Isolation and characterization of two O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in sacred lotus (Nelumbo nucifera)

JBC Papers in Press. Published on December 30, 2019 as Manuscript RA119.011547

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.011547

O-Methyltransferases from sacred lotus

Isolation and characterization of two O-methyltransferases involved in benzylisoquinoline alkaloids

biosynthesis in sacred lotus (Nelumbo nucifera)

Ivette M. Menéndez-Perdomo and Peter J. Facchini*

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

Running title: O-Methyltransferases from sacred lotus

*Correspondence: pfacchin@ucalgary.ca; Tel.: +1-403-220-7651

Keywords: benzylisoquinoline alkaloids; Nelumbo nucifera; O-methyltransferases; plant biochemistry;

plant molecular biology; regiospecificity; secondary metabolism; stereospecific; natural product;

secondary metabolism

methylation precedes BIA formation from 1-

ABSTRACT

benzylisoquinoline intermediates. K_mvalues for

Benzylisoquinoline alkaloids (BIAs) are a major

class of plant metabolites with many

NnOMT1 and NnOMT5 were 20 µM and 13

µM for (R,S)-norcoclaurine and (S)-N-

methylcoclaurine, respectively, similar to those

for OMTs from other BIA-producing plants.

Organ-based correlations of alkaloid content,

OMT activity in crude extracts, and OMT gene

expression supported physiological roles for

NnOMT1 and NnOMT5 in BIA metabolism,

occurring primarily in young leaves and

embryos of sacred lotus. In summary, our work

identifies two OMTs involved in BIA

metabolism in the medicinal plant N. nucifera.

pharmacological

benefits.

Sacred

lotus

(Nelumbo nucifera) is an ancient aquatic plant

of medicinal value owing to antiviral and

immunomodulatory activities linked to its

constituent BIAs. Although more than 30 BIAs

belonging

to

the

1-benzylisoquinoline,

aporphine, and bisbenzylisoquinoline structural

subclasses and displaying a predominant R

enantiomeric conformation have been isolated

from N. nucifera, its BIA biosynthetic genes and

enzymes remain unknown. Herein, we report

the isolation and biochemical characterization

of two O-methyltransferases (OMTs) involved

in BIA biosynthesis in sacred lotus. Five

homologous genes, designated NnOMT1–5 and

encoding polypeptides sharing > 40% amino

acid sequence identity, were expressed in

Escherichia coli. Functional characterization of

the purified recombinant proteins revealed that

NnOMT1 is a regiospecific 1-benzylisoquinoline

6-O-methyltransferase (6OMT) accepting both

R and S substrates, whereas NnOMT5 is mainly

INTRODUCTION

Benzylisoquinoline alkaloids (BIAs) are a major

class of plant specialized metabolites, many of

which possess significant pharmacological value.

The 1-benzylisoquinoline (S)-norcoclaurine has

been recognized as the common intermediate to all

biosynthetically characterized BIAs (Figure 1),

and is formed via the enantioselective

condensation of the tyrosine derivatives, dopamine

and 4-hydroxyphenylacetaldehyde, catalyzed by

norcoclaurine synthase (NCS) (1). Functional

group modifications, especially O- and N-

methylations, of the 1-benzylisoquinoline scaffold

combined with various C–C and C–O coupling

reactions yield additional structural subclasses

a

7-O-methyltransferase

(7OMT),

with

relatively minor 6OMT activity and a strong

stereospecific preference for S enantiomers.

Available aporphines were not accepted as

substrates by either enzyme, suggesting that O-

including

aporphines

(e.g.

magnoflorine),

bisbenzylisoquinolines (e.g. dauricine), pavine

1

O-Methyltransferases from sacred lotus

(e.g. eschscholtzidine), protoberberines (e.g.

of norcoclaurine (Figure 1). Since O-methylation

contributes substantially to the diversity of BIAs in

sacred lotus, we targeted the isolation and

functional characterization of OMTs.

berberine),

morphinans

(e.g.

morphine),

phthalideisoquinolines (e.g. noscapine), and

benzo[c]phenanthridines (e.g. sanguinarine) (2).

The natural occurrence of BIAs is

Methylations play a central role in the

restricted to a relatively small number of plant

families primarily in the order Ranunculales,

which includes the opium poppy (Papaver

somniferum), an extensively investigated model

species that remains the sole commercial source

for the narcotic analgesic morphine and the

antitussive and potential anticancer drug noscapine

(3). BIAs have also been detected in other plant

orders including the Cornales, Laurales,

Magnoliales, Piperales, Sapindales, and Proteales,

although BIA metabolism in members of these

taxa has not yet been empirically determined (4).

Sacred lotus (Nelumbo nucifera Gaertn.; Figure

2a), is an aquatic basal eudicot in the order

Proteales that has been cultivated as an edible crop

for nearly seven millennia, and is highly valued in

both Chinese and Ayurvedic traditional medicine

for its healing properties (5). The pharmacological

properties of sacred lotus are largely the result of

the BIA constituents in the plant. For instance, 1-

benzylisoquinolines such as norcoclaurine and its

6-O-methylated derivative coclaurine have

exhibited anti-HIV activity (6), whereas the 6,7-

O,O- and N-methylated derivative armepavine has

displayed promising immunomodulatory effects

for the treatment of autoimmune diseases such as

systemic lupus erythematosus (7). In addition,

functionalization of specialized metabolites (14).

In particular, OMTs involved in BIAs metabolism

catalyze methyl transfer to the hydroxyl group of

an alkaloid substrate, using S-adenosyl-L-

methionine (SAM) as the methyl donor group and

proceeding via a S_N2-like nucleophilic attack

yielding a methylated alkaloid product and S-

adenosyl-L-homocysteine (SAH) (15). OMTs are

typically homodimeric or, as recently discovered,

heterodimeric enzymes (16,17). Within each

monomer, the C-terminal domain adopts

a

canonical Rossmann fold (i.e. β-strand

a

sandwiched by two α-helices) and plays a

fundamental role in substrate binding and

catalysis, whereas the N-terminal domain is of

central importance in dimerization (15,18-20).

In opium poppy, the methylation of (S)-

norcoclaurine to (S)-coclaurine by (R,S)-

norcoclaurine 6-O-methyltransferase (Ps6OMT)

represents the first tailoring reaction in BIA

metabolism (21,22). Recent crystallographic

studies on 6OMT from meadow rue (Thalictrum

flavum, Tf6OMT) have revealed key determinants

for substrate recognition and turnover (18). In

opium poppy, at least six other OMTs involved in

BIA biosynthesis have been functionally

characterized,

methylcoclaurine

(Ps4'OMT2),

including

4'-O-methyltransferase

(R,S)-reticuline

(R,S)-3'-hydroxy-N-

major

aporphines

(e.g.

nuciferine)

and

2

7-O-

bisbenzylisoquinolines (e.g. neferine) have shown

potential for the treatment of lung, liver, ovarian

and breast cancers (8-12). However, none of the

biosynthetic genes and enzymes responsible for

the BIA biosynthesis in sacred lotus have been

characterized (13).

methyltransferase (Ps7OMT) and norreticuline 7-

O-methyltransferase (PsN7OMT), all involved in

1-benzylisoquinoline biosynthesis (i.e. reticuline,

laudanine and norlaudanine), as well as 4'-O-

desmethyl-3-O-acetylpapaveroxine

methyltransferase (PsOMT2:PsOMT3

4'-O-

and

Based on the reported alkaloid profile in

sacred lotus and well characterized pathways in

opium poppy, BIA metabolism is predicted to

begin with the formation of norcoclaurine and

proceed via a limited number of enzyme types

including one or more O-methyltransferases

(OMTs), N-methyltransferases, and cytochrome

P450 monooxygenases potentially belonging to

PsOMT2:Ps6OMT heterodimers) and scoulerine

9-O-methyltransferase (PsSOMT1), which

function in phthalideisoquinoline (e.g. noscapine)

metabolism (16,17,19,21-26). Although the

isolation of a specific 3'OMT has been elusive,

low 3'-O-methyltransferase activity has been

detected for PsSOMT1, as well as for SOMTs

from related species such as yellow horned poppy

(Glaucium flavum, GfOMT6) and California

poppy (Eschscholzia californica, EcSOMT) (26-

28). Finally, a columbamine O-methyltransferase

CYP80

and

CYP719

subfamilies

(13).

Specifically, the formation of known 1-

benzylisoquinoline alkaloids in sacred lotus would

involve only O-methylations and N-methylations

2

O-Methyltransferases from sacred lotus

has been isolated from Japanese goldthread

(Coptis japonica; CjCoOMT) (29). Although

several functionally related OMTs have been

investigated, these enzymes have been isolated

from fewer than ten species all from the

Ranunculales (20).

abundant in the embryos. In general, the rhizome

and roots contained only low BIA levels, although

no bisbenzylisoquinolines were detected. Notably,

the structural isomers N-methylisococlaurine and

norarmepavine were exclusively detected in the

Pink and White varieties, respectively. The

bisbenzylisoquinolines liensinine and neferine

were found only in the White variety, whereas

nelumboferine and isoliensinine were the only

bisbenzylisoquinolines identified in Pink and

accumulated at 20- (in folded leaves) and 4-fold

(in embryos) higher levels compared with the

White variety. In addition, alkaloids were detected

in the leaves (norarmepavine, nelumboferine and

isoliensinine), rhizome (norcoclaurine, coclaurine,

Sacred

benzylisoquinolines, and derived aporphines (via

C8–C2' intramolecular coupling) and

lotus

accumulates

1-

bisbenzylisoquinolines (via C8-C3'/5' and C7-O-

C3'/5' intermolecular coupling) (5), most of which

are O-methylated at C6, C7, and/or C4' (13). The

availability of a sacred lotus draft genome (30,31)

greatly facilitates gene mining for candidate OMTs

based on amino acid sequence similarity with

respect to characterized O-methyltransferases (32-

34). However, OMT candidates have so far been

investigated only in terms of gene expression, with

no functional characterization of the encoded

proteins. In addition, enzyme stereospecificity is

an intriguing feature of BIA metabolism in sacred

lotus, since norcoclaurine has been isolated from

the plant as both R and S enantiomers, and other

BIAs, including coclaurine, norarmepavine and N-

methylcoclaurine have been exclusively reported

as the R conformer (13), in contrasts with the

predominantly S stereochemistry of most BIAs in

opium poppy and related plants (3). Herein we

report the identification and characterization of O-

methyltransferases involved in BIA metabolism in

sacred lotus.

N-methylcoclaurine,

norarmepavine,

N-

methylisococlaurine, anonaine, roemerine, N-

nornuciferine, O-nornuciferine, nuciferine and

pronuciferine),

methylcoclaurine,

roots

(coclaurine,

N-

norarmepavine,

methylisococlaurine,

armepavine,

anonaine,

roemerine, N-nornuciferine and O-nornuciferine)

and embryo (norarmepavine and O-nornuciferine)

that had not been previously reported (5,13).

Overall, the alkaloid profile found for both

varieties supports the occurrence of 6-O-, 7-O- and

4'-O-methyltransferases in sacred lotus.

NnOMT identification and phylogeny

Sacred lotus transcripts encoding five putative

OMTs (NnOMT1-NnOMT5) were identified

based on amino acid sequence identity with opium

poppy OMTs (Table S2). The predicted

NnOMT1-NnOMT5 translation products displayed

predicted molecular weights of 38.4, 38.7, 38.8,

39.2 and 38.1 kDa and expected isoelectric points

of 6.5, 7.8, 6.1, 5.7 and 5.6, respectively. Four

sacred lotus OMTs shared ~80% amino acid

sequence identity, whereas NnOMT5 exhibited

<50% identity with respect to the others. NnOMT1

showed ~70% amino acid sequence identity

compared with functionally characterized 6OMTs,

whereas all candidates shared ~40-60% sequence

identity with previously reported 7OMTs and

4'OMTs (Table S3). Phylogenetic relationships

among NnOMTs and functionally characterized

BIA O-methyltransferases placed NnOMT1-

NnOMT4 in a single clade related to several

characterized 6OMTs, whereas NnOMT5 emerged

RESULTS

Sacred lotus BIA profile

Alkaloids corresponding to the leaf (folded and

unfolded developmental stages), rhizome, root and

embryo (lotus plumule) of two Nelumbo nucifera

cultivars, referred as Pink and White owing to

their distinct floral coloration, were extracted and

analyzed by mass spectrometry (MS) (Figure

2b,c, Table S1). Fifteen alkaloids (i.e. six 1-

benzylisoquinolines, four aporphines, one pro-

aporphine and four bisbenzylisoquinolines) were

identified based on their retention times and

collision-induced dissociation (CID) spectra

compared with available authentic standards or

previously published data. 1-Benzylisoquinoline

and aporphine alkaloids were prevalent in the

leaves, whereas bisbenzylisoquinolines were most

3

O-Methyltransferases from sacred lotus

on a distal clade that also contained CjCoOMT

(Figure 3).

to the corresponding substrate and were detected

in reactions containing NnOMT1 or NnOMT5

incubated with various 1-benzylisoquinoline

substrates (Figure 5, Figure S2). (R,S)-

Norcoclaurine and (S)-norlaudanosoline were O-

methylated by both NnOMT1 and NnOMT5,

whereas (R)-norlaudanosoline was only accepted

by NnOMT1. In addition, NnOMT5 catalyzed the

Amino acid sequence alignment of the

functionally characterized Tf6OMT and sacred

lotus OMT candidates showed a high conservation

of key amino acid residues, including the catalytic

determinants H²⁵⁶, D²⁵⁷and E³¹⁵(18), except for

NnOMT4, in which case D²⁵⁷was substituted with

a Y residue (Figure 4). Amino acids implicated in

O-methylation

of

(S)-coclaurine,

(S)-N-

BIA substrate binding (G¹⁶⁵, D¹⁶⁹, C²⁵³and D³⁰⁶

)

methylcoclaurine and (S)-reticuline with similar

efficiencies, but (R)-reticuline was not accepted.

Single peaks were detected for all the reactions,

indicating the formation of only one product.

Reaction products corresponding to the 6-

were also conserved among sacred lotus OMT

candidates except for G165V and C253W

substitutions in NnOMT2, G165A in NnOMT3,

G165S in NnOMT4, and G165A, D169H and

C253S in NnOMT5. The main residues involved

in SAM binding including T¹⁷⁰, G¹⁹⁵(motif I), D²¹⁸

(motif II), D²³⁸(motif III), and K²⁵²(motif IV)

were conserved across sacred lotus OMT

candidates, excluding D²¹⁸(substituted by G in

O-methylation

of

norcoclaurine

and

norlaudanosoline (coclaurine [m/z 286] and 6-O-

methylnorlaudanosoline [m/z 302], respectively)

and the 7-O-methylation of N-methylcoclaurine

(armepavine [m/z 314]), were unambiguously

identified by comparison with available authentic

standards. Enzyme assays with coclaurine and

reticuline were subjected to positive-mode

electrospray ionization (ESI[+]) high performance

liquid chromatography (HPLC)-tandem mass

spectrometry (MS/MS) for the accurate

characterization of reaction products by CID

fragmentation analysis to determine whether O-

methylation was associated with the isoquinoline

(C7) or benzyl moiety of coclaurine (C4') and

reticuline (C3') (Figure S3). ESI[+]-CID of 1-

benzylisoquinolines at low ionization energy

yielded isoquinoline and/or benzyl moieties as

major ion fragments; thus, the addition of 14 Da to

either fragment with respect to the parent ion

revealed the regiospecificity of the reaction. In this

regard, coclaurine showed two main fragments at

m/z 178 and m/z 107, corresponding to the

isoquinoline and benzyl moieties, respectively.

After incubation with NnOMT5, the m/z 107

fragment was retained in the reaction product,

indicating that the methyl group was added to the

isoquinoline moiety (i.e. 7-O-methylation)

resulting in the formation of norarmepavine, an

alkaloid found in sacred lotus (the other possible

reaction product, 4'-O-methylcoclaurine, has not

been reported in this plant). Similarly, the reaction

product of NnOMT5 incubated with (S)-reticuline

showed a mass increase of the isoquinoline moiety

from m/z 192 to m/z 206, corresponding to the 7-

O-methylated product laudanine, whereas the mass

of the benzyl moiety (m/z 137) remained

NnOMT4) and D²³⁸(substituted by

N

in

NnOMT3). Two methionine residues M¹⁶⁶and

M³⁰⁷assisting in the isoquinoline placing through

sulfur-aromatic interactions were also conserved in

lotus OMT candidates. The aromatic residues W¹⁴⁹

and F¹⁶², which interact with both SAM and BIA

substrates, were mutated, W¹⁴⁹to C, S and I in

NnOMT3, NnOMT4 and NnOMT5, respectively,

and F¹⁶²to L in NnOMT5. The conserved

GxGxGx sequence in motif I, a hallmark of SAM-

dependent methyltransferases (14), was fully

maintained in all the candidates except NnOMT5,

in which the middle G residue was mutated to C.

NnOMT purification and characterization

Sacred lotus recombinant His₆-tagged OMTs

produced in Escherichia coli were purified using

cobalt-affinity chromatography and detected on an

immunoblot using an anti-His₆antibody (Figure

S1). The molecular weights of the purified proteins

and the predicted translation products were

similar. Recombinant proteins were initially

screened for enzymatic activity using 15 potential

BIA

substrates

representing

the

1-

benzylisoquinoline, aporphine, protoberberine and

morphinan structural subclasses. The potential 1-

benzylisoquinoline

substrates

differentially

contained free hydroxyl groups at C6, C7, C3'

and/or C4', and both R and S enantiomers were in

some cases available (Table S4). Reaction

products showed an increase of 14 Da with respect

4

O-Methyltransferases from sacred lotus

unchanged. It is notable that neither reticuline nor

laudanine have been reported in sacred lotus.

4'OMT activity was not detected with any

combination of tested substrates and OMT

candidates.

embryo, rhizome and root, respectively. The Pink

cultivar was richer in N-methylcoclaurine, with the

folded leaves containing 3-, 40- and 90- fold

higher levels than the rhizome, root and embryo,

respectively.

NnOMT1 displayed strict regiospecificity

Crude plant protein extracts incubated

with respect to the C6 hydroxyl and showed

similar turnover using racemic norcoclaurine and

individual enantiomers of norlaudanosoline, the 3'-

hydroxylated derivative of norcoclaurine (Figure

5). NnOMT5 non-preferentially methylated the C7

hydroxyl of N-methylated (N-methylcoclaurine

and reticuline) or N-demethylated (coclaurine)

substrates, and also acted on the C6 hydroxyl of

(R,S)-norcoclaurine and (S)-norlaudanosoline. The

6-O-methylation activity of NnOMT5 was not

accompanied by secondary methylations (an

increase of 28 Da). Contrary to NnOMT1,

NnOMT5 showed a strict stereospecificity for the

substrates in the S conformation.

with (R,S)-norcoclaurine, (S)-N-methylcoclaurine

or (R)-armepavine revealed similarities between

the Pink and White varieties (Figure 6b, Table

S6). 6OMT activity was most abundant in folded

leaves and embryos, whereas 7OMT activity was

predominant in the unfolded leaves. 6OMT

activity was 5- and 4-fold higher than 7OMT

activity in folded leaves and embryos,

respectively. 7OMT activity was 8-fold higher

than 6OMT in the White variety, whereas 6OMT

activity was not detected in unfolded leaves of the

Pink variety. Rhizomes and roots showed only low

levels of 6OMT and 7OMT activities. 4'OMT

activity was not detected.

Both enzymes showed a pH optimum of

Gene expression profiles were similar for

8.0 using (R,S)-norcoclaurine (NnOMT1) or (S)-N-

methylcoclaurine (NnOMT5) as substrates and

maximum catalytic activity at 30 °C and 37 °C,

respectively (Figure S4). NnOMT1 and NnOMT5

displayed K_mvalues of 20 µM and 152 µM for

both the Pink and White varieties (Figure 6c,

Table S7). NnOMT1 and NnOMT5 transcripts

were most abundant in leaves and were 3- to 30-

fold higher compared with other OMT genes. The

levels of all five NnOMT transcripts were similar

in the embryos of both varieties.

(R,S)-norcoclaurine,

respectively,

whereas

NnOMT5 exhibited a K_mvalue of 13 µM and a K_i

of 870 µM for (S)-N-methylcoclaurine (Table 1,

Figure S5). Catalytic efficiencies (k_cat/K_m) were

similar for the 6OMT activity of NnOMT1 and the

7OMT activity of NnOMT5. The 7OMT activity

of NnOMT5 was 50-fold higher than the

corresponding 6OMT activity using (R,S)-

norcoclaurine as the substrate.

DISCUSSION

We have shown that two cDNAs from sacred lotus

efficiently catalyze the regiospecific 6-O-

methylation of norcoclaurine to coclaurine

(NnOMT1) and 7-O-methylation of coclaurine and

N-methylcoclaurine to norarmepavine and

armepavine, respectively (NnOMT5). NnOMT1

was not stereospecific whereas NnOMT5 accepted

only S enantiomers. Correlation between transcript

levels, enzyme activity and alkaloid content

supported a physiological role for NnOMT1 and

NnOMT5 in BIA metabolism.

NnOMT and BIA correlations in the plant

Alkaloid content, O-methyltransferase activity and

OMT gene expression were measured in leaves,

rhizome, roots and embryos for two sacred lotus

varieties. Alkaloid content was determined for

norcoclaurine, coclaurine, N-methylcoclaurine and

armepavine, for all of which authentic standards

were available (Figure S6). In both the Pink and

Despite sharing an apparently common

biosynthetic pathway to N-methylcoclaurine

(Figure 1), sacred lotus and opium poppy exhibit

substantial differences in their alkaloid profiles. In

this regard, whereas reticuline constitutes a key

branch-point intermediate in the biosynthesis of

most BIAs in opium poppy (3), sacred lotus does

not accumulate reticuline and other 1-

benzylisoquinolines containing a C3' functional

group, or reticuline-derived alkaloids including

White

varieties,

1-benzylisoquinoline

intermediates were predominantly detected in the

leaves (Figure 6a, Table S5). In the White

variety, norcoclaurine was the most abundant

alkaloid, with folded leaves accumulating 7-, 70-

and 1400-fold higher levels compared with the

5

O-Methyltransferases from sacred lotus

phthalideisoquinolines, benzo[c]phenanthridines,

protoberberines and morphinans, which occur in

members of the Ranunculales (13). In contrast,

aporphines and bisbenzylisoquinolines are the

major BIAs in sacred lotus, although alkaloid

profiles vary among nearly 600 known varieties,

and across different plant organs and

developmental stages (32-37). In addition, sacred

lotus aporphine alkaloids do not display

substitutions in the benzyl moiety (13), in contrast

with the reticuline-derived aporphines (e.g.

magnoflorine, corytuberine and glaucine) found in

members of the Ranunculales (27,38,39).

NnOMT5, the most phylogenetically

unique sacred lotus OMT, catalyzed the

methyltransfer to the C6 hydroxyl of (R,S)-

norcoclaurine and (S)-norlaudanosoline, but more

efficiently to the C7 hydroxyl of (S)-coclaurine,

(S)-N-methylcoclaurine and (S)-reticuline (Table

1). Despite the closest phylogenetic relationship

between NnOMT5 and CjCoOMT, their substrate

ranges are different. NnOMT5 did not accept the

tested protoberberines (Table S4), whereas

CjCoOMT preferred columbamine and other

protoberberines, but did not accept the 1-

benzylisoquinolines norlaudanosoline, coclaurine

and reticuline (29). NnOMT5 also showed a close

The sacred lotus BIA profile showed a

preferential accumulation of 1-benzylisoquinolines

relationship with

a

sister clade containing

and

aporphines

in

leaves,

and

PsOMT2, which together with PsOMT3 or

Ps6OMT forms the heterodimeric enzyme capable

of O-methylate a seco-berbine intermediate in

noscapine biosynthesis (16,17). Notably, this sister

clade also included Coptis chinensis 6OMT1

(Cc6OMT1), which was reported to catalyze either

6-O- or 7-O-methylation of (S)-norcoclaurine (43).

Dual 6-O- and 7-O-methyltransferase activity has

also been reported for Cc6OMT2, along with

enzymes from Coptis teeta (Ct7OMT), P.

somniferum (Ps6OMT and Ps7OMT) and G.

flavum (GfOMT1) (22,27,43).

bisbenzylisoquinolines in embryos (Figure 2b), in

agreement with previous investigations (36,37,40).

Alkaloids containing 6-O-, 7-O- and/or 4'-O-

methylations were detected in Pink and White

varieties, supporting the search for specific O-

methyltransferase activities in the plant. OMT gene

candidates have been predicted in sacred lotus

based on sequence similarity to functionally

characterized OMTs from BIA-accumulating

species (32-34), but the purported catalytic

activities has not been assessed. We show that two

of these candidates, NnOMT1 and NnOMT5,

Regardless of a nomenclature suggestive

catalyze

benzylisoquinolines.

the

O-methylation

of

1-

of regiospecific functions, OMTs involved in BIA

metabolism are generally promiscuous and exhibit

apparent redundancy in most plants (22,26-

28,43,44). In Tf6OMT co-crystalized with (S)-

norlaudanosoline the C7 hydroxyl group of the

BIA substrate was found in close proximity to the

catalytic H²⁵⁶, suggesting that minor adjustments

in substrate positioning could lead to productive 7-

O-methyltransfer (18) and the observed dual 6-O-

and 7-O-methyltransferase activity of some OMTs.

Based on the available crystal structure for

Tf6OMT (PDB 5ICE), we constructed a homology

model for NnOMT5 (Figure S7). In the resulting

structure, the key catalytic histidine (H²⁵¹in

NnOMT5) involved in hydrogen bonding with the

target C6-OH (3.0 Å), is also near the C7-OH (3.1

Å). Analysis of residues likely involved in BIA

substrate binding showed that G165A and C253S

mutations did not have a significant impact (i.e. the

main chain carbonyl involved in hydrogen bond

formation with the BIA substrate C3' and C6

hydroxyl groups, respectively, remained in a

similar position) in agreement with the observed

Fifteen BIA substrates possessing diverse

structural backbones were screened, including 1-

benzylisoquinolines with a free hydroxyl at C6, C7

and C4'. As described for several 6OMTs from the

Ranunculales (41,42), NnOMT1 displayed strict

C6 regiospecificity and accepted both R and S 1-

benzylisoquinoline conformers (Figure 5).

NnOMT1 contains all conserved catalytic and

substrate-binding residues occurring in Tf6OMT

(Figure 4). In Tf6OMT, the O-methyltransfer

reaction

occurs

via

H²⁵⁶

base-assisted

deprotonation of the substrate C6 hydroxyl,

followed by a nucleophilic attack on the SAM

methyl group. Interactions of H²⁵⁶and D²⁵⁷with

the C6 hydroxyl group provide proper orientation

and E³¹⁵contributes to the strong basicity in the

H²⁵⁶NE2 required for catalysis (18). Since

NnOMT1 and Tf6OMT possess equivalent key

catalytic residues (H²⁵⁶, D²⁵⁷and E³¹⁵) and share

~70% amino acid sequence identity, a similar

catalytic mechanism is expected for NnOMT1.

6

O-Methyltransferases from sacred lotus

variability of these residues in other 7OMTs (i.e.

G165A in Ps7OMT, PsN7OMT and Ct7OMT,

C253W in Ps7OMT and Ct7OMT, C253N in

PsN7OMT, and C253G in Ec7OMT). Conversely,

the D169H mutation disrupted a hydrogen bond

with the C4'-OH (2.6 Å), which stabilizes the

substrate in the active site. Interestingly, whereas

nitrogen of its only substrate norreticuline,

whereas Ps7OMT and Ec7OMT contain smaller

uncharged residues at equivalent positions

presumably to ease steric hindrance with (R,S)-

reticuline and other N-methylated substrates

(22,23,44), and Ct7OMT, with a D³⁰⁶to S

substitution, is able to 7-O-methylate (S)-

norcoclaurine (43). Notably, NnOMT5 contains a

D³⁰⁶residue (Figure S7, Figure S8), but catalyzes

similarly efficient 7-O-methylation of N-

methylated and N-demethyl substrates (Figure 5).

The catalytic activity of recombinants

D

¹⁶⁹is a conserved residue across all 6OMTs, or is

replaced by E in 4'OMTs, no consensus was found

in functionally characterized 7OMTs (i.e. D in

PsN7OMT, S in Ps7OMT and Ec7OMT, and T in

Ct7OMT); thus, this residue might not play a

significant role in 7OMT regiospecificity.

NnOMT1 and NnOMT5 with (R,S)-norcoclaurine

Interestingly, when both C6 and C7

and

exhibited similar reaction efficiency (k_cat/K_m),

whereas NnOMT5 showed 50-fold more

(S)-N-methylcoclaurine,

respectively,

hydroxyl groups were available, NnOMT5

exclusively catalyzed 6-O-methylation (Figure

S2). Similar results were reported for E.

californica and C. japonica 4'OMTs, which

a

efficient 7-O- versus 6-O-methylation (Table 1).

The K_mof NnOMT1 with (R,S)-norcoclaurine (20

µM) is similar to that reported for Ps6OMT (10

µM) and Tf6OMT (52 µM) (22,41), whilst the K_m

of NnOMT5 (152 µM) is substantially higher,

suggesting that NnOMT1 is the main 6OMT in

sacred lotus. On the other hand, the K_mof

NnOMT5 with (S)-N-methylcoclaurine (13 µM) is

substantially lower compared with that for (R,S)-

norcoclaurine (152 µM), but similar to that

reported for Ps7OMT with (S)-reticuline (16 µM)

(22) and PsN7OMT with (S)-norreticuline (44

µM) (23), suggesting that NnOMT5 acts primarily

as a 7OMT in the plant.

catalyze

norcoclaurine

6-O-methyltransfer,

although with lower efficiency than 4'-O-

methylation on already 6-O-methylated substrates

(45), in support of the importance of 6-O-

methylation as a purported rate-limiting step in

BIAs metabolism (21). However, since NnOMT5

catalyzed

methylcoclaurine 50-fold more efficiently than 6-

O-methylation of (R,S)-norcoclaurine, the

7-O-methylation

of

(S)-N-

physiological relevance of the latter activity is

questionable compared with NnOMT1 (Table 1).

It should be noted that, although coclaurine and N-

methylcoclaurine

are

almost

certainly

The apparent lack of recombinant NnOMT

physiological substrates of NnOMT5 (Figure 1),

other reported 1-benzylisoquinolines such as N-

methylisococlaurine and lotusine contain 7-O-

methyl but C6 hydroxyl groups, which does not

agree with the inability of NnOMT5 to 7-O-

methylate norcoclaurine in vitro. In this regard, it

is feasible that norcoclaurine N-methylation should

occur first for NnOMT5 to catalyze 7-O- rather

than 6-O-methylation, however the required

substrate to test this possibility was not available.

NnOMT5 also preferred norcoclaurine

over norlaudanosoline (Figure 5), whereas

NnOMT1 showed no substrate preference.

Nonetheless, the main catalytic activity detected

for NnOMT5 was the 7-O-methylation of several

N-methylated and N-demethylated substrates

(Figure 5). Although our understanding of 7OMT

substrate recognition and catalysis is limited, it has

been suggested that the conserved residue D³⁰⁶in

PsN7OMT is able to hydrogen bond with the

(Figure 5, Figure S2) or plant crude extract

activity (Figure 6b, Table S6) on substrates 3'- or

4'-hydroxyl groups partially agrees with the sacred

lotus BIA profile, which lacks C3'-substituted

alkaloids (13). In contrast, 4'-O-methylated BIAs

were detected (Figure 2b), including isoliensinine

and neferine (Figure 2c). Low 4'OMT activity has

been reported for enzymes with primarily 6-O- or

7-O-methyltransferase activity, such as GfOMT2

and Ps7OMT (22,27). However, NnOMT1 and

NnOMT5 did not show 4'-O-methyltransferase

activity on any substrate, including (R)-

armepavine, although the actual substrate(s) might

not have been available. The phylogenetic

relationships between NnOMT5 and PsOMT2, and

between NnOMT1 and PsOMT3 and Ps6OMT,

suggest that 4'OMT activity in sacred lotus could

also involve a heterodimeric enzyme. It is notable

that in the Ranunculales, a Berberis koetineana

4'OMT (Bk4'OMT), Cj4'OMT, Ec4'OMT and

7

O-Methyltransferases from sacred lotus

Ps4'OMT2 act only on 3'-hydroxylated substrates

(22,25,27,42,45,46), suggesting that sacred lotus

4'-O-methyltransferase activity could involve a

unique enzyme.

Assayed aporphine, protoberberine and

morphinan alkaloids were not accepted as

substrates (Table S4). OMTs shown to O-

characterized OMTs have either shown a lack of

stereospecificity (e.g. Cj6OMT, Ps6OMT and

Ps7OMT) or strict S stereospecificity (Bk4'OMT)

(22,42,53). Our results support the formation of

(R)-coclaurine and (S)-armepavine as previously

reported in lotus (6,54). However, despite

NnOMT5 showed strict stereospecificity toward S

enantiomers, neither norlaudanosoline nor

reticuline are physiologically relevant substrates

and it is possible that the enzyme catalyzes the

methylate

1-benzylisoquinolines

and

protoberberines occur in species accumulating

both alkaloid types (26-28), although specificity

for 1-benzylisoquinolines (e.g. Cj6OMT and

PsN7OMT) has also been reported (23,42).

Though sacred lotus does not accumulate

morphinans or protoberberines (13), aporphines

are major alkaloids in the leaves (34,35). The lack

of NnOMT activity on aporphines suggests that

the C8-C2' coupling yielding the aporphine

scaffold occurs after the O-methylation of 1-

turnover

methylcoclaurine in the formation of the reported

compounds (R)-norarmepavine and (R)-

of

(R)-coclaurine

and

(R)-N-

armepavine, respectively (55). Unfortunately, none

of the relevant R enantiomers were available as

potential substrates.

Previous work on the BIA profile in

sacred lotus focused on the quantification of

aporphines and bisbenzylisoquinolines in leaves

and embryos, respectively, but limited information

has been provided for 1-benzylisquinoline

intermediates (34,36). We quantified key alkaloids

produced in the early steps of the pathway (i.e.

norcoclaurine, coclaurine, N-methylcoclaurine and

armepavine) in different sacred lotus organs

(Figure 6a). Although 1-benzylisoquinolines were

predominant in leaves, the detected compounds

were likely intermediates leading to aporphine and

bisbenzylisoquinoline end products. The low

levels of 1-benzylisoquinolines in embryos could

also reflect their transformation to predominant

bisbenzylisoquinolines (36,37,40,56).

benzylisoquinoline

Dactylicapnos scandens 7OMT was recently

reported to O-methylate corytuberine,

intermediates.

However,

demonstrating the plausibility of aporphines as

OMTs substrates (47). It is notable that the

aporphines and bisbenzylisoquinolines of sacred

lotus were not available; thus, it remains possible

that certain intermediates within these BIA

structural types are NnOMT substrates.

The occurrence of most BIAs as R

conformers is another unique feature in sacred

lotus compared with members of the Ranunculales

(13). The importance of the stereochemistry of 1-

benzylisoquinoline substrates is evident in opium

poppy, in which reticuline conformation

determines the formation of major end-point

alkaloids, with morphine derived from (R)-

reticuline, and other alkaloids such as noscapine

and sanguinarine produced from (S)-reticuline.

BIA stereochemistry is initially established by the

stereoselectivity of norcoclaurine synthase (NCS).

Although all characterized NCS isoforms from the

Ranunculales yield exclusively (S)-norcoclaurine

(48-50), norcoclaurine in sacred lotus has been

detected as both S and R enantiomers (6,51,52) and

consequently, the presence of both enantiomeric

forms of norcoclaurine-derived alkaloids depends

on the stereospecificity of downstream enzymes.

We show that NnOMT1 catalyzed the 6-O-

methylation of (R)- and (S)-norlaudanosoline with

similar turnover efficiency, whereas NnOMT5

accepted only (S)-norlaudanosoline and (S)-

reticuline (Figure 5, Table S4). Previously

Assays containing crude plant extracts

incubated with (R,S)-norcoclaurine, (S)-N-

methylcoclaurine and (R)-armepavine showed 6-

O- and 7-O-methyltransferase activity in all organs

(Figure 6b), but mostly in leaves and embryos.

OMT transcripts were also expressed in all organs,

although NnOMT1 and NnOMT5 showed higher

expression levels, mainly in leaves (Figure 6c).

These results are consistent with a recent report on

NnOMT expression at different leaf developmental

stages, whereby NnOMT1 and NnOMT5 transcript

levels were up to 10-fold higher compared with

NnOMT2-NnOMT4 (34). A lag between NnOMT

expression and aporphine alkaloid accumulation

was also noted in this study, which is potentially

associated with the involvement of OMTs early in

BIA metabolism and a requirement for the

expression of additional genes to complete

aporphine and bisbenzylisoquinoline biosynthesis.

8

O-Methyltransferases from sacred lotus

It is notable that since this earlier study did not

perform a functional characterization of candidate

genes, NnOMT5 was reported as a 4'OMT (34). In

agreement with the suggested order of O-

methylations in other plants (21,45), NnOMT1

appears to act earlier than NnOMT5 in the leaves

since 6OMT activity decreased in unfolded leaves

whereas 7OMT activity increased (Figure 6b).

Similarly, NnOMT1 transcript levels were higher

in folded leaves than unfolded leaves, whereas

NnOMT5 transcript levels remained constant

(Figure 6c), consistent with a previous report (34).

methyltransferase,

and

aporphine

and

bisbenzylisoquinoline synthases) will provide

additional key insights into the evolutionary

history of BIA biosynthesis in sacred lotus.

EXPERIMENTAL PROCEDURES

Plant material

Seeds for two sacred lotus (Nelumbo nucifera)

varieties, named Pink and White owing to their

distinct flower colors, were germinated in water

according to the instructions of the vendor

(www.rarexoticseeds.com). After the emergence

of embryogenic leaves, the seedlings were planted

in pots containing heavy loam soil and submerged

in an artificial pond. Plants were grown at 30 °C

and under high-intensity lighting with a 16 h

photoperiod. Alternatively, seeds were carefully

opened with a bench press and the embryos were

isolated. All harvested plant samples were

immediately frozen in liquid nitrogen and stored at

-80 °C until used.

Although

NnOMT1

and

NnOMT5

transcript levels were 5- to 15-fold lower in

embryos compared with folded leaves, specific 6-

and 7-O-methyltransferase activities were similar

in both organs, likely owing to the prevalence of

photosynthetic leaf proteins. Interestingly, the

levels of transcripts encoding CYP80G,

purportedly involved in aporphine biosynthesis, in

leaves were 5 times higher than levels of

transcripts

encoding

CYP80A,

potentially

involved in bisbenzylisoquinoline biosynthesis, in

embryos (33), suggesting that lower biosynthetic

gene transcript levels in embryos might be

sufficient in this organ. However, neither CYP80G

or CYP80A orthologs in sacred lotus were

functionally characterized, so it is not certain that

these transcripts are relevant to BIA metabolism.

Bisbenzylisoquinolines biosynthesis has been

proposed to occur in leaves, with alkaloids

subsequently transported to embryos (36). NCS

activity (4) and purported NnNCS transcripts (57)

were detected in leaves, suggesting that BIA

biosynthesis occurs in this organ. The detection of

O-methyltransferase activity indicates that

embryos are also involved in alkaloid biosynthesis.

From an evolutionary perspective, it is not

known whether OMT recruitment occurred

repeatedly or if an ancestrally promiscuous OMT

gave rise to functionally distinct enzymes (20).

The 6-O-methyltransferase activity of NnOMT1

was correctly predicted based on similarity to

Tf6OMT and other 6OMTs. However, the 7-O-

methyltransferase activity of NnOMT5 was

unexpected owing to its relatively low similarity to

characterized 7OMTs from the Ranunculales

(Figure 3, Table S3), suggesting a possible

independent recruitment in sacred lotus. The

Chemicals and reagents

(R,S)-Norcoclaurine, (R)-norlaudanosoline, (S)-

norlaudanosoline, (S)-coclaurine and (13S,14R)-

1,13-dihydroxy-N-methylcanadine were purchased

from Toronto Research Chemicals (ON, Canada);

(R)-reticuline was purchased from Santa Cruz

Biotechnology (CA, USA); (R)-armepavine was

purchased from MuseChem (NJ, USA); (S)-

scoulerine was purchased from ChromaDex (CA,

USA); (+)-bulbocapnine and (+)-isocorydine were

purchased from Sequoia Research Products

(Pangbourne, UK); and boldine and S-adenosyl-L-

methionine (SAM) were purchased from Sigma-

Aldrich. (S)-Reticuline was a gift from Tasmanian

Alkaloids Pty Ltd (Westbury, Australia), and

morphine and codeine were gifts from Sanofi-

Aventis (Paris, France). (S)-N-Methylcoclaurine

was generated from (S)-coclaurine using purified

recombinant NMT1 from Glaucium flavum (58).

Enzymatic conversion was performed at 30 °C,

with 1 mM (S)-coclaurine, 1 mM SAM, and 20 µg

recombinant GfNMT1. Reactions were extracted

by ethyl acetate three times and the product

identity and purity confirmed by HPLC-MS/MS.

characterization

norcoclaurine

of

other

enzymes

coclaurine

(e.g.

N-

synthase,

9

O-Methyltransferases from sacred lotus

All other chemicals were purchased from Sigma-

Aldrich or Bioshop Canada (ON, Canada).

methylcoclaurine and armepavine was performed

using authentic standards.

Plant alkaloid extraction and LC-MS analysis

NnOMT identification and phylogeny

Nelumbo nucifera samples corresponding to folded

and unfolded leaves, rhizomes, roots and embryos

were ground to a fine powder under liquid nitrogen

using a mortar and pestle. Alkaloid extraction was

A previously reported Nelumbo nucifera draft

genome (30,31) was searched to identify

sequences encoding potential O-methyltransferase

involved in BIA biosynthesis using the tBLASTn

algorithm and functionally characterized OMTs

from Papaver somniferum as queries (Ps6OMT,

Ps7OMT, PsN7OMT and Ps4'OMT2). Five

candidates (NnOMT1-NnOMT5) sharing >40%

amino acid sequence identity with query sequences

were isolated. The GenBank accession codes were

as follows: XM_010245752, XM_010249599,

performed using

a

modified version of

a

previously reported protocol for sacred lotus (36).

Briefly, 1 mL methanol was added to ~100 mg of

plant material, and samples were sonicated in an

ultrasonic bath for 30 min at room temperature,

centrifuged at 14,000 g for 10 min, and the

supernatant was filtered (0.2 µm Acrodisc syringe

filter, Pall Corporation, NY, USA). The insoluble

material was dried and weighed. Samples (5 µL)

were fractionated on a Zorbax C18 column, 2.1 x

50 mm, 1.8 µm (Agilent, California, USA) using a

Dionex UltiMate 3000 HPLC system (Thermo

Scientific) at a flow rate of 500 µL/min. The

mobile phase consisted of solvent A (10 mM

ammonium acetate, pH 5.5, 5% (v/v) acetonitrile)

and solvent B (100% acetonitrile), starting at

100% (v/v) Solvent A, and ramping from 0 to 20%

(v/v) Solvent B over 5 min, 20 to 50% (v/v)

Solvent B over 3 min, 50 to 100% (v/v) Solvent B

over 3 min, remaining isocratic at 100% (v/v)

Solvent B over 2 min, ramping from 100 to 0%

(v/v) Solvent B over 0.1 min, and remaining

isocratic at 100% (v/v) Solvent A over 1.9 min.

The total run time was 15 min and data were

collected for the first 10 min. Heated ESI source

and interface conditions were operated in positive

ion mode as follows: vaporizer temperature 400

°C, source voltage 3kV, sheath gas 60 au, auxiliary

gas 20 au, capillary temperature 380 °C, capillary

voltage 6 V, tube lens 45 V. A LTQ-Orbitrap-XL

(Thermo Fisher) mass spectrometer was operated

using LTQ Tune Plus v. 2.5.5 SP1 and Xcalibur

software (version 2.1.0.1140), with additional

analyses using the QualBrowser feature of

Xcalibur. Internal and external calibration, tuning

and general operations were performed as

previously reported (27,59). Error was maintained

at < 2 ppm. Exact masses, retention times and CID

spectra of available authentic standards, or

previously published data, was used for alkaloid

XM_010249600,

XM_010273389,

and

XM_010277761) (Table S2). Protein molecular

weight and isoelectric point predictions were made

using the Geneious software package (Biomatters,

NJ, USA). Amino acid sequence alignments were

performed using the default parameters of the

MUSCLE algorithm (60) implemented in MEGA

X (61). Evolutionary history was inferred using the

Maximum Likelihood method established on the

JTT matrix-based model (62). The tree with the

highest log likelihood was drawn, with branch

lengths proportional to the number of substitutions

per site, and nodes labelled according to the

percentage of trees in which the associated taxa

clustered together in the bootstrap test based on

1000 replicates. The amino acid percent identity

matrix among Nelumbo nucifera (Nn) and other

OMTs was performed using Clustal Omega (63)

The GenBank accession codes were as follows:

Cc6OMT1

(MH165875);

Cc6OMT2

(MH165876); Cj4'OMT (D29812); Cj6OMT

(D29811); CjCoOMT (AB073908); CjSOMT

(D29809); Ct7OMT (MH165877); CtSOMT

(MH165874); Ec4'OMT (AB745041); Ec7OMT

(AB232153); EcSOMT (LC171865); GfOMT1

(KP176693); GfOMT2 (KP176694); GfOMT6

(KP176698); GfOMT7 (KP176699); Ps4'OMT2

(AY217334); Ps6OMT (AY217335); Ps7OMT

(AY268893); PsN7OMT (FJ156103); PsSOMT1

(JN185323); PsSOMT2 (MH029292); PsSOMT3

(MH029294); Tf6OMT (AY610507); Tt6OMT1

(AF064693);

Tt6OMT2

(AF064694).

Abbreviations were as follows: Cc, Coptis

chinensis; Cj, Coptis japonica; Ct, Coptis teeta;

Ec, Eschscholzia californica; Gf, Glaucium

identification

(Table

S1,

Figure

S6).

Quantification of norcoclaurine, coclaurine, N-

10

O-Methyltransferases from sacred lotus

flavum; Nn, Nelumbo nucifera; Ps, Papaver

somniferum; Tf, Thalictrum flavum; Tt, Thalictrum

encoding an N-terminal His₆-tag using specific

primers (Table S8a).

tuberosum;

4'OMT,

3'-hydroxy-N-

Recombinant protein purification

methylcoclaurine 4’-O-methyltransferase; 6OMT,

norcoclaurine 6-O-methyltransferase; 7OMT,

The pRSET-A-NnOMT expression plasmids were

used to transform the E. coli Rosetta (DE3) pLysS

(EMD Millipore, MA, USA) strain and single

colonies were used to inoculate 50 mL LB medium

supplemented with 100 µg/mL ampicillin and 35

µg/mL chloramphenicol. Cultures were grown at

30 °C with orbital shaking at 200 rpm for 16 h and

used to inoculate 1 L LB media (100 µg/mL

ampicillin and 35 µg/mL chloramphenicol) to a

starting OD₆₀₀of 0.1. Cultures were grown at 30

°C until OD₆₀₀~ 0.6, cooled to 16 °C and the

production of recombinant proteins was induced

by the addition of isopropyl β-D-thiogalactoside

(IPTG) to a final concentration of 0.1 mM.

Cultures were kept at 16 °C with shaking at 200

rpm for 16 h and cells harvested by centrifugation

at 5,000 g for 20 min at 4 °C. Cell pellets were

resuspended in 40 mL protein extraction buffer

(100 mM Tris-HCl pH 7.5, 300 mM NaCl, 10%

[v/v] glycerol) supplemented with 1 mg/mL

lysozyme and sonicated on ice for 3 min (10 sec

on, 30 sec off). The crude lysate was centrifuged at

16,000 g for 20 min at 4 °C to remove cellular

debris. The cleared supernatant was loaded onto 1

mL of equilibrated Talon cobalt affinity resin

(Clontech, CA, USA) and incubated at 4 °C for 30

min with gentle shaking. The resin was washed

with 20 mL of protein extraction buffer, followed

by 5 mL of protein extraction buffer containing 20

mM imidazole. Purified protein was eluted using 5

mL of protein extraction buffer containing 200

mM imidazole, and subsequently concentrated and

desalted by repeated ultrafiltration on an Amicon

Ultra 30K column (EMD Millipore, MA, USA) in

storage buffer (100 mM Tris-HCl pH 7.5, 10%

(v/v) glycerol, 1 mM β-mercaptoethanol). Purified

His₆-tagged protein concentration was determined

using the Bradford reagent according to the

instructions of the manufacturer (Thermo Fisher,

IL, USA) and using bovine serum albumin as the

standard. Protein purity was assessed by SDS-

PAGE using a 10% (w/v) gel, which was

reticuline

columbamine O-methyltransferase; N7OMT,

norreticuline 7-O-methyltransferase; SOMT,

scoulerine 9-O-methyltransferase. The amino acid

sequences of functionally characterized OMTs

involved in BIAs biosynthesis used in the present

work are provided in Table S9.

7-O-methyltransferase;

CoOMT,

NnOMT cDNA isolation and expression

Plant tissues were ground with a mortar and pestle

to a fine powder under liquid nitrogen. Total RNA

was extracted using the cetyltrimethylammonium

bromide (CTAB) method, as previously reported

for Nelumbo nucifera tissues (64). RNA quality

was confirmed by A_260/280and A_260/230absorbance

measurements using

a

NanoDrop ND-1000

(Thermo Fisher Scientific) spectrophotometer, and

visualization by agarose gel electrophoresis. After

genomic DNA removal (AccuRT Genomics DNA

Removal kit, Applied Biological Materials, BC,

Canada), first-strand cDNA synthesis was

performed on 1 µg of RNA using 5X All-in-One

RT MasterMix kit, according to the instructions of

the manufacturer (Applied Biological Materials,

BC, Canada). NnOMT open reading frames were

amplified from cDNA using Q5 High Fidelity

DNA polymerase (New England Biolabs, MA,

USA) and sequence-specific primers (Table S8a)

under the following conditions: 98 °C for 30 sec;

35 cycles of 98 °C for 10 sec, 52-65 °C (optimized

for each NnOMT) for 30 sec, and 72 °C for 30 sec;

72 °C for 2 min. Amplicons were cloned in a

pMiniT 2.0 vector, according to the instructions of

the manufacturer (New England Biolabs, MA,

USA) and used to transform E. coli TOP 10 strain

for colony PCR screening. Plasmids from positive

colonies were purified (GeneJet Plasmid Miniprep

kit, Thermo-Fisher Scientific, MA, USA) and

sequenced. PCR products were purified (GeneJet

Gel Extraction Kit, Thermo-Fisher Scientific, MA,

USA) and ligated into pRSET-A (Invitrogen, CA,

USA) to construct the expression vectors using

one-step sequence- and ligation-independent

cloning (SLIC) (65). Full-length NnOMT coding

regions were cloned in frame with sequences

subsequently transferred to

a

nitrocellulose

membrane. Protein blots were blocked overnight

with 5% (w/v) skim milk and incubated for 1 h

with a 1:1 000 dilution of His₆-antibody. After

11

O-Methyltransferases from sacred lotus

washing with TBST buffer (200 mM Tris-HCl pH

7.6, 1.5 M NaCl, 0.1% (v/v) Tween 20), the blots

were incubated for 1 h with a 1:10 000 dilution of

goat anti-mouse IgG antibodies conjugated with

horseradish peroxidize (Bio-Rad). Immunoblots

were washed and developed using SuperSignal

West Pico chemiluminescent substrate, according

to the instructions of the manufacturer (Thermo

Scientific, IL, USA) and the bands were visualized

using an Amersham Imager 600 (GE Healthcare,

IL, USA).

confirmed by comparison of retention times and

CID spectra with respect to authentic standards, or

data reported in the literature. Substrate conversion

rates were calculated based on substrate loss.

NnOMT1 and NnOMT5 pH-optimum assays were

conducted in 25 mM sodium citrate (pH 5.0-6.0),

50 mM sodium phosphate (pH 6.0-7.5), and 100

mM Tris-HCl (pH 7.5-9.0) using 100 µM (R,S)-

norcoclaurine or 100 µM (S)-N-methylcoclaurine,

respectively, 200 µM SAM and 1 µg purified

recombinant protein in a reaction volume of 50

µL. Assays were incubated within the linear range

for product formation at 30 °C for 5 min and

stopped with 100 µL acetonitrile. Temperature

optima were determined under identical conditions

except that 100 mM Tris-HCl, pH 8 and

incubation temperatures between 4-60 °C were

used. Kinetic parameters were measured for 5 min

under the same conditions except that NnOMT1

was incubated with (R,S)-norcoclaurine at 30 °C

and NnOMT5 with either (R,S)-norcoclaurine or

(S)-N-methylcoclaurine at 37 °C, using alkaloid

concentrations from 5 to 400 µM and a fixed SAM

concentration of 400 µM. Product formation for

NnOMT1 and NnOMT5 was quantified by

comparison with authentic standards of coclaurine

and armepavine, respectively. All assays were

performed in triplicates. Saturation curves and

kinetic parameters were determined based on the

Michaelis-Menten equation (NnOMT1 and

NnOMT5 incubated with (R,S)-norcoclaurine) or a

substrate-inhibition equation (NnOMT5 incubated

Plant protein extraction

Plant tissues were ground to a fine powder under

liquid nitrogen using a mortar and pestle. Total

proteins were extracted by adding 5 mL of ice-cold

extraction buffer A containing 100 mM Tris-HCl

pH 7.5, 10% (v/v) glycerol, 1% (w/v)

polyvinylpyrrolidone 40, 5 mM dithiothreitol

(DTT) and 1 mM phenylmethylsulfonyl fluoride

(PMFS) to ~1 g of plant material, and sonicated in

an ultrasonic bath for 5 min at 4 °C. Plant debris

was eliminated by centrifugation at 14,000 g for

10 min, and the supernatant was desalted in a PD-

10 column (GE Healthcare, IL, USA) and eluted in

extraction buffer without DTT and PMFS. The

solution

was

concentrated

by

repeated

ultrafiltration using an Amicon Ultra 30K column

(EMD Millipore, MA, USA) in storage buffer (100

mM Tris-HCl pH 7.5, 10% glycerol, 1 mM β-

mercaptoethanol).

Crude

plant

protein

concentration was determined using the Bradford

reagent according to the instructions of the

manufacturer (Thermo Fisher, IL, USA) and using

bovine serum albumin as the standard.

with (S)-N-methylcoclaurine), using Prism

(GraphPad, CA, USA).

5

Plant protein assays were performed using

50 µg of crude protein in 100 mM Tris-HCl pH

7.5, using 200 µM alkaloid and 400 µM SAM in a

100 µL reaction. Negative control assays were

performed using crude plant protein extracts

denatured in boiling water for 10 min. Reactions

were incubated overnight at 30 °C, quenched with

200 µL acetonitrile and analyzed by LC-MS/MS.

Product formation, determined as an increase of 14

Da with respect to the substrate, was confirmed by

comparison of retention times and CID spectra

with respect to authentic standards.

Enzyme assays

Substrate-acceptance assays were performed in

100 mM Tris-HCl pH 7.5, using 100 µM alkaloid,

200 µM SAM and 5 µg of purified recombinant

protein in a reaction volume of 50 µL. Negative

control assays were performed using purified

recombinant protein denatured in boiling water for

10 min. Assays were incubated at 30 °C and

quenched after 4 h with 100 µL acetonitrile,

centrifuged at 17,000 g for 40 min to precipitate

proteins, and the supernatant analyzed by LC-

MS/MS. Product formation, determined as an

increase of 14 Da with respect to the substrate, was

HPLC-MS/MS

HPLC-MS/MS was performed using a 1200 HPLC

instrument coupled to a 6410 triple quadrupole MS

12

O-Methyltransferases from sacred lotus

(Agilent, CA, USA). Samples (5 µL) were injected

onto a Poroshell 120 SB-C18 HPLC column, 2.1 x

50 mm, 2.7-µm particle size (Agilent, CA, USA).

Analytes were eluted using a mobile phase

gradient of Solvent A (10 mM ammonium acetate,

pH 5.5, 5% (v/v) acetonitrile) and Solvent B

(100% acetonitrile) at a flow rate of 600 µL/min.

The gradient started at 100% (v/v) Solvent A,

ramped linearly to 60% (v/v) Solvent B by 8 min,

further increased linearly to 99% (v/v) Solvent B

over 2 min, remained isocratic at 99% (v/v)

Solvent B from 10 to 11 min, and returned to

100% (v/v) Solvent A at 11.1 min for a 3 min re-

equilibration period. Analytes were applied to the

mass analyzer using an electrospray ionization

probe operating in positive mode with the

following conditions: capillary voltage 4 000 V,

fragmentor voltage 110 V, source temperature 350

°C, nebulizer pressure 50 psi, gas flow 10 L/min.

For full-scan analysis, quadrupole 1 and 2 were set

to radio frequency only, whereas the third

quadrupole scanned from m/z 200 to m/z 700.

ESI[+] and CID spectra were analyzed, the

precursor m/z was selected in quadrupole 1 and

collision energy of 25 eV and an argon collision

gas pressure of 1.8 x 10^-3torr were applied in

quadrupole 2. The resulting MS²fragments were

resolved by quadrupole 3 scanning from 30 m/z to

5 m/z greater than the precursor ion m/z.

Compounds were identified based on retention

times and ESI[+]-CID spectra compared with

authentic standards or published spectral data.

ramp rate of 1.6 °C/sec to 95 °C maintained for 15

sec, 1.6 °C/sec to 60 °C maintained for 1 min, and

0.15 °C/sec to 95 °C, which was maintained for 15

sec. Primer efficiency was verified using

LinRegPCR software (66) and samples with values

between 1.8-2 were selected for analysis. Relative

transcript abundance was calculated by the 2^-ΔΔCt

method (67), using β-actin as the endogenous

reference gene (68). Transcript levels were

normalized with respect to the gene showing the

lowest expression level.

Homology modeling

The previously reported crystal structure from

Thalictrum

flavum

norcoclaurine

6-O-

methyltransferase (Tf6OMT) in complex with (S)-

norlaudanosoline and S-adenosyl-L-homocysteine

(PDB 5ICE) (18) was used as template to generate

a NnOMT5 homology model using SWISS-

MODEL (69). The resulting NnOMT5 model and

Tf6OMT crystal structure were visualized, and

figures were generated, using PyMOL Molecular

Graphic System (v2.3.1, Schrödinger, LLC).

Acknowledgements: We thank Dr. Jillian Hagel

for assistance with LTQ-Orbitrap data analysis and

for creating original artwork, and Dr. Jeremy

Morris and Dr. Xue Chen for guidance on the

experimental work and critical comments. Funding

for this research was provided by a Natural

Sciences and Engineering Research Council of

Canada Discovery grant to PJF. IMMP is the

recipient of the Alberta Innovates Technology

Futures Scholarship.

Quantitative real-time PCR

RNA extraction and cDNA synthesis were

performed as described above. qRT-PCR analysis

was performed using PowerUp SYBR Green

Master Mix and a QuantStudio-3 Real-Time PCR

System (Applied Biosystems, MA, USA).

Reactions (10 µL) contained diluted cDNA (~8

ng), 5 µL SYBR Green Master Mix (2X) (Applied

Biosystems), and 500 nM of each primer (Table

S8b). Thermal conditions were 50 °C for 2 min, 95

°C for 2 min, 40 cycles of 95 °C for 1 sec and 60

°C for 30 sec. Amplification specificity was

confirmed by melt-curve analysis, generated at a

Conflict of interest: The authors have no conflicts

to declare.

Author contributions: Conceptualization, IMMP,

PJF; Methodology, IMMP, PJF; Formal Analysis,

IMMP; Investigation, IMMP; Resources, PJF;

Data curation, IMMP; Writing – Original draft,

IMMP; Visualization IMMP, PJF; Funding

Acquisition, PJF. All authors reviewed the results

and approved the final version of the manuscript.

13

O-Methyltransferases from sacred lotus

REFERENCES

1. Dastmalchi, M., Park, M. R., Morris, J. S., and Facchini, P. J. (2018) Family portraits: the enzymes behind

benzylisoquinoline alkaloid diversity. Phytochem Rev. 17, 249-277

2. Hagel, J. M., and Facchini, P. J. (2013) Benzylisoquinoline alkaloid metabolism: a century of discovery and a

brave new world. Plant Cell Physiol. 54, 647-672

3. Singh, A., Menéndez-Perdomo, I. M., and Facchini, P.J. (2019) Benzylisoquinoline alkaloid biosynthesis in

opium poppy: and update. Phytochem. Rev. 18, 1457-1482.

4. Liscombe, D. K., MacLeod, B. P., Loukanina, N., Nandi, O. I., and Facchini, P. J. (2005) Evidence for the

monophyletic evolution of benzylisoquinoline alkaloid biosynthesis in angiosperms. Phytochemistry. 66, 2501-

2520

5. Chen, G., Zhu, M., and Guo, M. (2019) Research advances in traditional and modern use of Nelumbo nucifera:

phytochemicals, health promoting activities and beyond. Crit Rev Food Sci Nutr. 1-21

6. Kashiwada, Y., Aoshima, A., Ikeshiro, Y., Chen, Y. P., Furukawa, H., Itoigawa, M., Fujioka, T., Mihashi, K.,

Cosentino, L. M., Morris-Natschke, S. L., and Lee, K. H. (2005) Anti-HIV benzylisoquinoline alkaloids and

flavonoids from the leaves of Nelumbo nucifera, and structure-activity correlations with related alkaloids. Bioorg

Med Chem. 13, 443-448

7. Liu, C. P., Tsai, W. J., Shen, C. C., Lin, Y. L., Liao, J. F., Chen, C. F., and Kuo, Y. C. (2006) Inhibition of (S)-

armepavine from Nelumbo nucifera on autoimmune disease of MRL/MpJ-lpr/lpr mice. Eur J Pharmacol. 531,

270-279

8. Liu, W., Yi, D. D., Guo, J. L., Xiang, Z. X., Deng, L. F., and He, L. (2015) Nuciferine, extracted from Nelumbo

nucifera Gaertn., inhibits tumor-promoting effect of nicotine involving Wnt/beta-catenin signaling in non-small

cell lung cancer. J Ethnopharmacol. 165, 83-93

9. Kang, E. J., Lee, S. K., Park, K. K., Son, S. H., Kim, K. R., and Chung, W. Y. (2017) Liensinine and nuciferine,

bioactive components of Nelumbo nucifera, inhibit the growth of breast cancer cells and breast cancer-associated

bone loss. Evid Based Complement Alternat Med. 2017, 1583185

10. Poornima, P., Weng, C. F., and Padma, V. V. (2014) Neferine, an alkaloid from lotus seed embryo, inhibits

human lung cancer cell growth by MAPK activation and cell cycle arrest. Biofactors. 40, 121-131

11. Yoon, J. S., Kim, H. M., Yadunandam, A. K., Kim, N. H., Jung, H. A., Choi, J. S., Kim, C. Y., and Kim, G. D.

(2013) Neferine isolated from Nelumbo nucifera enhances anti-cancer activities in Hep3B cells: molecular

mechanisms of cell cycle arrest, ER stress induced apoptosis and anti-angiogenic response. Phytomedicine. 20,

1013-1022

12. Xu, L., Zhang, X., Li, Y., Lu, S., Li, J., Wang, Y., Tian, X., Wei, J. J., Shao, C., and Liu, Z. (2016) Neferine

induces autophagy of human ovarian cancer cells via p38 MAPK/ JNK activation. Tumour Biol. 37, 8721-8729

13. Menendez-Perdomo, I. M., and Facchini, P. J. (2018) Benzylisoquinoline alkaloids biosynthesis in sacred lotus.

Molecules. 23

14. Liscombe, D. K., Louie, G. V., and Noel, J. P. (2012) Architectures, mechanisms and molecular evolution of

natural product methyltransferases. Nat Prod Rep. 29, 1238-1250

15. Lam, K. C., Ibrahim, R. K., Behdad, B., and Dayanandan, S. (2007) Structure, function, and evolution of plant

O-methyltransferases. Genome. 50, 1001-1013

16. Park, M. R., Chen, X., Lang, D. E., Ng, K. K. S., and Facchini, P. J. (2018) Heterodimeric O-methyltransferases

involved in the biosynthesis of noscapine in opium poppy. Plant J. 95, 252-267

17. Li, Y., and Smolke, C. D. (2016) Engineering biosynthesis of the anticancer alkaloid noscapine in yeast. Nat

Commun. 7, 12137

18. Robin, A. Y., Giustini, C., Graindorge, M., Matringe, M., and Dumas, R. (2016) Crystal structure of

norcoclaurine-6-O-methyltransferase, a key rate-limiting step in the synthesis of benzylisoquinoline alkaloids.

Plant J. 87, 641-653

19. Cabry, M., Offen, W. A., Saleh, P., Li, Y., Winzer, T., Graham, I. A., and Davies, G. J. (2019) Structure of

Papaver somniferum O-Methyltransferase 1 reveals initiation of noscapine biosynthesis with implications for

plant natural product methylation. ACS Catal. 9, 3840-3848

20. Desgagne-Penix, I., and Facchini, P. J. (2012) Systematic silencing of benzylisoquinoline alkaloid biosynthetic

genes reveals the major route to papaverine in opium poppy. Plant J. 72, 331-344

21. Ounaroon, A., Decker, G., Schmidt, J., Lottspeich, F., and Kutchan, T. M. (2003) (R,S)-Reticuline 7-O-

methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum - cDNA cloning and

characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J. 36, 808-819

14

O-Methyltransferases from sacred lotus

22. Pienkny, S., Brandt, W., Schmidt, J., Kramell, R., and Ziegler, J. (2009) Functional characterization of a novel

benzylisoquinoline O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy

(Papaver somniferum L). Plant J. 60, 56-67

23. Facchini, P. J., and Park, S. U. (2003) Developmental and inducible accumulation of gene transcripts involved in

alkaloid biosynthesis in opium poppy. Phytochemistry. 64, 177-186

24. Ziegler, J., Diaz-Chavez, M. L., Kramell, R., Ammer, C., and Kutchan, T. M. (2005) Comparative macroarray

analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-

methyltransferase involved in benzylisoquinoline biosynthesis. Planta. 222, 458-471

25. Dang, T. T., and Facchini, P. J. (2012) Characterization of three O-methyltransferases involved in noscapine

biosynthesis in opium poppy. Plant Physiol. 159, 618-631

26. Chang, L., Hagel, J. M., and Facchini, P. J. (2015) Isolation and characterization of O-methyltransferases

involved in the biosynthesis of glaucine in Glaucium flavum. Plant Physiol. 169, 1127-1140

27. Purwanto, R., Hori, K., Yamada, Y., and Sato, F. (2017) Unraveling additional O-methylation steps in

benzylisoquinoline alkaloid biosynthesis in California poppy (Eschscholzia californica). Plant Cell Physiol. 58,

1528-1540

28. Morishige, T., Dubouzet, E., Choi, K. B., Yazaki, K., and Sato, F. (2002) Molecular cloning of columbamine O-

methyltransferase from cultured Coptis japonica cells. Eur J Biochem. 269, 5659-5667

29. Facchini, P. J., and Morris, J. S. (2019) Molecular origins of functional diversity in benzylisoquinoline alkaloid

methyltransferases. Front Plant Sci.

30. Gui, S., Peng, J., Wang, X., Wu, Z., Cao, R., Salse, J., Zhang, H., Zhu, Z., Xia, Q., Quan, Z., Shu, L., Ke, W.,

and Ding, Y. (2018) Improving Nelumbo nucifera genome assemblies using high-resolution genetic maps and

BioNano genome mapping reveals ancient chromosome rearrangements. Plant J. 94, 721-734

31. Ming, R., VanBuren, R., Liu, Y., Yang, M., Han, Y., Li, L. T., Zhang, Q., Kim, M. J., Schatz, M. C., Campbell,

M., Li, J., Bowers, J. E., Tang, H., Lyons, E., Ferguson, A. A., Narzisi, G., Nelson, D. R., Blaby-Haas, C. E.,

Gschwend, A. R., Jiao, Y., Der, J. P., Zeng, F., Han, J., Min, X. J., Hudson, K. A., Singh, R., Grennan, A. K.,

Karpowicz, S. J., Watling, J. R., Ito, K., Robinson, S. A., Hudson, M. E., Yu, Q., Mockler, T. C., Carroll, A.,

Zheng, Y., Sunkar, R., Jia, R., Chen, N., Arro, J., Wai, C. M., Wafula, E., Spence, A., Xu, L., Zhang, J., Peery,

R., Haus, M. J., Xiong, W., Walsh, J. A., Wu, J., Wang, M. L., Zhu, Y. J., Paull, R. E., Britt, A. B., Du, C.,

Downie, S. R., Schuler, M. A., Michael, T. P., Long, S. P., Ort, D. R., Schopf, J. W., Gang, D. R., Jiang, N.,

Yandell, M., dePamphilis, C. W., Merchant, S. S., Paterson, A. H., Buchanan, B. B., Li, S., and Shen-Miller, J.

(2013) Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.). Genome Biol. 14, R41

32. Meelaph, T., Kobtrakul, K., Chansilpa, N. N., Han, Y., Rani, D., De-Eknamkul, W., and Vimolmangkang, S.

(2018) Coregulation of biosynthetic genes and transcription factors for aporphine-type alkaloid production in

wounded lotus provides insight into the biosynthetic pathway of nuciferine. ACS Omega. 3, 8794-8802

33. Deng, X., Zhao, L., Fang, T., Xiong, Y., Ogutu, C., Yang, D., Vimolmangkang, S., Liu, Y., and Han, Y. (2018)

Investigation of benzylisoquinoline alkaloid biosynthetic pathway and its transcriptional regulation in lotus.

Hortic Res. 5, 29

34. Yang, M., Zhu, L., Li, L., Li, J., Xu, L., Feng, J., and Liu, Y. (2017) Digital gene expression analysis provides

insight into the transcript profile of the genes involved in aporphine alkaloid biosynthesis in lotus (Nelumbo

nucifera). Front Plant Sci. 8, 80

35. Chen, S., Zhang, H., Liu, Y., Fang, J., and Li, S. (2013) Determination of lotus leaf alkaloids by solid phase

extraction combined with high performance liquid chromatography with diode array and tandem mass

spectrometry. Anal Lett. 46, 2846-2859

36. Deng, X., Zhu, L., Fang, T., Vimolmangkang, S., Yang, D., Ogutu, C., Liu, Y., and Han, Y. (2016) Analysis of

isoquinoline alkaloid composition and wound-induced variation in Nelumbo using HPLC-MS/MS. J Agric Food

Chem. 64, 1130-1136

37. Zhou, M., Jiang, M., Ying, X., Cui, Q., Han, Y., Hou, Y., Gao, J., Bai, G., and Luo, G. (2013) Identification and

comparison of anti-inflammatory ingredients from different organs of lotus Nelumbo by UPLC/Q-TOF and PCA

coupled with a NF-kappaB reporter gene assay. PLoS One. 8, e81971

38. Morris, J. S., and Facchini, P. J. (2016) Isolation and characterization of reticuline N-methyltransferase involved

in biosynthesis of the aporphine alkaloid magnoflorine in opium poppy. J Biol Chem. 291, 23416-23427

39. Ikezawa, N., Iwasa, K., and Sato, F. (2008) Molecular cloning and characterization of CYP80G2, a cytochrome

P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from

cultured Coptis japonica cells. J Biol Chem. 283, 8810-8821

40. Lin, Z., Yang, R., Guan, Z., Chen, A., and Li, W. (2014) Ultra-performance LC separation and quadrupole time-

of-flight MS identification of major alkaloids in Plumula nelumbinis. Phytochem Anal. 25, 485-494

15

O-Methyltransferases from sacred lotus

41. Do, T. C., Nguyen, T. D., Tran, H., Stuppner, H., and Ganzera, M. (2013) Analysis of alkaloids in lotus

(Nelumbo nucifera Gaertn.) leaves by non-aqueous capillary electrophoresis using ultraviolet and mass

spectrometric detection. J Chromatogr A. 1302, 174-180

42. Morikawa, T., Kitagawa, N., Tanabe, G., Ninomiya, K., Okugawa, S., Motai, C., Kamei, I., Yoshikawa, M., Lee,

I. J., and Muraoka, O. (2016) Quantitative determination of alkaloids in lotus flower (flower buds of Nelumbo

nucifera) and their melanogenesis inhibitory activity. Molecules. 21

43. Hara, M., Yazaki, K., Tanaka, S., and Tabata, M. (1995) S-adenosil-_L-methionine:norcoclaurine 6-O-

methyltransferase from Thalictrum minus cell cultures. Phytochemistry. 38, 1131-1135

44. Morishige, T., Tsujita, T., Yamada, Y., and Sato, F. (2000) Molecular characterization of the S-adenosyl-L-

methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase involved in isoquinoline alkaloid

biosynthesis in Coptis japonica. J Biol Chem. 275, 23398-23405

45. He, S. M., Liang, Y. L., Cong, K., Chen, G., Zhao, X., Zhao, Q. M., Zhang, J. J., Wang, X., Dong, Y., Yang, J.

L., Zhang, G. H., Qian, Z. L., Fan, W., and Yang, S. C. (2018) Identification and characterization of genes

involved in benzylisoquinoline alkaloid biosynthesis in Coptis species. Front Plant Sci. 9, 731

46. Fujii, N., Inui, T., Iwasa, K., Morishige, T., and Sato, F. (2007) Knockdown of berberine bridge enzyme by

RNAi accumulates (S)-reticuline and activates a silent pathway in cultured California poppy cells. Transgenic

Res. 16, 363-375

47. Inui, T., Tamura, K., Fujii, N., Morishige, T., and Sato, F. (2007) Overexpression of Coptis japonica

norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in benzylisoquinoline alkaloid

biosynthesis in cultured Eschscholzia californica. Plant Cell Physiol. 48, 252-262

48. Frenzel, T., and Zenk, M. H. (1990) S-Adenosyl-L-methionine:3'-hydroxy-N-methyl-(S)-coclaurine-4'-O-

methyltransferase, a regio- and stereoselective enzyme of the (S)-reticuline pathway. Phytochemistry. 29, 3505-

3511

49. He, S. M., Song, W. L., Cong, K., Wang, X., Dong, Y., Cai, J., Zhang, J. J., Zhang, G. H., Yang, J. L., Yang, S.

C., and Fan, W. (2017) Identification of candidate genes involved in isoquinoline alkaloids biosynthesis in

Dactylicapnos scandens by transcriptome analysis. Sci Rep. 7, 9119

50. Lee, E. J., and Facchini, P. (2010) Norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1

protein family. Plant Cell. 22, 3489-3503

51. Li, J., Lee, E. J., Chang, L., and Facchini, P. J. (2016) Genes encoding norcoclaurine synthase occur as tandem

fusions in the Papaveraceae. Sci Rep. 6, 39256

52. Sheng, X., and Himo, F. (2019) Enzymatic Pictet-Spengler reaction: Computational study of the mechanism and

enantioselectivity of norcoclaurine synthase. J Am Chem Soc. 141, 11230-11238

53. Hong, H., Lee, Y. I., and Jin, D. (2010) Determination of R-(+)-higenamine enantiomer in Nelumbo nucifera by

high-performance liquid chromatography with a fluorescent chiral tagging reagent. Microchemical Journal. 96,

374-379

54. Kato, E., Iwata, R., and Kawabata, J. (2017) Synthesis and detailed examination of spectral properties of (S)- and

(R)-Higenamine 4'-O-β-d-Glucoside and HPLC analytical conditions to distinguish the diastereomers.

Molecules. 22

55. Sato, F., Tsujita, T., Katagiri, Y., Yoshida, S., and Yamada, Y. (1994) Purification and characterization of S-

adenosyl-L-methionine: norcoclaurine 6-O-methyltransferase from cultured Coptis japonica cells. Eur J

Biochem. 225, 125-131

56. Ka, S. M., Kuo, Y. C., Ho, P. J., Tsai, P. Y., Hsu, Y. J., Tsai, W. J., Lin, Y. L., Shen, C. C., and Chen, A. (2010)

(S)-armepavine from Chinese medicine improves experimental autoimmune crescentic glomerulonephritis.

Rheumatology (Oxford). 49, 1840-1851

57. Itoh, A., Saitoh, T., Tani, K., Uchigaki, M., Sugimoto, Y., Yamada, J., Nakajima, H., Ohshiro, H., Sun, S., and

Tanahashi, T. (2011) Bisbenzylisoquinoline alkaloids from Nelumbo nucifera. Chem Pharm Bull (Tokyo). 59,

947-951

58. Vimolmangkang, S., Deng, X., Owiti, A., Meelaph, T., Ogutu, C., and Han, Y. (2016) Evolutionary origin of the

NCSI gene subfamily encoding norcoclaurine synthase is associated with the biosynthesis of benzylisoquinoline

alkaloids in plants. Sci Rep. 6, 26323

59. Hagel, J. M., Morris, J. S., Lee, E. J., Desgagne-Penix, I., Bross, C. D., Chang, L., Chen, X., Farrow, S. C.,

Zhang, Y., Soh, J., Sensen, C. W., and Facchini, P. J. (2015) Transcriptome analysis of 20 taxonomically related

benzylisoquinoline alkaloid-producing plants. BMC Plant Biol. 15, 227

60. Morris, J. S., Dastmalchi, M., Li, J., Chang, L., Chen, X., Hagel, J. M., and Facchini, P. J. (2016) Plug-and-play

benzylisoquinoline alkaloid biosynthetic gene discovery in engineered yeast. Methods Enzymol. 575, 143-178

16

O-Methyltransferases from sacred lotus

61. Edgar, R. C. (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity.

BMC Bioinformatics. 5, 113

62. Kumar, S., Stecher, G., Li, M., Knyaz, C., and Tamura, K. (2018) MEGA X: Molecular evolutionary genetics

analysis across computing platforms. Mol Biol Evol. 35, 1547-1549

63. Jones, D. T., Taylor, W. R., and Thornton, J. M. (1992) The rapid generation of mutation data matrices from

protein sequences. Comput Appl Biosci. 8, 275-282

64. Sievers, F., and Higgins, D. G. (2018) Clustal Omega for making accurate alignments of many protein

sequences. Protein Sci. 27, 135-145

65. Zhang, Y. J., Hao, X. Y., Liang, Z. S., Ke, W. D., and Guo, H. B. (2013) Efficient isolation of high-quality RNA

from lotus Nelumbo nucifera ssp nucifera tissues. Genet Mol Res. 12, 223-229

66. Jeong, J. Y., Yim, H. S., Ryu, J. Y., Lee, H. S., Lee, J. H., Seen, D. S., and Kang, S. G. (2012) One-step

sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics

studies. Appl Environ Microbiol. 78, 5440-5443

67. Ruijter, J. M., Pfaffl, M. W., Zhao, S., Spiess, A. N., Boggy, G., Blom, J., Rutledge, R. G., Sisti, D., Lievens, A.,

De Preter, K., Derveaux, S., Hellemans, J., and Vandesompele, J. (2013) Evaluation of qPCR curve analysis

methods for reliable biomarker discovery: bias, resolution, precision, and implications. Methods. 59, 32-46

68. Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative

-

PCR and the 2 ^CtMethod. Methods. 25, 402-408

ΔΔ

69. Dong, C., Li, G., Li, Z., Zhu, H., Zhou, M., and Hu, Z. (2009) Molecular cloning and expression analysis of an

Mn-SOD gene from Nelumbo nucifera. Appl Biochem Biotechnol. 158, 605-614

FOOTNOTES

Abbreviations: 4'OMT, 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase; 6OMT, norcoclaurine 6-

O-methyltransferase; 7OMT, reticuline 7-O-methyltransferase; BIA, benzylisoquinoline alkaloid; CID,

collision-induced dissociation; CoOMT, columbamine O-methyltransferase; CYP, cytochrome P450

monooxygenase; ESI, electrospray ionization; IPTG, isopropyl β-D-thiogalactoside; N7OMT,

norreticuline 7-O-methyltransferase; NCS, norcoclaurine synthase; NMT, N-methyltransferase; OD,

optical density; OMT, O-methyltransferase; PR10, pathogenesis-related 10 protein; SOMT, scoulerine 9-

O-methyltransferase.

17

O-Methyltransferases from sacred lotus

Table 1: Kinetics parameters for recombinant NnOMT1 (6OMT) and NnOMT5 (6OMT and 7OMT)

determined at a fixed saturating concentration of SAM. Values represent the mean ± standard deviation of

three independent measurements.

18

O-Methyltransferases from sacred lotus

FIGURE LEGENDS

+

4-Hydroxyphenylacetaldehyde

Dopamine

5

6

4

1

4α

3

7

8α

8

*

2'

3'

α

1'

6'

4'

5'

Norcoclaurine

NnOMT1

6-Demethyl-4'-O-methyl

N-methylcoclaurine

N-Methylisococlaurine

Lotusine

NnOMT5

Norarmepavine

Coclaurine

Isolotusine

NnOMT5

4'-O-Methyl-N-methylcoclaurine N-Methylcoclaurine

Armepavine

NCS

6OMT

7OMT

4’OMT

NMT

4'-O-Methylarmepavine

Figure 1: Schematic representation of the proposed 1-benzylisoquinoline alkaloid biosynthetic pathway

in sacred lotus (Nelumbo nucifera). The enzymes functionally characterized in this study (NnOMT1 and

NnOMT5) are indicated, and detected alkaloids are shown in bold. Asterisk denotes the chiral center in

norcoclaurine. Pink arrow, norcoclaurine synthase (NCS); green arrows, 6-O-methyltransferase (6OMT);

red arrows, 7-O-methyltransferase (7OMT); blue arrow, 4'-O-methyltransferase (4'OMT); black arrows,

N-methyltransferase (NMT).

19

O-Methyltransferases from sacred lotus

Folded Leaf (FL)

Unfolded Leaf (UF)

(a)

Embryo (E)

Rhizome (Rh)

Root (Ro)

Pink

FL UL Rh Ro

White

FL UL Rh Ro E

(b)

Alkaloid

E

Norcoclaurine

Coclaurine

N-Methylcoclaurine

Norarmepavine

N-Methylisococlaurine

Armepavine

Anonaine

Roemerine

N-Nornuciferine

O-Nornuciferine

Nuciferine

Pronuciferine

Nelumboferine

Liensinine

Isoliensinine

Neferine

0

100

Relative abundance (%)

(c)

R₁

R₂

-CH₂-

CH₃

R₃

Anonaine

H

Roemerine

CH₃

H

N-Nornuciferine

O-Nornuciferine

N-Nornuciferine

Nuciferine

CH₃

H

CH₃

H

CH₃

Pronuciferine

R₁

H

R₂

R₃

R₄

Nelumboferine

Liensinine

H

CH₃CH₃

H

CH₃CH₃CH₃

CH₃CH₃

CH₃CH₃CH₃CH₃

Isoliensinine

Neferine

CH₃

H

Figure 2: (a) Diagram of sacred lotus. (b) Heatmap showing the organ-specific profile of BIAs in two

varieties of sacred lotus (Pink and White). Relative abundance corresponds to the mean value of three

independent replicates. Values were normalized to the sample with the highest level for each compound.

(c) Structures corresponding to detected aporphine, pro-aporphine and bisbenzylisoquinoline alkaloids

isolated from sacred lotus (for 1-benzylisoquinoline alkaloid structures refer to Figure 1). FL, folded leaf;

UL, unfolded leaf; Rh, rhizome; Ro, roots; E, embryos.

20

O-Methyltransferases from sacred lotus

NnOMT1

NnOMT4

53

100

NnOMT2

99

NnOMT3

Tf6OMT

Cc6OMT2

Cj6OMT

GfOMT2

99

100

96

98

86

95

Ps6OMT

PsSOMT3

PsN7OMT

100

99

Cj4'OMT

100

99

Ec4'OMT

GfOMT1

98

57

Ps4'OMT2

Cc6OMT1

PsSOMT2

99

81

64

NnOMT5

81

CjCoOMT

Ps7OMT

Ct7OMT

EcSOMT

99

70

Ec7OMT

TtOMT1

TtOMT2

GfOMT7

100

97

99

CjSOMT

CtSOMT

100

GfOMT6

PsSOMT1

90

0.20

Figure 3: Phylogenetic relationships among sacred lotus O-methyltransferase (NnOMT) candidates and

functionally characterized OMTs from BIA-accumulating plants in the Ranunculales. The evolutionary

history was inferred using the Maximum Likelihood and the JTT matrix-based model. The percentage of

replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is

shown next to the branches. The tree with the highest log likelihood is drawn to scale, with branch lengths

measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGAX. Cc,

Coptis chinensis; Cj, Coptis japonica; Ct, Coptis teeta; Ec, Eschscholzia californica; Gf, Glaucium

flavum; Nn, Nelumbo nucifera; Ps, Papaver somniferum; Tf, Thalictrum flavum; Tt, Thalictrum

tuberosum. 4'OMT: 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase; 6OMT: norcoclaurine 6-O-

methyltransferase; 7OMT: reticuline 7-O-methyltransferase; CoOMT: columbamine O-methyltransferase;

N7OMT: norreticuline 7-O-methyltransferase; OMT: O-methyltransferase; SOMT: scoulerine 9-O-

methyltransferase. Sacred lotus NnOMT1 (green box) and NnOMT5 (red box) are indicated.

21

O-Methyltransferases from sacred lotus

Tf6OMT

NnOMT1

NnOMT2

NnOMT3

NnOMT4

NnOMT5

MEMINKENLSSQAKLWNFIYGFADSLVLKSAVQLDLANIIHNHGSPMTLSELSLHLPSQP

60

59

56

ME-IQKEGQAAAAKIWKFVYGFADCLVLRCAIDLGIADIIHKQGEPLTLSELGAQIPVQP

ME-IPKEVQADEVEIWKFGYDFADTLVLRCAIEFGIADIIHKQGEPLTLFELGAQIPVQP

ME-IQKEVQAADVEIRKFGYGFADILVIRCAIQLGIADIIHKQGEPLTLSELEAQIPVKP

ME-NQKEVQAAEAKIWNFVYGFADTLVLRCAIELGIADIIHKQGEPLTLSELGAQIPLKS

ME----EDMKAQAQVWKHIYGFVESFTLKCAIELGIADILYEHGQPMTLSELASSIPLPS

Tf6OMT

NnOMT1

NnOMT2

NnOMT3

NnOMT4

NnOMT5

VNQDALYRVLRYLVHMKLFTKS-SIDGELRYGLAPPAKFLVKGWD-KCMLGAILTITDKD

VNTDHLHRLMRYLVHMKIFTKE-TLDGEARYGLAPPAKFIVKGWD-KSIVSIILVVTDKD

VNTDHLHRLMRYMVHMKIFTKE-TLGGEEXYGLSPHGKFLVKGWD-KSMASAILAITDED

VNTDHLHRLMRYMVHMKIFTKE-TPDGEERYGLAPLGKFLVNGWD-RNMVSAILAVTDKD

VNTDHLHRLMRYLVHMKLFTKE-TLDGEARYGLAPPAKLLVKWWEDKGLASIIFGITDKD

VSQDGLYRVLRYLVHMKLFDLQVDSDGLKKYRLTPASKLLVKNQE-KNLASFVLL--QLY

118

117

118

113

*

**

Tf6OMT

NnOMT1

NnOMT2

NnOMT3

NnOMT4

NnOMT5

FMAPWHYLKEGILNDGSTSTAFEKALG-TNIWDYMAEHPEKNQLFNEGMANDTR-LIMSA

FMAPWHCLKDSLCGEG---TAFEKALG-RSIWTYMADHPEKNKLFNEGMACDTK-LLISA

FFAPWHCLKDVLAGEG---TAFEKALG-KSIWAYVADHPEKNKLFNEVMACDTS-FITSV

FMVPWYRLKDSLVGEG---TAFEKALG-KTICECMADHPEKKKPFNEAMACDTTRLLTSA

FIAPWHHLKDSLAGDGE-ETTFEKVLG-KSISTYMADHLEKSMLFNESMVHDTR-LFTSV

EIDTWNHLSAAVEGTV---TPWEKCHGGVDYIEYFKKDSVANQLLSDAMTSHTS-MVTDA

176

172

173

175

169

*

Tf6OMT

NnOMT1

NnOMT2

NnOMT3

NnOMT4

NnOMT5

LVKECSS--MFDGITTIVDVGGGTGTAVRNIAKAFPHIKCTVYDLPHVIADSPGYTEINS

LVQDCKD--LFQGIMSLVDVGGGTGTAMRAIAKAFPHLKCTIYDLPHVIADSPDYPEVDR

LIQDCKD--VFQGIKSVVDVGGGTGTAMRDIAKAFPHLKCTIYDLPHVIADSPDYPEVDR

LIQDCKD--LFQGIMSLVDVGGGTGTAMRDIAKTFPHLKCTIYDLPHVIADSPDYPEVDR

LIQDFKD--VFQGIKSLVDVGGGSGTDMGAIAKAFPHLKCTIYGLPHVIADSPDYPEVDR

LVKGCKKAHILDGVGSLIDVGGCTGVAARAIAKWFPSIKCAVFDLPHVVANAPECPEVTR

234

230

231

233

229

I

II

*

** **

Tf6OMT

NnOMT1

NnOMT2

NnOMT3

NnOMT4

NnOMT5

IQGDMFKYIPNADAIMMKCILHDWDDKECIEILKRCKDAVPRDGGKVIIIDIILDVKSEH

IAGDMFKHIPSADAILLKCILHDWDDGECIEILKRCKESVPREGGKVIIVDIVVDLESKH

IAGDMFKHIPSADAILLKWILHDWDDGECIEILKRCKESVPREGGKVIIVDIVLDPESKD

IAGNMFKHIPSADGILLKCILHDLGDRQCIEILQRCKESVPREGGKVIIVDIVLDPESTD

ISGDMFKHIPSADAILLKCILHYWGDGQCIEILKRCKESVPREGGIVIIADAVVDLESKH

IGGDMFVSIPKTDVVFMKSVLHDWGDEDCVKILKKCKEAISEKGGKAVIVDIVMDVESSP

294

290

291

293

289

IV

III

*

Tf6OMT

NnOMT1

NnOMT2

NnOMT3

NnOMT4

NnOMT5

PY-TKMRLTLDLDMMLNTGGKERTEEEWKKLIHDAGYKGYKITHISAVQ-SVIEAYPY

PL-TKTRLSLDLDMMVTTGGKERTEAEWKKLLNAAGFPVFKITHISAVQ-SVIVAYPY

PL-TKARLRLDLDMMVYTGGKERSEAEWKKLLNAAGFPGYKILHVAAVQ-SVIMAYPY

PL-TKARLRLDLDMMVYTGGKERSEAEWKKLLNAAGFPRYKILHIAAVQ-SVIEAYPY

PYLTKTLLSTDLDMMLNTGGKERTEAEWKKLFNAAGFPAYKITHVADVEYSVIEAYPY

NEFTGARLGMEMDMLVAVGGKERSEKEWHKLFKEAGYSGYKITPIVAIE-SIIEVFP-

350

346

347

351

345

Figure 4: Multiple sequence alignment among Nelumbo nucifera O-methyltransferase (NnOMT)

candidates and Thalictrum flavum 6OMT (Tf6OMT). Residues shared by Tf6OMT and sacred lotus

OMTs are shaded in grey, and fully conserved residues are shaded in black. Asterisks indicate catalytic

(H²⁵⁶, D²⁵⁷and E³¹⁵) and other key residues implicated in BIA (G¹⁶⁵, D¹⁶⁹, C²⁵³and D³⁰⁶) and SAM (T¹⁷⁰

G¹⁹⁵, D²¹⁸, D²³⁸and K²⁵²) binding, as reported for Tf6OMT. Conserved motives I-IV are underlined.

,

22

O-Methyltransferases from sacred lotus

Figure 5: Substrate range for recombinant NnOMT1 and NnOMT5. Values represent the mean ±

standard deviation of three independent replicates. The structure of the corresponding substrates is shown;

nd, not detected.

23

O-Methyltransferases from sacred lotus

Figure 6: Organ-specific correlational analysis of 1-benzylisoquinoline alkaloids metabolism in two

varieties (Pink and White) of sacred lotus. (a) Content of selected 1-benzylisoquinoline alkaloids. (b)

6OMT, 7OMT and 4'OMT specific activity of plant crude extracts when incubated with (R,S)-

norcoclaurine, (S)-N-methylcoclaurine and (R)-armepavine, respectively. (c) Relative abundance of

NnOMT1-NnOMT5 transcripts. Values represent the mean ± standard deviation of three independent

measurements. FL, folded leaf; UL, unfolded leaf; Rh, rhizome; Ro, roots; E, embryos.

24

Isolation and characterization of two O-methyltransferases involved in

benzylisoquinoline alkaloids biosynthesis in sacred lotus ( Nelumbo nucifera)

Ivette M. Menéndez-Perdomo and Peter J. Facchini

J. Biol. Chem. published online December 30, 2019

Access the most updated version of this article at doi: 10.1074/jbc.RA119.011547

Alerts:

• When this article is cited

• When a correction for this article is posted

Click here to choose from all of JBC's e-mail alerts

Article Doi

Isolation and characterization of two O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in sacred lotus (Nelumbo nucifera)

DOI: 10.1074/jbc.RA119.011547

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.RA119.011547

Full text of DOI:10.1074/jbc.RA119.011547

Products guided by the article

R&D Labs maybe for 3423-07-2

Relevant to this article

Hot Product