Purification and characterization of a novel O-methyltransferase from Flammulina velutipes

Article abstract of DOI:10.1080/09168451.2014.912117

An enzyme catalyzing the methylation of phenolic hydroxyl groups in polyphenols was identified from mycelial cultures of edible mushrooms to synthesize O-methylated polyphenols. Enzyme activity was measured to assess whether methyl groups were introduced

Full text of DOI:10.1080/09168451.2014.912117

This article was downloaded by: [University of Birmingham]
On: 22 March 2015, At: 17:37
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbbb20
Purification and characterization of a novel O-
methyltransferase from Flammulina velutipes
a
a
a
a
Masanobu Kirita , Yoshihisa Tanaka , Motoyuki Tagashira , Tomomasa Kanda & Mari
b
Maeda-Yamamoto
a
Research & Development-Production Headquarters, Asahi Breweries Limited, Moriya-
shi, Japan
b
National Food Research Institute, National Agriculture and Food Research Organization,
Tsukuba-shi, Japan
Published online: 28 May 2014.
Click for updates
To cite this article: Masanobu Kirita, Yoshihisa Tanaka, Motoyuki Tagashira, Tomomasa Kanda & Mari Maeda-Yamamoto
2014) Purification and characterization of a novel O-methyltransferase from Flammulina velutipes, Bioscience,
(
Biotechnology, and Biochemistry, 78:5, 806-811, DOI: 10.1080/09168451.2014.912117
To link to this article: http://dx.doi.org/10.1080/09168451.2014.912117
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Bioscience, Biotechnology, and Biochemistry, 2014
Vol. 78, No. 5, 806–811
Purification and characterization of a novel O-methyltransferase from
Flammulina velutipes
1
,
1
1
1
Masanobu Kirita *, Yoshihisa Tanaka , Motoyuki Tagashira , Tomomasa Kanda and
2
Mari Maeda-Yamamoto
1
2
Research & Development-Production Headquarters, Asahi Breweries Limited, Moriya-shi, Japan; National Food
Research Institute, National Agriculture and Food Research Organization, Tsukuba-shi, Japan
Received November 22, 2013; accepted January 20, 2014
http://dx.doi.org/10.1080/09168451.2014.912117
An enzyme catalyzing the methylation of phenolic
hydroxyl groups in polyphenols was identified from
mycelial cultures of edible mushrooms to synthesize
O-methylated polyphenols. Enzyme activity was mea-
sured to assess whether methyl groups were intro-
duced into (−)-epigallocatechin-3-O-gallate (EGCG)
using SAM as a methyl donor, and (−)-epigallocate-
chin-3-O-(3-O-methyl)-gallate (EGCG3″Me), (−)-epi-
gallocatechin-3-O-(4-O-methyl)-gallate (EGCG4″Me),
and (−)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate
AUC (area under the drug concentration time curve;
−
1
min l g mL ) of EGCG was 6.72 ± 2.87 and EGCG3″
Me was 8.48 ± 2.54 in healthy human volunteers.
Though the dose of EGCG was 5.1 times the dose of
EGCG3″Me, the AUC of EGCG3″Me was higher than
4
)
that of EGCG’s. The antiallergic effects of EGCG and
other O-methylated EGCGs were measured using
histamine release assays in bone marrow-derived mouse
mast cells, and the order of potencies was (−)-3′-
O-methyl-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate
(EGCG3′,3″,5″triMe) = (−)-epigallocatechin-3-O-(3,5-
O-dimethyl)-gallate (EGCG3″,5″diMe) > EGCG3″Me
(
EGCG3″,5″diMe) peaks were detected using crude
enzyme preparations from mycelial cultures of Flam-
mulina velutipes. The enzyme was purified using
chromatographic and two-dimensional electrophore-
sis. The purified enzyme was subsequently analyzed
on the basis of the partial amino acid sequence using
LC–MS/MS. Partial amino acid sequencing identi-
fied the 17 and 12 amino acid sequences,
VLEVGTLGGYSTTWLAR and TGGIIIVDNVVR.
In database searches, these sequences showed high
identity with O-methyltransferases from other
mushroom species and completely matched 11 of 17
and 9 of 12 amino acids from five other mushroom
O-methyltransferases.
5
)
> EGCG. The inhibition of histamine release was
related to an increase in the number of methyl groups
in EGCG, particularly the galloyl moiety. The meta-
bolic stability of the methylated flavones 7-methoxyf-
lavone, 7,4′-dimethoxyflavone, 5,7-dimethoxyflavone,
and 5,7,4′-trimethoxyflavone in pooled human liver S9
fractions was investigated which showed their intestinal
absorption in caco-2 cells to be more effective than that
6
)
of unmethylated flavones. However, these O-methyl-
ated polyphenols are present at extremely low levels in
plants.
These reports indicate that the bioavailability and
efficacy of polyphenols are improved by methylation of
the phenolic hydroxl group. Therefore, enzymes cata-
lyzing such polyphenol-methylating reactions may be
useful tools for enhancing the efficacy of naturally
occuring polyphenols. In plants, Caffeoyl-Coenzyme A
O-methyltransferases (CCoAOMT) play important roles
Key words: Flammulina velutipes; O-methyltransferase;
(−)-epigallocatechin-3-O-gallate; EGCG
Dietary polyphenols, generally contained in our diet,
contribute to human health and prevent disease. Low
bioavailability and instability of metabolic functions
7
,8)
in the biosynthesis of lignin.
In our previous report,
O-methyltransferase isolated from tea catalyzed the
1
,2)
hamper the potential of several native polyphenols
;
methylation of EGCG, which is highly homologous to
5
)
however, O-methylated polyphenols have improved
bioavailability and metabolic stability.
In a previous study, the O-methylated (−)-epigalloca-
techin-3-O-gallate (EGCG), (−)-epigallocatechin-3-O-
CCoAOMT. Similarly, mushroom including white rot
fungi have the ability to degrade lignin; this degrada-
tion involves O-methyltransferases. In Phanerochaete
chrysosporium, this multistep lignin degredation path-
(
‘
3-O-methyl)-gallate (EGCG3″Me) was isolated from
Benifuuki’ cultivars of Camellia sinensis L. After
way has been shown to involve oxidation, reduction,
3
)
9,10)
and methylation reactions.
Therefore, we predicted
drinking O-methylated EGCG-rich green tea containing
3.5 mg of EGCG and 8.5 mg of EGCG3″Me, the
that such mushroom species would express enzymes
that catalyze the methylation of polyphenols. However,
4
*Corresponding author. Email: masanobu.kirita@asahibeer.co.jp
Abbreviations: EGCG, (−)-epigallocatechin-3-O-gallate; SAM, S-adenosyl-L-methionine; CCoAOMT, Caffeoyl-Coenzyme A O-methyltransferas;
PAGE, polyacrylamide gel electrophoresis.
©
2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

O-Methyltransferase from Flammulina velutipes
807
till date, very few reports demonstrate methylase activi-
ties in mushrooms such as white rot fungi. Among
these studies, 3-O- and 4-O-methyltransferases were
velutipes, Hypsizygus marmoreus, Pleurotus ostreatus,
Armillaria mellea subsp. nipponica, Pleurotus cornuco-
piae var. citrinopileatus, Pleurotus eryngii, and Pleuro-
tus nebrodensis.
11,12)
purified from P. chrysosporium.
The 3-O-methyl-
transferases catalyze the methylation of isovanillic acid,
whereas 4-O-methyltransferases catalyzes the methyla-
tion of acetovanilline using S-adenosyl-L-methionine
Assay of O-methyltransferase activity.
Mycelial
cultures were harvested and sonicated in a suspension
buffer containing 20 mM Tris–HCl (pH 7.5), 1 mM
dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic
acid (EDTA), and 10% glycerol. Supernatants were
collected and centrifuged to extract the crude
enzyme, which was assayed for O-methyltransferase
activity by assessing EGCG methylation rates. The
(
SAM) as a donor. Therefore, we searched for novel
enzymes in mycelial cultures of edible mushroom that
can be applied as functional foods. Subsequently, we
demonstrated the potential for large-scale synthesis
of O-methylated EGCGs using crude enzyme
preparations.
3-mL reaction mixture contained 100 mM Tris–HCl
(
pH 7.4), 0.2 mM MgCl , 0.25 mM EGCG, 0.5 mM
2
Materials and methods
SAM as a methyl donor, and 1.5 mL of crude
enzyme solution. The mixture was incubated at 37 °C
for 14 h, and the reaction was stopped by the addi-
tion of 1 N HCl. The reaction mixture was extracted
with 5 mL of ethyl acetate, and centrifugation was
used to collect the organic phase, which was dried
under N2 gas and resuspended in 30% methanol
containing 1% ascorbic acid. Enzyme activity was
determined using HPLC or LC–TOF–MS according
Reagents. All chemicals and solvents were of high
or high performance liquid chromatography (HPLC)
grade. EGCG3″Me, (−)-epigallocatechin-3-O-(4-O-
methyl)-gallate (EGCG4″Me), and EGCG3″,5″diMe
were prepared according to a previous method with
5
)
minor modifications (Fig. 1). Edible mushrooms were
purchased from a Japanese supermarket.
5
)
to a previously described method.
Isolation and mycelial culture of edible mush-
Enzyme purification. To purify the enzyme, myce-
lial cultures of F. velutipes were lyophilized and ground
and subsequently resuspended in the above suspension
buffer. After sonication, the supernatant was collected
by centrifugation and fractionated using 60–80%
ammonium sulfate saturation. The precipitate was sus-
pended in suspension buffer and desalted using PD-10
columns (GE Healthcare, Japan). The sample was
loaded onto a DEAE column (HiPrep 16/10, GE
Healthcare, Japan) that had been previously
equilibrated with 20 mM Tris–HCl (pH 7.5) containing
rooms.
Stalks of edible mushrooms were cut, and
mushroom surfaces were disinfected with 0.5% sodium
hypochlorite solution for 15 min. Internal stalks were cut
after rinsing with sterile distilled water and placed on
potato dextrose agar medium at 25 °C to isolate mycelia.
Isolated mycelia were cultured in a broth containing
0
0
.02% glucose, 0.01% peptone, 0.002% yeast extract,
.002% KH PO , and 0.001% MgSO at 28 °C while
2
4
4
shaking. Edible mushroom species included Lentinus ed-
odes, Lyophyllum shimeji, Grifola frondosa, Flammulina
OH
3
'
4
'
OH
OH
5
'
HO
O
O
OH
3'' R1
O
4
''
R2
5''
R3
Compounds
R1
R2
OH
OH
R3
OH
OH
(
(
(
(
−)-epigallocatechin-3-O-gallate (EGCG)
OH
−)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3''Me)
−)-epigallocatechin-3-O-(4-O-methyl)-gallate (EGCG4''Me)
OCH3
OH
OCH3
OH
OH
−)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3'',5''diMe)
OCH3
OCH3
Fig. 1. Chemical structures of EGCG and O-methylated EGCGs.

808
M. Kirita et al.
1
mM DTT. The enzyme was eluted at a flow rate of 2
The de novo sequence analysis was performed using
a Serveyo-LC-system (Thermo Fisher Scientific K.K.,
Japan) with a MS system LTQ-orbitrap mass spectrom-
eter (Thermo Fisher Scientific K.K., Japan). LC–MS/
MS was performed using a Zorbax SB-300 column
(0.075 mm i.d. × 50 mm, Agilent Technologies, Japan)
at a flow rate of 200 μL min . Mixtures of A (0.1%
(v/v) formic acid) and B (80% (v/v) methanol contain-
ing 0.1% (v/v) formic acid) were used as mobile
phases. The initial eluent used was 5% mobile phase
B, followed by a linear gradient from 5 to 55% B for
65 min, 100% B for 10 min, and finally 5% B for 15
min.
−
1
mL min using a linear gradient of 0–1 M NaCl in the
same buffer. Active fractions were combined, desalted,
and concentrated by ultrafiltration. Subsequently, the
sample was prepared with 1.5 M ammonium sulfate
and loaded on to a Phenyl Sepharose HP column
−
1
(
Hiload 26/10, GE Healthcare, Japan) that had been
previously equilibrated with 100 mM phosphate buffer
pH 7.5) containing 1 mM DTT and 1.5 M ammonium
sulfate. Elution was performed at a flow rate of 2 mL
(
−
1
min using a linear gradient of 1.5–0 M ammonium
sulfate. Active fractions were collected, desalted, and
concentrated as above. For further purification, an ali-
quot of the eluate from the Phenyl Sepharose HP col-
umn was loaded onto an anion exchange column
Optimal pH and temperature of the enzyme. Opti-
mal pH and temperature were assessed using 0.25 mL
of crude enzyme and 0.05 mM EGCG as a substrate, in
(
TSK-gel BioAssist Q, 4.6-mm i.d. × 5 cm; TOSHO
corporation, Japan) that had been previously equili-
brated with 20 mM Tris–HCl (pH 7.5) containing
1
1
the same buffer. The active fractions were collected,
desalted, and concentrated as above. Each purified sam-
ple was subsequently analyzed using 12% SDS-PAGE.
a solution containing 2.5 mM MgCl , 0.04% ascorbic
mM DTT. The enzyme was eluted at a flow rate of
2
−
1
acid, and 0.5 mM SAM. Optimal pH was determined in
the range of 3–10 using 20 mM acetate buffer (pH,
mL min using a 0–0.5 M linear gradient of NaCl in
3
2
.0–5.5), 20 mM phosphate buffer (pH, 6.0–7.0), and
0 mM Tris–HCl (pH, 7.5–10). The mixture was incu-
bated at 37 °C for 6 h. The optimal temperature was
determined by incubation with 20 mM phosphate buffer
SDS-PAGE, 2D gel electrophoresis, and trypsin
(pH 7.0) for 6 h and O-methylated EGCGs were ana-
digestion.
Active fractions from the Phenyl
lyzed using HPLC.
Sepharose HP column were separated using 12%
SDS-PAGE and 2D gel electrophoresis. Bands of
interest were excised from SDS-PAGE gels. For 2D
gel electrophoresis, isoelectric focusing was per-
formed using an Ettan IPGphor3 IEF System (GE
Healthcare, Japan). Immobiline Dry Strip linear
immobilized pH gradient gel strips (7 cm) with a pH
range of 3–10 (GE Healthcare, Japan) were rehy-
drated in a solution containing 9 M urea, 2% (w/v)
CHAPS, 0.5% (v/v) ampholytes (pH 3–10), 0.002%
bromophenol blue, and 20 μg of protein. Proteins
were focused at 500 V for 1 h, 1000 V for 1 h, 8000
V for 2 h, and 8000 V for 12 h at 20 °C. After focus-
ing, Immobiline Dry Strips were embedded onto 4–
Culture of mycelia in a jar fermentor and production
of EGCG3″Me.
Mycelia were cultured in 300 mL
Erlenmeyer flasks containing 150 mL of 0.02% glu-
cose, 0.01% peptone, 0.002% yeast extract, 0.002%
KH PO , and 0.001% MgSO . The culture was pre-
2
4
4
pared in a rotary shaker at 130 rpm for 6 days at 20 °C.
Cultured mycelia were inoculated into a 3-L working
volume in a 5-L jar fermentor (B. E. Marubishi Co.,
Ltd., Japan) of 165-mm diameter and 265-mm height.
Fermentation was carried out at 50 rpm using a stan-
dard turbine-type impeller at 20 °C and an aeration rate
of 1.5 vvm for 5 days. Mycelial cultures were lyophi-
lized before measuring enzyme activity.
2
0% SDS-PAGE gels. Protein spots that confirmed
active fractions were excised from 2D electrophoresis
gels. These excised proteins were purified and sub-
jected to in-gel trypsin digestion according to a pre-
Results
O-methyltransferase activity assays
1
3)
viously reported method.
Methylation activity for EGCG was determined in
mycelial cultures of edible mushroom. In these experi-
ments, EGCG3″Me, EGCG4″Me, and EGCG3″,5″diMe
peaks were detected in F. velutipes crude enzyme
preparations at retention times that matched those of
O-methylated EGCGs’ standards (Fig. 2). Theoretical
values of molecular weights for these O-methylated
EGCGs were confirmed using LC–TOF-MS. No peaks
corresponding to O-methylated EGCGs were detected
in enzyme extracts from any of the other mushroom
species.
LC–MS/MS peptide sequence analysis.
Purified
protein samples from SDS-PAGE or 2D gel electropho-
resis were analyzed using LC–MS/MS. LC–MS/MS
was performed using a nano-LC system paradigm
AMC Inc., Camden) with a MS system LTQ (Thermo
Fisher Scientific K.K., Japan). LC–MS/MS was per-
formed using a Magic C18, 3-μm, 200-Å column
(
(
0.2 mm i.d. × 50 mm, Michrom Bioresources, CA) at a
−
1
flow rate of 1.5 μL min . Mixture of A (0.1% (v/v)
formic acid/2% (v/v) acetonitrile) and B (0.1% (v/v)
formic acid/90% (v/v) acetonitrile were used as the
mobile phase. The initial eluent was 5% mobile phase
B, followed by a linear gradient from 5 to 45% B for
Enzyme purification and peptide sequence analyses
Enzyme purification protocols are summarized in
Table 1. After sonication, supernatants from mycelial
cultures were fractionated using 40%–80% ammonium
sulfate saturation in 10% increments. After desalting,
enzyme activity was determined in each fraction.
2
0 min. Survey full scan spectra were acquired in the
m/z range of 400–2000. Data were analyzed using Bio-
works software version 3.1 (Thermo Fisher Scientific
K.K., Japan).

O-Methyltransferase from Flammulina velutipes
809
purified. After loading onto anion exchange columns
BioAssist Q), a few bands were detected on SDS-
(
PAGE gels. Although only a small amount of sample
was purified after anion exchange chromatography, the
same bands were excised from the SDS-PAGE gels
after elution from Phenyl Sepharose HP column. Bands
of interest were analyzed for partial amino acid
sequences using LC–MS/MS after treatment with in-gel
trypsin digestion. In database searches, the identified
amino acid sequence VLEVGTLGGYSTTWLAR was
highly homologous with O-methyltransferase sequences
from Laccaria bicolor S238 N-H82 (XP_001878803.1;
Fig. 3). This L. bicolor O-methyltransferase had a
molecular weight of 25.3 kDa and an isoelectric point
of 5.24. Similarly, the sequence of F. velutipes was
confirmed from an excised SDS-PAGE band of approx-
imately 24–25 kDa. To further purify the enzyme,
active fractions from the Phenyl Sepharose HP column
were separated using 2D gel electrophoresis and spots
of similar molecular weight and isoelectric point were
excised from the marginal zone as above (Fig. 4). The
above amino acid sequence was confirmed in one of
these protein spots, and subsequent de novo sequence
analyses identified the sequence TGGIIIVDNVVR.
Database searching indicated a high degree of homol-
ogy with another region of O-methyltransferase from L.
bicolor. Taken together, these results suggest purifica-
tion of an EGCG O-methyltransferase (Fv-OMT) from
F. velutipes.
Optimal pH and temperature for enzyme activity
The optimal pH and temperature for enzyme activity
were determined using the O-methylated EGCG synthe-
sis index. The enzyme was stable for 6 h in a pH range
of 6.5–8.5 at 37 °C (Fig. 5(A)) and its activity was
greatest at pH 7.0. The enzyme was active for 6 h
between 20 and 42 °C at pH 7.0 (Fig. 5(B)), and the
optimal temperature was 37 °C. Enzymatic activity dra-
matically decreased as the temperature was increased
from 42 °C. Each ratio of EGCG3″Me, EGCG4″Me,
and EGCG3″,5″diMe production by pH and tempera-
ture was same.
Fig. 2. HPLC analysis of the enzymatic reaction products using
crude enzyme from F. velutipes.
Note: (A) Standard catechins. Peak identification: 1, EGCG; 2,
EGCG4″Me; 3, EGCG3″Me; 4, EGCG3″,5″diMe. (B) Enzymatic
reaction products. But enzyme was added after thermal denaturation.
EGCG3″Me production
Mycelium pellets were grown in a jar fermentor and
(
C) Enzymatic reaction products.
49 g of lyophilized mycelium was collected. Subse-
quently, 50 mg of lyophilized mycelium was sonicated
in 3 mL of suspension buffer, and 1 mL of this enzyme
suspension produced 150.7 μM EGCG3″Me in 14 h
(71.2 μg).
Fractions from 60%–80% ammonium sulfate
saturations contained more than 90% of total EGCG
O-methylase activity and were collected and further
Table 1. Purification of the Fv-OMT from F. velutipes.
Total protein Yield Purification Total activity
Specific activity
Step no.
(mg)
(%)
fold
(unit)
(unit/mg)
1
2
3
4
5
Crude extract
173.28
21.00
9.48
2.19
0.07
100.0
91.8
46.6
16.7
0.9
1
715.92
657.35
333.96
119.84
6.52
4.13
31.30
35.20
54.70
90.56
60–80% ammonium sulfate saturation
Anion-exchange chromatography (DEAE)
Hydrophobic interaction chromatography (Phenyl Sepharose HP)
Anion-exchange chromatography (BioAssist Q)
7.58
8.52
13.24
21.93
Note: One unit of enzyme activity is defined as the amount of enzyme producing the formation of 1 mmol of EGCG3″Me, following 14 h of incubation at 37 °C.

810
M. Kirita et al.
Fv-OMT1
L.bicolor
T.versicolor
D.squalens
C.cinerea okayama I
S.hirsutum
V L E V G T L G G Y
S
S
S
S
S
S
T
S
T
A
T
A
T W L A R
I
I
L
L
E V G T L G G Y
E V G T L G G Y
I
I
I
I
I
W L A R
W F A R
W L A R
W L A R
V L E V G T L G G Y
E V G T L G G Y
V L E V G T L G G
L
S
C F A R
Fv-OMT1
L.bicolor
T.versicolor
D.squalens
C.cinerea okayama S G G V
S.hirsutum K G G
T G G
K G G V
R G G
K G G
I
I
I
I
I
I
I
I
I
I
I
I
I
V D N V V R
V D N V V R
V D N V I R
V D N V V R
V D N V V R
V D N V V R
I
I
I
Fig. 3. Comparison of partial amino acid sequences of O-metyltransferase with the mushroom species L. bicolor, T. versicolor, D. sequalens, C.
cinerea okayama, and S. hirsutum.
Note: Completely matched residues are indicated with gray boxes.
1
1
2
0
8
6
4
2
0
pH3
pH10
EGCG3''Me
(
A)
EGCG4''Me
EGCG3'',5''diMe
(
kDa)
7
2
5
5
Fv-OMT
3
4
5
6
7
8
9
10
pH
1
2
0
8
6
4
2
0
(B)
EGCG3''Me
EGCG4''Me
1
EGCG3'',5''diMe
Fig. 4. Representative 2D gel electrophoresis of Fv-OMT in active
fractions from phenyl sepharose columns.
Note: Proteins were stained with SYPRO Red Protein (Takara Bio
Inc., Japan). The immobilized pH gradient (pH, 3–10) is indicated on
top. Molecular mass markers are indicated on the right. The arrow
mark indicates the spot from which O-methyltransferase amino acid
sequence was confirmed.
0
10
20
30
40
50
60
temperature (˚C)
Fig. 5. Optimal pH (A) and Temperature (B) of Fv-OMT.
Note: Activity of the enzyme is indexed according to the production
Discussion
of EGCG3″Me, EGCG4″Me, and EGCG3″,5″diMe.
We identified, isolated, and measured the activity of
an enzyme from F. velutipes that methylates EGCG.
Although three kinds of columns were used to purify
this enzyme, multiple bands were identified on SDS-
PAGE gels. Therefore, candidate bands were excised
and amino acid sequences were determined using LC–
MS/MS and de novo sequence analyses. The partial
amino acid sequence VLEVGTLGGYSTTWLAR had
high identity with O-methyltransferases from other
mushrooms that were identified in database searches. In
comparisons with five other mushroom species, 11 of
regions among plant enzymes of the CCoAOMT fam-
ily. Partial amino acid sequences of Fv-OMT were
positioned in the motif E (GVXTGYS) that shows
some similarity with the putative SAM-binding
domains. Hence, the sequence GTLGGYS of Fv-OMT
may serve as a SAM-binding domain.
The other partial Fv-OMT amino acid sequence
TGGIIIVDNVVR showed high identity with sequences
from other mushrooms, and 9 of 11 amino acids were
completely mached. This sequence was positioned in
motif A (LVXXGGXI) of the plant CCoAOMT family,
which also shows some homology to putative SAM-
binding domains. Amino acid sequencing of this region
in the other five mushrooms indicated the common
sequence (L/M)VRXGG(I/V)I. Similarly, the partial
Fv-OMT sequence TGGIIIVDNVVR may include a
SAM-binding domain. On the basis of these sequence
homologies, we speculate that Fv-OMT comprises
1
7 amino acids completely matched and the other resi-
dues were extremely similar. SAM-dependent methyl-
transferases of plants belonging to the CCoAOMT
1
4)
group are categorized into two major classes. Class I
and II enzymes comprise 231–248 and 344–383 amino
acids, respectively. Mushroom O-methyltransferases,
which showed high homology with the O-methyltrans-
ferase idenitified in this study, comprised 222–234
1
4)
amino acids. Joshi et al. identified several conserved

O-Methyltransferase from Flammulina velutipes
811
approximately 230 residues and has a molecular weight
of 25 kDa.
We thank Daisuke Higo (Thermo Fisher Scientific
K.K., Japan) for de novo sequence analysis.
Only a few reports demonstrate methylation activities
in mushrooms such as white rot fungi. Among these,
Coulter et al. and Jeffers et al. purified 3-O- and 4-
References
1
2,15)
O-methyltransferases from P. chrysosporium
indicated dimeric and monomeric proteins of 36 and
4 kDa, respectively. In experiments using several ben-
and
[
1] Manach C, Williamson G, Morand C, Scalbert A, Rémésy C.
Bioavailability and bioefficacy of polyphenols in humans. I.
Review of 97 bioavailability studies. Am. J. Clin. Nutr. 2005;
81:230S–242S.
5
zoic acids, the substrate specificity of 3-O-methyltrans-
ferase was highly restricted to hydroxyl groups at the
[
[
2] Williamson G, Manach C. Bioavailability and bioefficacy of
polyphenols in humans. II. Review of 93 intervention studies.
Am. J. Clin. Nutr. 2005;81:243S–255S.
3] Sano M, Suzuki M, Miyase T, Yoshino K, Maeda-Yamamoto M.
Novel antiallergic catechin derivatives isolated from oolong tea.
J. Agric. Food Chem. 1999;47:1906–1910.
3
rd position. In contrast, 4-O-methyltransferase cata-
lyzed methylation at both 2nd and 4th positions. The
present data indicate that Fv-OMT has a distinctly dif-
ferent molecular weight and low substrate preference
for hydroxyl group positions. Nonetheless, the optimal
pH for Fv-OMT activity was between 6.5 and 8.5, sim-
ilar to that of P. chrysosporium 3-O-methyltransfer-
[4] Maeda-Yamamoto M. In vitro and in vivo anti-allergic effect of
`benifuuki` green tea containing O-methylated catechin and gin-
ger extract enhancement. Cytotechnology. 2007;55:135–142.
[5] Kirita M, Honma D, Tanaka Y, Usui S, Shoji T, Sami M,
Yokota T, Tagashira M, Muranaka A, Uchiyama M, Kanda T, Ma-
eda-Yamamoto M. Cloning of a novel O-methyltransferase from
camellia sinensis and synthesis of O-methylated EGCG and
evaluation of their bioactivity. J. Agric. Food Chem.
2010;58:7196–7201.
1
2)
ase’s. However, the optimal temperature for Fv-OMT
activity was between 20 and 42 °C, whereas that
of 3-O-methyltransferase of P. chrysosporium was
between 15 and 55 °C. Fv-OMT was denatured at
5
0 °C.
[
6] Wen X, Walle T. Methylated flavonoids have greatly improved
intestinal absorption and metabolic stability. Drug Metab. Dis-
pos. 2006;34:1786–1792.
Mycelia from Erlenmeyer flasks cultures were pel-
leted, but when fermentation was conducted at 200
rpm, mycelia were extremely finely sheared and gave
no pellet, and the ensuing enzyme preparation failed to
produce EGCG3″Me. In contrast, fermentation at 50
rpm produced mycelium pellets from Erlenmeyer flask
cultures and enzymatic activity was obtained. Hence,
formation of the pellet was important for fermentation.
In conclusion, we searched for enzymes that methyl-
ate EGCG and identified Fv-OMT from F. velutipes.
Subsequently, we characterized the partial amino acid
sequence of Fv-OMT and determined optimal catalytic
pH and temperature. EGCG3″Me was synthesized using
the enzyme prepared from mycelial fermentation. This
enzyme may be a useful tool for the production of
O-methylated polyphenols and elucidation of their func-
tion. In future studies, we will isolate the gene for Fv-
OMT using degenerated primers that will be designed
on the basis of partial amino acid sequences and will
confirm the methylation activities for other polyphenols.
[
7] Diana L, Meyer L, Chapple L, Douglas CJ. Antisense suppres-
sion of 4-coumarate:coenzyme A ligase activity in Arabidopsis
leads to altered lignin subunit composition. Plant Cell.
1997;9:1985–1998.
[8] Zhong R, Herbert Morrison III W, Ye Z-H. Dual methylation
pathways in lignin biosynthesis. Plant Cell. 1998;10:2033–2045.
[
9] Valli K, Gold MH. Degradation of 2,4-dichlorophenol by the
lignin-degrading fungus Phanerochaete chrysosporium. J. Bacte-
riol. 1991;173:345–352.
[
[
[
[
10] Valli K, Wariishi H, Gold MH. Degradation of 2,7-dic-
hlorodibenzo-p-Dioxin by the lignin-degrading basidiomycete
Phanerochaete chrysosporium. J. Bacteriol. 1992;174:2131–
2137.
11] Coulter C, Hamilton JTG, Harper DB. Evidence for the existence
of independent chloromethane- and S-adenosylmethionine-utiliz-
ing systems for methylation in Phanerochaete chrysosporium.
Appl. Environ. Microbiol. 1993;59:1461–1466.
12] Jeffers MR, McRoberts WC, Harper DB. Identification of a
phenolic 3-O-methyltransferase in the lignin-degrading fungus
Phanerochaete chrysosporium. Microbiology. 1997;143:1975–
1
981.
13] Rahman MA, Shinjo R, Oomori T, Wörheide G. Analysis of the
Proteinaceous Components of the Organic Matrix of Calcitic
Sclerites from the Soft Coral Sinularia sp. PLOS ONE. 2013;8
(3):e57871.
Supplemental material
The supplemental material for this paper is available
at http://dx.doi.org/10.1080/09168451.2014.912117.
[14] Joshi CP, Chiang VL. Conserved sequence motifs in plant
S-adenosyl-L-methionine-dependent methyltransferases. Plant.
Mol. Biol. 1998;37:663–674.
Acknowledgments
[
15] Coulter C, Kennedy JT, McRoberts WC, Harper DB. Purifica-
tion and properties of an S-adenosylmethionine: 2,4-Disubsti-
tuted phenol O-methyltransferase from Phanerochaete
chrysosporium. Appl. Environ. Microbiol. 1993;59:706–711.
We thank Tadashi Muto (LB Co., Ltd., Japan) for
helpful enzyme purification and LC–MS/MS analysis.