Characterization of a Flavonoid 3’/5’/7-O-Methyltransferase from Citrus reticulata and Evaluation of the in Vitro Cytotoxicity of Its Methylated Products

Article abstract of DOI:10.3390/molecules25040858

O-methylation of flavonoids is an important modification reaction that occurs in plants. O-methylation contributes to the structural diversity of flavonoids, which have several biological and pharmacological functions. In this study, an O-methyltransferase gene (CrOMT2) was isolated from the fruit peel of Citrus reticulata, which encoding a multifunctional O-methyltransferase and could effectively catalyze the methylation of 3’-, 5’-, and 7-OH of flavonoids with vicinal hydroxyl substitutions. Substrate preference assays indicated that this recombinant enzyme favored polymethoxylated flavones (PMF)-type substrates in vitro, thereby providing biochemical evidence for the potential role of the enzyme in plants. Additionally, the cytotoxicity of the methylated products from the enzymatic catalytic reaction was evaluated in vitro using human gastric cell lines SGC-7901 and BGC-823. The results showed that the in vitro cytotoxicity of the flavonoids with the unsaturated C2-C3 bond was increased after being methylated at position 3’. These combined results provide biochemical insight regarding CrOMT2 in vitro and indicate the in vitro cytotoxicity of the products methylated by its catalytic reaction.

Full text of DOI:10.3390/molecules25040858

molecules
Article
Characterization of a Flavonoid
3’/5’/7-O-Methyltransferase from Citrus reticulata and
Evaluation of the In Vitro Cytotoxicity of Its
Methylated Products
Xiaojuan Liu 1,2,3,†, Yue Wang 1,2,3,† , Yezhi Chen 1,2,3, Shuting Xu 1,2,3, Qin Gong 1,2,3
,
Chenning Zhao 1,2,3, Jinping Cao 1,2,3 and Chongde Sun 1,2,3,
*
1
College of Agriculture & Biotechnology, Zhejiang University, Zijingang Campus, Hangzhou 310058, China;
11416048@zju.edu.cn (X.L.); fruit@zju.edu.cn (Y.W.); yzchen000@163.com (Y.C.); shutingxu@zju.edu.cn (S.X.);
21816138@zju.edu.cn (Q.G.); 11616041@zju.edu.cn (C.Z.); 0017165@zju.edu.cn (J.C.)
Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Zhejiang University,
Zijingang Campus, Hangzhou 310058, China
The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality
Improvement, Zhejiang University, Zijingang Campus, Hangzhou 310058, China
Correspondence: adesun2006@zju.edu.cn; Tel.: +86-0571-8898-2229
2
3
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 January 2020; Accepted: 12 February 2020; Published: 15 February 2020
Abstract: O-methylation of flavonoids is an important modification reaction that occurs in plants.
O-methylation contributes to the structural diversity of flavonoids, which have several biological
and pharmacological functions. In this study, an O-methyltransferase gene (CrOMT2) was isolated
from the fruit peel of Citrus reticulata, which encoding a multifunctional O-methyltransferase
and could effectively catalyze the methylation of 3’-, 5’-, and 7-OH of flavonoids with vicinal
hydroxyl substitutions. Substrate preference assays indicated that this recombinant enzyme favored
polymethoxylated flavones (PMF)-type substrates in vitro, thereby providing biochemical evidence
for the potential role of the enzyme in plants. Additionally, the cytotoxicity of the methylated products
from the enzymatic catalytic reaction was evaluated in vitro using human gastric cell lines SGC-7901
and BGC-823. The results showed that the in vitro cytotoxicity of the flavonoids with the unsaturated
C2-C3 bond was increased after being methylated at position 3’. These combined results provide
biochemical insight regarding CrOMT2 in vitro and indicate the in vitro cytotoxicity of the products
methylated by its catalytic reaction.
Keywords: citrus; flavonoids; O-methyltransferase; bioactivity; cytotoxicity
1. Introduction
Flavonoids are secondary metabolites widespread in plants, and are considered to be important
small molecules participating in various metabolic pathways. Flavonoids are compounds with
C6-C3-C6 skeletons. Based on the saturation of C2-C3 bond and the presence or absence of the
hydroxy group at C3, flavonoids can be further divided into several types, including flavones,
flavonols, dihydroflavones, and dihydroflavonols [
play important roles in plant biology [ ]. For example, flavonoids can form specific pigments to
serve as pollinator attractants, help in seed germination, and act as phytoalexins, signal molecules,
allopathic compounds, and antimicrobials in plants [ ]. Flavonoids can protect plants against various
types of damage including that induced by ultraviolet (UV) irradiation, freezing, drought, and nitrogen
1]. It has been well documented that flavonoids
1–6
4
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deficiency [
1
]. Flavonoids are also thought to be important dietary ingredients and have also been
]. Flavonoids produced by plants
], antioxidant [10], and anti-cancer
intensively studied for their health benefits to humans and animals [7,8
have diverse biological activities, including anti-inflammatory [
activities [11].
9
The functional diversity of flavonoids is based on their structural diversity. It has been estimated
that there are more than 10,000 plant-derived flavonoids with diverse structures [12]. The structural
diversity of flavonoids is achieved by a number of modifications, including hydroxylation, methylation,
glycosylation, and malonylation [12]. In plants, flavonoid O-methylation on the hydroxy groups
enhances the chemical stability and transportability of flavonoids, and is thus one of the most important
modifications [13]. It has also been suggested that methylated flavonoids possess higher biological
activity than unmethylated compounds [13]. O-methyltransferases (OMTs) can transfer the methyl
group to specific hydroxy moieties of flavonoids by utilizing S-Adenosyl-l-methionine (SAM) as a
methyl donor; thereby, the regioselective O-methylation of flavonoids can be catalyzed. Plant OMTs
are a large family; based on their bivalent ion dependency and molecular mass, they are mainly
divided into two categories: caffeic acid OMTs (COMTs) and caffeoyl-CoA OMTs (CCoAOMTs) [14].
Both categories of OMTs have been demonstrated to be involved in the methylation of flavonoids [13].
3’-OMTs responsible for 3’-O-methylation of flavonoids are the most common flavonoid
O-methyltransferases (FOMTs) [12]. The 3’-OMTs from different species exhibit distinct catalytic
properties. For example, OsOMT1 from Oryza sativa [15] and SlMOMT4 from Solanum lycopersicum [16
]
display stringent specificity toward 3’-OH of flavonoids, while other plant 3’-OMTs show broader
methylation sites not restricted to the 3’-OH of the B-ring. The following examples are all methylated
at both the 3’ and 5’ hydroxy groups of flavonoids: ZmOMT2 from Zea mays and HvOMT1 from
Hordeum vulgare [17], ShMOMT1 from Solanum habrochaites [18], CrOMT2 from Catharanthus roseus [19],
and SlOMT3 from Solanum lycopersicum [20]. In particular, TaOMT1 [21] and TaOMT2 [22] from Triticum
aestivum are both active with all of the hydroxy groups on the B-ring, and can sequentially methylate
the 3’, 4’, 5’-OH of tricetin. Published evidence shows that 3’-OMTs from different species display
diverse regioselectivity and specificity, indicating that unique 3’-OMTs may exist in species that are rich
in various methylated flavonoids, such as citrus. The peel of citrus fruit is also known to accumulate
abundant methylated flavonoids, especially polymethoxylated flavones (PMFs) [23].
In silico analysis of the OMT family has been comprehensively performed in Citrus sinensis,
and candidate genes for flavonoid O-methylation have been predicted based on the phylogenetic
tree [24]. CdFOMT5 from Citrus depressa has been demonstrated to be a multifunctional OMT and
can methylate the 3-, 5-, 6-, and 7-OH of flavones by in vitro experiment [25]. Cell-free extracts of
citrus tissues exhibit 3’-O-methylation against several flavonoids containing 3’-OH and 4’-OH, such as
quercetin, luteolin, and quercetagetin [26,27]. However, the 3’-OMT responsible for this enzymatic
reaction has not been experimentally identified in citrus.
In this study, we isolated and characterized a 3’-OMT from Citrus reticulata, termed C. reticulata
O-methyltransferase gene (CrOMT2). The recombinant CrOMT2 was found to be a 3’/5’/7-OMT
by in vitro experiments. To evaluate the potential role of this enzyme in citrus, an analysis was
performed for the substrate preference of the recombinant enzyme in combination with the phylogeny.
Additionally, flavonoid substrates and their corresponding methylated products were evaluated for
their in vitro cytotoxicity activity on two human gastric cell lines, SGC-7901 and BGC-823, respectively.
2. Results
2.1. CrOMT2 Isolation and Bioinformatic Analysis
It has been proposed that the peel of “Ougan” fruit is rich in PMFs, and 3’-O-methylation was
common in PMFs [23]. Here, the coding gene of a putative 3’-OMT was isolated from the cDNA
library of the Ougan fruit peel. Specifically, the amino acid sequence of a flavonoid 3’-OMT (AtOMT1)
from Arabidopsis thaliana [28] was used as a query in the local blast against the protein database of

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Citrus clementina to screen for the most promising 3’-OMT in citrus. As shown in Supplementary
Table S1, Ciclev10020814m was the most identical OMT, with 73.35% identity to AtOMT1. Based on
the Citrus clementina genome, gene-specific primers were designed to amplify the coding region of
Ciclev10020814m. A 1101 bp DNA sequence was then yielded with 98.79% identity to its template DNA
sequence (1074 bp) (Supplementary Figure S1). The deduced amino acid sequence of the amplified gene
was identical to that of a putative caffeic acid O-methyltransferase (GenBank: ADK97702.1) isolated
from Citrus aurantium, which is a sequence directly submitted to NCBI, without experimental study.
Only four nucleotides were not identical between the coding sequences of the two genes (Supplementary
Figure S1). The cloned OMT in our research was termed CrOMT2. It encoded 366 amino acid residues,
which can in theory generate a protein with the molecular mass of 39.99 kDa (ExPASy Compute,
https://web.expasy.org/compute_pi/). It also included two conserved domains, Methyltranferase_2
(pfam00891) and dimerization (pfam08100). This in silico analysis together indicated that CrOMT2
belongs to the COMT family and is a candidate flavonoid 3’-OMT.
2.2. Substrate Specificity and Kinetic Parameters of CrOMT2
The CrOMT2 gene was expressed by infusing with two histidine tags in Escherichia coli, and the
theoretically calculated molecular weight for the recombinant enzyme was 58.75 kDa. SDS-PAGE
analysis showed that the recombinant enzyme was successfully purified, with its molecular weight
consistent with the theoretical one (Supplementary Figure S2). The pH and temperature dependency
assays showed that the purified CrOMT2 had high activity at a broad pH range from 8 to 9 (in Tris-HCl
buffer), indicating that it preferred◦an alkaline environment to a neutral or acidic one. The optimum
◦
temperature for CrOMT2 was 55 C and 87.9% activity was lost at 25 C (Supplementary Figure
S2). The following tests were carried out at pH 8.0 and 37 ◦C to balance the substrate stability and
enzyme activity.
To screen the substrate specificity of the purified recombinant CrOMT2, caffeic acid and four
representative classes of flavonoids were tested using SAM as the methyl donor (Figure 1). The results of
relative activity assays showed that the purified enzyme could methylate a wide array of catechol-type
flavonoids except for 7,8-dihydroxyflavone and eriodictyol glycosides (eriocitrin and neoeriocitrin)
(Figure 1). Flavonoids without vicinal hydroxy groups were not converted (e.g., apigenin, naringenin,
hesperetin) or were only slightly converted (e.g., kaempferol). It could also be concluded that flavones,
flavonols, and dihydroflavones were preferred categories of the recombinant CrOMT2 compared with
dihydroflavonols (Figure 1). The purified CrOMT2 could also methylate phenylpropanoid caffeic acid
with lower relative activity.
Substrates with relative activity over 7% were used to further investigate the kinetic properties of the
recombinant CrOMT2 (Figure 2). The results indicated that the enzyme displayed the highest catalytic
efficiencies (Kcat/Km value: 2707.9 and 2450.8 M−1S−1) against luteolin and tricetin (two flavones), and
had a high affinity for both substrates (Km values: 7.6 µM and 8.7 µM). Flavone baicalein was also
methylated by the enzyme with a similar affinity constant to luteolin and tricetin, but a Kcat value
was ca. 5-fold lower than that of these two flavones. The enzyme had a much higher affinity for
quercetin than for myricetin. However, due to the much higher turnover rates of the enzyme with
myricetin, the catalytic efficiencies for these two flavonols were similar. Dihydroflavone eriodictyol
was methylated by the protein with a relatively high catalytic efficiency (Kcat/Km value 1650.8 M−1S−1
and an affinity constant of 4.6 M. The enzyme could also methylate the simple phenolic acid caffeic
acid, as a substrate, albeit with a lower affinity (Figure 2).
)
µ

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Figure 1. Relative activity of Citrus reticulata O-methyltransferase 2 (CrOMT2) against four
representative flavonoids and caffeic acid in vitro. The recombinant CrOMT2 purified from Escherichia
coli was incubated with a number of phenolic substrates, and the relative activity against different
substrates was estimated. The methylation sites identified for substrates are indicated in red in their
structural formula. ND represents “not detectable”. R- in eriocitrin represents rutinoside and N- in
neoeriocitrin represents neohesperidoside. The data are shown as means ± SE (n = 3).

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Figure 2. Kinetic analysis of Citrus reticulata O-methyltransferase 2 (CrOMT2) using seven substrates.
Seven substrates with relative higher activity were selected for kinetic analysis. Apparent Km and Kcat
were estimated using non-linear fit to Michaelis–Menten. Change in RLU (relative light units) indicates
background-subtracted RLU. The data are shown as means ± SE (n = 3).
2.3. Identification of Methylated Products
The methylated products generated by the recombinant CrOMT2 were identified by comparing
the retention times and MS/MS data with the corresponding commercial standards (Figure 3, Figure 4,
and Supplementary Figure S3). The enzyme could convert luteolin and tricetin into their corresponding
3’-methyl ether (chrysoeriol) and 3’,5’-dimethyl ether (tricin), respectively (Figure 3A,B). Baicalein,
(i.e., 6,7,8-dihydroxyflavone) was methylated at 7-OH, yielding baicalein 7-methyl ether (Figure 3C).
Quercetin and myricetin exhibited the same methylation pattern as luteolin and tricetin, and their
corresponding 3’-methyl ether (isorhamnetin) and 3’,5’-dimethyl ether (syringetin) were produced
by the enzyme, respectively (Figure 3D,E). When quercetin 3-methyl ether served as the substrate,
its 3’-O-methylation product (quercetin 3,3’-dimethyl ether) was obtained (Figure 3F). The enzyme
also displayed a negligible activity to kaempferol, yielding its 3-methylated product (isokaempferide)

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(Figure 3G). With eriodictyol as a substrate, its 3’-methylation product (homoeriodictyol) was identified
(Figure 3H). The recombinant CrOMT2 could also separately methylate the three dihydroflavonols
(dihydroquercetin, dihydromyricetin, and dihydrokaempferol) into a single product based on the
HPLC chromatogram (Supplementary Figure S4); however, the structures of the methylated products
remained to be verified. As a simple phenolic acid, caffeic acid was also converted into ferulic acid
(caffeic acid 3-methyl ether), as determined by comparing the retention time of the methylated product
with the commercial standard (Supplementary Figure S4).
Figure 3. HPLC analysis of products catalyzed by Citrus reticulata O-methyltransferase 2 (CrOMT2).
The methylated products produced by the recombinant CrOMT2 were identified by comparison of the
retention times and MS/MS data with corresponding standards (see Supplementary Figure S3 for the
MS/MS data of standards). HPLC chromatograms of enzyme reactions with luteolin (
baicalein ( ), quercetin ( ), myricetin ( ), quercetin 3-methyl ether ( ), kaempferol ( ), and eriodictyol
) are indicated. Substrates catalyzed by CrOMT2 indicated in red; substrates incubated with empty
A), tricetin (B),
C
D
E
F
G
(
H
vector (pET32a) indicated in blue, commercial standards of the methylated products indicated in black.
Lut: luteolin; Chr: chrysoeriol; Tri: tricetin; Bai: baicalein; Bai 7-OMe: baicalein 7-methyl ether; Que:
quercetin; Isor: isorhamnetin; Myr: myricetin; Syr: syringetin; Que 3-OMe: quercetin 3-methyl ether;
Que 3,3’-diOMe: quercetin 3,3’-dimethyl ether; Kae: kaempferol; Isok: isokaempferide; Eri: eriodictyol;
Hom: homoeriodictyol.

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A luteolin methylated product
E myricetin methylated product
B tricetin methylated product
C baicalein methylated product
D quercetin methylated product
F quercetin 3-methyl ether methylated product
G kaempferol methylated product
H eriodictyol methylated product
Figure 4. MS/MS spectra of the methylated products catalyzed by Citrus reticulata O-methyltransferase
2 (CrOMT2). Substrates utilized were luteolin ( ), tricetin ( ), baicalein ( ), quercetin ( ), myricetin ( ),
quercetin 3-methyl ether ( ), kaempferol ( ), and eriodictyol ( ). ( ) were operated in negative
A
B
C
D
E
F
G
H
A, F, H
ion mode and the rest were in positive ion mode. The y axis represents the percent intensity.
2.4. Phylogenetic Relationship of CrOMT2 to other COMTs
The phylogenetic tree was constructed using the deduced amino acid sequences of CrOMT2 and
other COMTs. CrOMT2 was clustered with the clade in which most members catalyze the hydroxy
groups on the B-ring (3’/4’/5’) or C-ring of flavonoids (Figure 5). The other clade consisted of OMTs that
methylate the hydroxy moieties on the A-ring (6/7/8) or B-ring (4’) except for CrOMT2 from C. roseus
(Figure 5). In addition to the expected meta-methylation pattern (3’/5’) on the B-ring, the recombinant
CrOMT2 also displayed unusual 7-OH methylation on baicalein. To the best of our knowledge, this is
the first report of a 3’/5’ flavonoid O-methyltransferase with the capacity of 7-OH methylation. Another
unique OMT was CdFOMT5 from C. depressa, which also exhibited 7-OH methylation [25] (Figure 5).
The residues associated with the catalytic function of CrOMT2 were also inferred according to previous

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studies [29] (Supplementary Figure S5). It could be indicated that the residues and motifs (indicated
with red asterisks) responsible for SAM binding were conserved across the aligned COMTs.
Figure 5. Phylogenetic tree of Citrus reticulata O-methyltransferase 2 (CrOMT2) and selected plant caffeic
acid O-methyltransferases (COMTs) at the protein level. The preferred substrates and methylation
positions are indicated for each protein. Percent bootstrap values (n = 1000) greater than 70 are shown
in the middle of each branch. The evolutionary history was analyzed using the neighbor-joining
method with SaDMPM from Streptomyces alboniger acting as the outgroup. Two COMTs from citrus are
indicated with arrows. The small clade including CrOMT2 from C. reticulata is boxed in black. To avoid
ambiguity, CrOMT2 from Catharanthus roseus was marked with a triangle. See Supplementary Table S3
for UniProt entries and species of the OMTs.
2.5. In Vitro Cytotoxicity of Flavonoids on Human Gastric Cancer Cells
Given the diverse methylation sites of the recombinant CrOMT2 against different flavonoids,
six substrates and their corresponding methylation products were used to assess their cytotoxicity
against human gastric cancer cells in vitro. Two human gastric cancer cell lines, i.e., SGC-7901 and
BGC-823, were used for the cytotoxicity assay. The flavonoid concentration gradients were set in a
range from 3.13
µM to 50 µM (Supplementary Figure S6). Results showed that two products catalyzed
by CrOMT2 produced stronger inhibitory effects on the human gastric cancer cells compared to

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luteolin and quercetin substrates in vitro (Table 1). Specifically, the inhibitory effects of chrysoeriol
on SGC-7901 and BGC-823 cells were 1.15- and 1.68-fold higher than those of luteolin, respectively,
while the inhibitory effects of isorhamnetin on SGC-7901 and BGC-823 were 3.54- and 6.68-fold higher
than those of quercetin, respectively. As for the substrates with improved activities after methylation,
their structures were characterized by the double bond at the C-ring and the methylation site of the
B-ring toward the 3’ position compared to the flavonoids with no increase in activities. Paclitaxel was
used as a positive control, and its IC50 values for SCG-7901 and BGC-823 cells were 0.06 and 0.07 µM,
respectively (data not shown). DMF was used as a solvent control and showed no inhibitory effect on
the two cell lines at the tested concentration.
Table 1. Inhibitory effects of flavonoids catalyzed by Citrus reticulata O-methyltransferase 2 (CrOMT2)
on the human gastric cancer cells in vitro.
Substrate
IC50 (µM)
Product
IC50 (µM)
SCG-7910
BGC-823
tricetin
luteolin
baicalein
quercetin
myricetin
eriodictyol
78.8
15.1
37.5
37.7
\
tricin
chrysoeriol
baicalein 7-methyl ether
isorhamnetin
\
13.3
40.2
10.7
\
syringetin
homoeriodictyol
66.4
166
tricetin
luteolin
baicalein
quercetin
myricetin
eriodictyol
\
tricin
chrysoeriol
baicalein 7-methyl ether
isorhamnetin
\
24.2
\
41.2
86.9
322
\
48.4
\
syringetin
homoeriodictyol
188
\
3. Discussion
In this study, the coding gene of a putative flavonoid 3’-O-methyltransferase, named CrOMT2,
was isolated from citrus and successfully expressed in vitro in E. coli. The purified CrOMT2 could
methylate a number of flavonoids with vicinal hydroxy groups in vitro, with the methylation sites
occurring at 3’-OH, 5’-OH, or 7-OH. The inhibitory effect of the specific methylation on human gastric
cancer cells was examined. The in vitro cytotoxicity of the two substrates (i.e., luteolin and quercetin)
showed a significant increase after being methylated at 3’-OH.
The recombinant CrOMT2 could methylate numerous flavonoids with vicinal B-ring hydroxy
moieties in vitro; this finding was consistent with the catalytic activity of cell-free extract from citrus
tissues [27]. This substrate specificity was also similar to AtOMT1 from A. thaliana [28], which clustered
closely to CrOMT2 on the phylogenetic tree (Figure 5). Two flavones, i.e., luteolin and tricetin, were the
most preferred substrates for CrOMT2 among other tested substrates (Figures 1 and 2). This strong
preference to the two flavones also occurred in other COMTs: TaOMT1 [21] and TaOMT2 [22] from
T. aestivum, ZmOMT2 from Z. mays, and HvOMT1 from H. vulgare [17]. In contrast to CrOMT2,
which catalyzed luteolin as effectively as tricetin (Figure 2), all of the above four OMTs exhibited a
preference to tricetin over luteolin as a substrate. As these four OMTs effectively converted tricetin
into its 3’,5’-dimethyl ether (tricin), they were considered to be involved in the synthesis of tricin,
a characteristic flavone of cereal grain plants [17,21,22]. As a flavone with three hydroxy groups at
the A-ring, baicalein was also methylated by the recombinant CrOMT2, with a lower turnover rate
compared to the luteolin and tricetin (Figure 2). Thus, CrOMT2 might be involved in the synthesis
of PMFs in citrus due to its preference for flavones, which were exactly the PMF-type substrates [23].
In contrast to many other FOMTs capable of 3’-methylation with lower [17,20,30] or no activities [28,31]
on eriodictyol (a dihydroflavone), CrOMT2 showed high affinity and catalytic efficiency with this
substrate and produced its 3’-methyl ether derivative, homoeriodictyol (Figures 2 and 3H). Given the
ubiquitous eriodictyol glycosides in citrus [23], CrOMT2 was determined a promising enzyme involved

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in the homoeriodictyol synthesis in vivo. As two widespread flavonols in plants [32], quercetin and
myricetin, were also methylated by the recombinant CrOMT2, their products were confirmed as
3’-methyl quercetin (isorhamnetin) and 3’,5’-dimethyl myricetin (syringetin), respectively (Figure 3D,E).
When myricetin serves as a substrate, similarly to CrOMT2, flavonoid 3’-OMTs often sequentially
methylate both 3’-OH and 5’-OH due to the similar chemical properties of the hydroxy groups in
the two positions [18–20]. However, there were several flavonoid 3’-OMTs that only transferred one
methyl group into the 3’-OH of myricetin, e.g., OsOMT1 from O. sativa [15] and SlMOMT4 from
S. lycopersicum [16]. Except for the activity toward flavonoids, the purified CrOMT2 also showed
relatively lower activity toward caffeic acid (Figure 1), which is an important intermediate compound
involved in lignin biosynthesis [33]. This was consistent with many characterized FOMTs belonging to
COMTs [15,19,21,28,30,34,35]. CrOMT2 was closely clustered within a small clade in which both typical
caffeic acid 3-OMTs and flavonoid 3’-OMTs were located (Figure 5). AtOMT1, sister to CrOMT2 in this
clade, has been identified as a flavonol 3’-OMT. This identification was confirmed by the substrate
specificity assay in vitro [28] and was further verified by the analysis of flavonoid profile in the mutant
omt1 [36]. Moreover, it was reported that AtOMT1 was involved in the biosynthesis of lignin and
soluble sinapate esters [37]. There were two typical caffeic acid 3-OMTs in this clade (Figure 5). One was
MsCOMT1 from alfalfa (Medicago sativa), which has a strong impact on lignin biosynthesis that has
been verified by downregulating its coding gene in transgenic alfalfa [38]. The other was CrCOMT1
from C. roseus. It also favored phenylpropanoids compared to flavonoids in vitro, and this substrate
preference indicated the potential role of the enzyme in the lignin pathway [39]. The two lignin-related
OMTs together with the bifunctional AtOMT1 in this clade indicated that CrOMT2 could participate in
the lignin pathway in addition to flavonoid metabolism.
7-O-methylation was detected when using baicalein as a substrate for CrOMT2 (Figure 3C).
7-FOMTs usually have stringent specificity and cluster closely in the phylogenetic tree (Figure 5). Several
plant 7-FOMTs have been characterized, e.g., ObFOMT1 and ObFOMT2 from Ocimum basilicum [40],
MpOMT1A and MpOMT1B from Mentha x piperita [35], Hv7OMT from H. vulgare [41], and OsNOMT
from O. sativa [34]. Of the B-ring (3’/4’/5’) or C-ring FOMTs, only two citrus OMTs exhibited activity on
7-OH. One is CrOMT2, reported in our study, and the other one is CdFOMT5 from C. depressa, which
can methylate 3-, 5-, 6-, and 7-hydroxy groups of flavonoids [25] (Figure 5). Cell-free extracts from
citrus also showed 7-O-methylation on several flavonoids, including baicalein [26,27]. The unique
7-O-methylation catalytic activity of citrus OMTs indicated the significance of 7-O-methylation in
synthesizing PMFs in citrus. It has been reported that the 7-O-glycosides of flavonoids (e.g., narirutin,
naringin, hesperidin, and neohesperidin) are the most common flavonoid derivatives in citrus [23],
whereas PMFs in citrus usually accumulate in the form of free aglycones instead of glycosides [23].
Since the 7-O-glycosylation reaction can be completely blocked by the 7-O-methylation, the flux toward
PMFs aglycones was likely determined by the 7-O-methylation. Similarly, in plants that did not
produce 3-O-glycosylated flavonoids, the 3-OH methylation also appeared to be the first procedure [42].
Further experiments are needed to verify the hypothesis on the methylation order of flavonoids in
citrus. Given the high affinity of CrOMT2 towards baicalein (a PMF-type substrate), CrOMT2 remains
a promising 7-OMT in citrus.
4. Materials and Methods
4.1. Materials
Ougan (Citrus reticulata cv. Suavissima) fruits were randomly collected for the subsequent
experiments at 160 days after flowering. The peels of the fruits were ground in liquid nitrogen to
isolate RNA. Human gastric cancer cell lines SGC-7901 and BGC-823 were kindly provided by the
Second Affiliated Hospital of Zhejiang University.

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4.2. Chemicals and Reagents
Mobile phases of acetonitrile and methanol for HPLC (high-performance liquid chromatography)
and phenolic acids, including caffeic acid, ferulic acid, naringenin, neoeriocitrin, and hesperetin
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dihydroflavonols (dihydroquercetin
dihydromyricetin and dihydrokaempferol), eriodictyol, homoeriodictyol, quercetin 3,3’-dimethyl ether,
quercetin 3-methyl ether, chrysoeriol, and isokaempferide were obtained from BioBioPha Co., Ltd.
(Kunming, China). Eriocitrin, baicalein, luteolin, quercetin, kaempferol, isorhamnetin, and SAM
were from Aladdin (Shanghai, China). Apigenin, myricetin, and 7,8-dihydroxyflavone hydrate were
from TCI (Shanghai, China) and tricin was purchased from ChromaDex (Irvine, CA, USA). Tricetin
and syringetin were purchased from Extrasynthese (Lyon, France) and baicalein 7-methyl ether was
purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). RPMI-1640 medium,
fetal bovine serum (FBS), HEPES, and trypsin-EDTA were purchased from Gibco (Waltham, MA, USA).
Cell Counting Kit-8 was purchased from Dojindo Technology (Shanghai, China).
4.3. Isolation, Cloning, and Heterologous Expression of CrOMT2
The extraction of total RNA from the peel of Ougan fruit was conducted as described by
Kiefer et al. [43], and cDNA was synthesized from 1 µg RNA using a PrimeScript™RT Reagent
Kit (Takara, Dalian, China). The full coding sequence (CDs) of CrOMT2 was amplified by PCR.
The cDNA from Ougan fruit peel was used as a template and the annealing primers were designed
based on the coding sequence of Ciclev10020814m on the Citrus clementina reference genome (http:
//www.citrusgenomedb.org/species/clementina/genome1.0). The cloned CDs was further inserted
into the pET32a vector and the expression plasmid CiOMT2-pET32a was transformed to the E. coli
BL21(DE3)pLysS cells (Promega, Madison, WI, USA) for protein expression. Bacterial cultures were
incubated in Luria–Bertani medium (0.1 g
expression was then induced by adding 1 mM isopropyl
The induced bacterial cells were lysed by sonication in 1
subjected to Co2+-affinity chromatography to purify recombinant protein, as described in the user
manual of HisTALON Gravity Columns Purification Kit (Takara, Dalian, China). Eluted fractions
·
L−1 ampicillin) at 37 ◦C until OD600 reached 0.6. Protein
-thiogalactopyranoside at 18 ◦C for 24 h.
PBS buffer, and the supernatants were then
β
×
™
were applied to a PD-10 desalting column (GE Healthcare, Uppsala, Sweden) to store the purified
◦
protein in a Tris-HCl buffer (50 mM, 10% glycerol, 2 mM dithiothreitol, pH 8.0) at
nucleotides used above are listed in Supplementary Table S2.
−
80 C. Oligo
4.4. Phylogenetic Tree and Protein Alignments
CrOMT2 and several identified COMTs from plant were aligned using ClustalW. The evolutionary
history was constructed by MEGA X tool [44] using the neighbor-joining method [45], and the
phylogenetic tree was assessed with 1000 bootstrap replicates [46]. Accession numbers of proteins
used are given in Supplementary Table S3. The structure-based alignment of the OMTs was inferred by
T-Coffee (Expresso) [47] and deciphered via ESpript 3.0 [48].
4.5. Enzyme Activity Measurements and Kinetic Analysis
A wide range of flavonoids along with caffeic acid were incubated with recombinant
CrOMT2-pET32a to explore the substrate specificity. A final volume of 200
mM SAM as methyl donor, 200 M phenolic substrates, and 25 L purified enzyme in 50 mM Tris-HCl
buffer, pH 8.0. Assays were conducted for 2 h at 37 ◦C and quenched with 200
L methanol. The reaction
m filter and were analyzed by HPLC and mass spectrometry as
µL was used with 1
µ
µ
µ
products were filtered through a 0.22
described below.
µ
Tris-HCl buffer (pH 8.0–9.0) and potassium phosphate buffer (pH 5.5–7.5) were used to investigate
the effect of pH on CrOMT2 activity. The temperature dependency was assessed between 25 ◦C and
60 ◦C.

Molecules 2020, 25, 858
12 of 15
Relative activity on different substrates was determined using an MTase-Glo
™
Methyltransferase
Assay kit (Promega, Madison, WI, USA). It should be noted that the demethylated product of SAM,
i.e., S-adenosyl homocysteine (SAH), in the reaction was correlated to luminescence detected by a
luminometer. Specifically, 12.5 µM substrate, 250 µM SAM, and diluted protein were incubated in
Tris-HCl buffer (pH 8.0) at 37 ◦C for 30 min. The reaction was terminated by adding 0.5% trifluoroacetic
acid, and then the luminescence converted by SAH was assessed via a plate-reading luminometer.
The kinetic properties were assessed as described in the method using 2 mM SAM and a set of
substrates at different concentrations. The apparent Km and KCat were calculated using a nonlinear
regression curve. The calculation was fitted to Michaelis–Menten via GraphPad Prism version 7
(GraphPad Software, San Diego, CA, USA).
4.6. Cell Culture and Cell Viability Assay
Two human gastric cancer cell lines, i.e., SGC-7901 and BGC-823, were used in the cell viability
assay. These classic cell lines are commonly used to explore the effect of proliferation inhibition
of gastric cancer cells [11
and 1
HEPES) and maintained at 37 ◦C in a humidified incubator containing 5% CO2. Cells were
passaged using trypsin-EDTA before reaching 90% confluence. A Cell Counting Kit-8 was used to
evaluate the cell viability according to the manufacturer’s instructions. Briefly, 4
103 cells per well
for SGC-7901 and 8
103 cells per well for BGC-823 were seeded onto a 96 well plate and cultured
,23,49]. The two cell lines were cultured in RPMI-1640 medium (10% FBS
×
×
×
overnight. The cells were replenished with the fresh medium the next day and different concentrations
of flavonoids dissolved in DMF (N,N-dimethyl-formamide) were added. The DMF was used as a
solvent control with a final concentration of 0.1%. Paclitaxel was used as a positive control. After 48 h
incubation, the supernatant was discarded and the cells were washed twice with 1
amount of cck-8 solution (dissolved in FBS-free RPMI-1640 medium) was added into the well and
cultured for 1 h. The light absorbance values at 450 nm and 620 nm were measured using a microplate
×
PBS. An equal
reader and the inhibition ratio was calculated as (A450 A620)/A450. IC50 values were calculated using
−
SPSS 19.0 software (IBM, Armonk, NY, USA). The cell viabilities under different treatments were
compared using the Tukey test in GraphPad Prism version 7 (GraphPad Software, San Diego, CA, USA).
4.7. HPLC and MS/MS Analysis
HPLC analysis was performed on an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara,
CA, USA) with a Sunfire C18 ODS column (4.6
×
250 mm, 5 µm, Waters Corp., Milford, MA, USA).
The mobile phases consisted of acetonitrile (A) and water with 0.1% formic acid (B). The compounds
were segregated with linear gradient run as follows: 20% A (0–5 min), 20%–27% A (5–10 min), 27% A
(10–15 min), 27%–40% A (15–25 min), 40%–60% A (25–35 min), 60%–80% A (35–40 min), 80%–100%
A (40–42 min), 100%–20% A (42–45 min), 20% A (45–50 min). The flow rate was 1 mL
·
min−1 and
injection volume was 10 L. Chromatographic detection was done using a VWD detector at the
µ
wavelength of 280 nm for dihydroflavones, dihydroflavonols, and caffeic acid, and at 350 nm for
flavones and flavonols.
High-resolution mass spectrometry was conducted with an AB TripleTOF 5600plus System
(AB SCIEX, Framingham, MA, USA). MS/MS analysis was carried out in the positive ion mode (ESI) or
negative ion mode, and the exact mass was then obtained.
4.8. Statistical Analyses
All results were from at least three replicates and expressed as means
±
standard errors (SE).
Graphics were made with OriginPro 2019b Learning Edition (Microcal Software Inc., Northampton, MA,
USA). Structural formulas were generated using ChemBioDraw Ultra 12.0 (PerkinElmer Informatics,
Waltham, MA, USA).

Molecules 2020, 25, 858
13 of 15
5. Conclusions
CrOMT2 from citrus was a typical FOMT belonging to COMTs that transferred methyl groups
into the 3’/5’/7-hydroxy groups of the flavonoids backbone in vitro. It was found that the enzyme
favored PMF-type substrates, and its methylation sites tended to be critical for PMFs synthesis in citrus.
This recombinant CrOMT2 also displayed a broad substrate spectrum and various methylation sites
in vitro. The 3’-methylation that occurred in flavones or flavonols (having unsaturated C2-C3 bonds)
all increased the in vitro cytotoxicity. This was the first evaluation of the effects of citrus enzymes on
human health-related biological activities. The implications of this study may provide a new strategy
for the directional biosynthesis of bioactive substances.
Supplementary Materials: The following are available online. Figure S1: Sequence alignment of CrOMT2 and
two paralogous genes from citrus, Figure S2: biochemical characterization of the recombinant CrOMT2, Figure
S3: MS/MS spectra of the authentic standards corresponding to the methylated products catalyzed by CrOMT2,
Figure S4: HPLC analysis of dihydroflavonols and caffeic acid methylation by recombinant CrOMT2, Figure
S5: Sequence alignment of CrOMT2 and other COMTs from plants, Figure S6: Cell viability after treated with
flavonoids substrates or methylated products of CrOMT2 in vitro, Table S1: Percent identity of AtOMT1 against
OMTs in Citrus clementina, Table S2: Primers used for amplification, Table S3: UniProt entries of COMT proteins
used in the phylogenetic tree.
Author Contributions: Conceptualization, C.S.; Data curation, X.L., Y.W. and C.Z.; Formal analysis, X.L., Y.W.,
Y.C., S.X., Q.G. and C.Z.; Funding acquisition, C.S.; Investigation, X.L., Y.W., Y.C., S.X., J.C. and C.S.; Methodology,
X.L., Y.W., Y.C., S.X., Q.G., C.Z., J.C. and C.S.; Resources, C.S.; Software, X.L., Y.W., Y.C., S.X., Q.G. and J.C.;
Supervision, C.S.; Validation, X.L. and Y.W.; Visualization, X.L. and Y.W.; Writing – original draft, X.L. and
Y.W.; Writing – review & editing, X.L. and C.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (31571838),
Natural Science Foundation of Zhejiang Province (LR17C200001), the National Key R&D Program of China
(2017YFD0400200), the 111 project (B17039), and the Agricultural Outstanding Talents and Innovation Team of the
State Agricultural Ministry on Health and Nutrition of Fruit.
Acknowledgments: The authors thank Zhiwei Ge for assistance with the LC/MS analyses.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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