization of o-methyltransferases …...isolation and characterization...

Documents