Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana

Article abstract of DOI:10.1271/bbb.60006

Most flavonoids found in plants exist as glycosides, and glycosylation status has a wide range of effects on flavonoid solubility, stability, and bioavailability. Glycosylation of flavonoids is mediated by Family 1 glycosyltransferases (UGTs), which use UDP-sugars, such as UDP-glucose, as the glycosyl donor. AtGT-2, a UGT from Arabidopsis thaliana, was cloned and expressed in Escherichia coli as a gluthatione S-transferase fusion protein. Several compounds, including flavonoids, were tested as potential substrates. HPLC analysis of the reaction products indicated that AtGT-2 transfers a glucose molecule into several different kinds of flavonoids, eriodictyol being the most effective substrate, followed by luteolin, kaempferol, and quercetin. Based on comparison of HPLC retention times with authentic flavonoid 7-O-glucosides and nuclear magnetic resonance spectroscopy, the glycosylation position in the reacted flavonoids was determined to be the C-7 hydroxyl group. These results indicate that AtGT-2 encodes a flavonoid 7-O-glucosyltransferase.

Full text of DOI:10.1271/bbb.60006

Biosci. Biotechnol. Biochem., 70 (6), 1471–1477, 2006
Characterization of Flavonoid 7-O-Glucosyltransferase
from Arabidopsis thaliana
Jeong Ho KIM, Bong Gyu KIM, Younghee PARK, Jae Hyung KO, Chae Eun LIM,
y
Jun LIM, Yoongho LIM, and Joong-Hoon AHN
Bio/Molecular Informatics Center, Division of Bioscience and Biotechnology,
Konkuk University, Seoul 143-701, Korea
Received January 6, 2006; Accepted February 2, 2006; Online Publication, June 23, 2006
[doi:10.1271/bbb.60006]
Most flavonoids found in plants exist as glycosides,
and glycosylation status has a wide range of effects on
flavonoid solubility, stability, and bioavailability. Gly-
cosylation of flavonoids is mediated by Family 1 glyco-
syltransferases (UGTs), which use UDP-sugars, such as
UDP-glucose, as the glycosyl donor. AtGT-2, a UGT
from Arabidopsis thaliana, was cloned and expressed in
Escherichia coli as a gluthatione S-transferase fusion
protein. Several compounds, including flavonoids, were
tested as potential substrates. HPLC analysis of the
reaction products indicated that AtGT-2 transfers a
glucose molecule into several different kinds of flavo-
noids, eriodictyol being the most effective substrate,
followed by luteolin, kaempferol, and quercetin. Based
on comparison of HPLC retention times with authentic
flavonoid 7-O-glucosides and nuclear magnetic reso-
nance spectroscopy, the glycosylation position in the
reacted flavonoids was determined to be the C-7
hydroxyl group. These results indicate that AtGT-2
encodes a flavonoid 7-O-glucosyltransferase.
78 families, GT Family 1 consists of uridine-diphos-
phate glucose (UDP) glycosyltransferases (UGTs),12)
and the glucose donors for Family 1 GTs include not
only UDP-glucose, but also UDP-glucuronic acid, UDP-
xylose, UDP-galactose, and UDP-rhamnose. UGT ac-
ceptors include small compounds including flavonoids,
alkaloids, and hormones. Arabidopsis contains 120
UGTs,13) and functional analysis of the individual genes
associated with these UGTs is progressing with the help
of genomics and metabolomics.14) In vivo studies of
individual UGTs is not an easy task due to the diversity
of metabolites and the overlapping functions of different
UGTs.15) In vitro studies provide clues as to which
metabolites are observed during in vivo analysis, and
thus in vitro analysis of UGTs proceeds with in vivo. We
were interested in functional analysis of Arabidopsis
UGTs. As an initial step, we carried out in vitro analysis
of one of the UGTs, AtGT-2 and determined the
substrate of AtGT-2.
Materials and Methods
Key words: Arabidopsis thaliana; flavonoid; glycosyl-
transferase
Cloning and expression of AtGT-2. A full length
cDNA clone of AtGT-2 (At4g34138) was obtained from
the Arabidopsis Stock Center and sequencing was
performed to verify the cDNA identity. The AtGT-2
cDNA was digested with EcoRI and Not I, and
subcloned into the corresponding sites of a pGEX 5X-
2 vector (Amersham Biotech, Piscataway, NJ) as a
glutathione S-transferase (GST) gene fusion system.
Induction and purification of the expressed protein was
performed as described in Kim et al.16)
Flavonoids are secondary metabolites found in plants.
They play a variety of roles such as UV protection,1,2)
pathogen defense3,4) and coloration.5) Flavonoids also
have an effect on humans as antioxidants or phytoes-
trogen.6,7) Many flavonoids share a common phenyl
benzopyrane skeleton and differ from one another only
by the numbers and positions of hydroxyl or methylated
functional groups.8) The flavonoid aglycones, which
have a variety of glycosylation sites, are converted into
glycon by glycosyltransferases (GTs) in the last step of
flavonoid biosynthesis. Flavonoids are stored as glycon
forms.9,10)
Enzyme assay and analysis of reaction products. The
UGT assay contained a reaction mixture of 10 mg of
recombinant protein, 250 mM MgCl2, 250 mM UDP-
Glucose, and 70 mM substrate in phosphate buffer
(pH 7.0). The substrates analyzed were apigenin, caffeic
acid, diadzein, eriodictyol, esculetin, genistein, kaemp-
ferol, luteolin, naringenin, and quercetin, all of which
GTs responsible for the glycosylation reaction are
categorized into 78 families according to sequence
similarity, signature motifs, stereochemistry of the
glucoside linkage, and target specificity.11) Among the
y
To whom correspondence should be addressed. Fax: +82-2-3437-6106; E-mail: jhahn@konkuk.ac.kr

1472
J. H. KIM et al.
were purchased from either Indofinechemicals (Hills-
borough, NJ) or Sigma (St. Louis, MO). The reaction
mixture was incubated at 37 ꢀC for 3 h and extracted
twice with ethylacetate, and then the ethylacetate was
evaporated completely. The reaction product was dis-
solved with methanol and analyzed by thin layer
chromatography (TLC, MERCK, silica gel 60F254)
using a butanol–acetic acid–water mixture (5:3:1, v/v/
v). Plates were visualized by UV.
Reaction products and flavonoid substrates were
analyzed using a Varian HPLC (Series 100, Palo Alto,
CA) equipped with a photo diode array (PDA) detector
and a Waters Symmetry C18 column (3.5 mm particle
size, 4:6 ꢁ 250 mm, Milford, MA). For generation of an
analytical scale, the mobile phase consisted of 50 mM
potassium phosphate buffer (pH 3.0) and was program-
med as follows: 20% acetonitrile at 0 min; 40%
acetonitrile at 10 min; 70% acetonitrile at 20 min; 90%
acetonitrile at 30 min; 90% acetonitrile at 35 min; 20%
acetonitrile 40 min. The flow rate was 1 ml/min and UV
detection was performed at 270 nm.
higher in the flowers and leaves than in roots and stems
(data not shown).
Determination of substrate and analysis of reaction
product
The open reading frame of AtGT-2 was subcloned into
pGEX 5X-2 vector and expressed in E. coli as a
gluthatione S-transferase fusion protein. The recombi-
nant AtGT-2 protein was purified. The molecular weight
of the expressed recombinant protein was approximately
82 kDa, consistent with the molecular weight sum of
GST (26 kDa) and AtGT-2 (54.8 kDa) (Fig. 2). Several
compounds, including caffeic acid, esculetin, and sev-
eral flavonoids (apigenin, eriodictyol, luteolin, kaemp-
ferol, naringenin, and quercetin) were tested as potential
substrates for AtGT-2. The reaction products were
initially analyzed by thin layer chromatography (TLC).
All the substrates, except for caffeic acid, and esculetin
gave a new product with a different Rf value than the
substrate. Reaction products were further analyzed with
HPLC, and each product produced a peak having a
different retention time from the substrate. In order to
decide the number of glucose molecules added during
the reaction, the products from apigenin, naringein,
eriodictyol, and kaempferol were subject to liquid
chromatography–mass spectrometry (LC–MS). The mo-
lecular mass of four reaction products increased 162 Da
relative to the original substrates, indicating that one
glucose molecule was attached during the reaction.
The glucosylation positions of reaction products were
determined by three methods: comparison of HPLC
retention time and UV spectra with authentic flavonoid
glucosides, change in hypsochromic shift in the reaction
product, and NMR spectroscopy. The commercial
availability of flavonoid glucosides is limited, and hence
the comparison of retention time and UV-spectra was
carried out only where authentic flavonoid glucosides
were commercially available. Additional assignments
were made by hypsochromic shift data based on the
observation that a shift occurs only when the glycosy-
lated positions were either at the C-3 or the C-40
hydroxyl group of flavone or flavonol; in contrast,
glycosylation at the 7-hydroxyl group does not result in
a hypsochromic shift.20,22)
Nuclear magnetic resonance spectroscopy (NMR)
analysis of the reaction product was performed as
described by Yoon et al.17)
Results and Discussion
Cloning and characterization of AtGT-2
Following the completion of several genome projects
for various organisms, a number of websites that deal
with particular sets of genes have appeared. The CAZY
website (carbohydrate-active enzymes; http://afmb.cnrs-
mrs.fr/CAZY) provides classification of GTs from
several organisms, and according to the CAZY database,
A. thaliana contains 122 UGTs. Because we are inter-
ested in flavonoid glycosylation of A. thaliana, A. thali-
ana UGTs sequences were compared with flavonoid 7-
O-glycosyltransferases from Scutellaria baicalensis,18)
and AtGT-2 showed the highest homology with the
compared sequence. A full-length cDNA clone was
obtained from the Arabidopsis Gene stock center and
sequenced for verification. The sequence was consistent
with GenBank accession no. AY090273 and was named
AtGT-2. The clone was further classified as UGT73B1
based on the UDP-glycosyltransferase annotation sys-
tem.19)
The open reading frame of AtGT-2 consisted of 1,467
bp and was predicted to encode a 54.8 kDa protein.
AtGT-2 contained an amino acid sequence among plant
secondary product glucosyltransferases (PSPG box,
Fig. 1). It had sequence similarities of 44.2% with
betanidin-5-O-glucosyltransferase from Dorotheanthus
bellidiformis,20) 43.8% with flavonoid 7-O-glucosyl-
transferase from Scutellaria baicalensis,18) and 42.1%
with anthocyanin 30-O-glucosyltransferase from Genti-
ana triflora (Fig. 1).21)
The retention times of the reaction products from
apigenin and eriodictyol were 7.43 min and 6.43 min
respectively. The retention times were similar to those
of authentic compounds corresponding 7-O-glucoside.
In addition, UV spectra of the reaction products from
apigenin and eriodictyol were indistinguishable from
that of the corresponding authentic 7-O-glucoside
(Fig. 3B, D), indicating that AtGT-2 transferred a
glucose molecule to the 7-hydroxyl group of the two
flavonoids. Two more commercially available flavonoid
7-O-glucosides, luteolin 7-O-glucoside and naringenin
7-O-glucoside, were purchased and used to confirm the
glucosylation position of the reaction products. The
retention times and UV spectra of the two flavonoid 7-
AtGT-2 was expressed in the root, stem, flower, and
leaf tissue of Arabidopsis, although expression was

Flavonoid 7-O-Glycosyltransferase from Arabidopsis thaliana
1473
Fig. 1. Alignments of the Deduced Site Amino Acid Sequence among AtGT-2 and Several Other Plant UGTs.
above an amino acid indicates a conserved sequence among plant secondary product glucosyltransferase (PSPG box). At 2, AtGT-2
(AY090273.1); B5GT, betanidin-5-O-glucosyltransferase from Dorotheanthus bellidiformis (Y18871.1); UF7GT, flavonoid 7-O-glucosyltrans-
ferase from Scutellaria baicalensis (AB031274.1); UA30GT, anthocyanin 30-glucosyltransferase from Gentiana triflora (AB076697.1).
O-glucosides were similar to those from the correspond-
ing reaction products (data not shown). To further
confirm the glucosylation position, the reaction product
of eriodicytiol was subjected to NMR analysis. Eriodic-
tyol 7-O-glucoside is commercially available, and hence
ation is determined as the hydroxyl group of carbon 7.
Glucosylation positions of the kaempferol and querce-
tin reaction products were assigned based on the
hypsochromic shift data. The maximum absorbances of
kaempferol were 265.8 and 366.9 nm, and those of the
kaempferol reaction product were 265.7 and 366.0 nm.
The lack of a hypsochromic shift between substrate and
reaction product strongly suggested that glycosylation
occurred at the hydroxyl group of C-7. Likewise,
quercetin as well was likely to be glucosylated at the
1
the associated H NMR data was compared with those
of the AtGT-2 eriodicytiol reaction product. As shown
in Table 1, the chemical shifts of eriodictyol 7-O-
goucoside and the reaction product were identical.
Together, the data indicate that the position of glycosyl-

1474
J. H. KIM et al.
Table 1. Assignments of the 1H NMR Data of the Reaction Product
of Eriodictyol Produced by AtGT-2
M
1
2
kDa
OH
4'
116.3
97.4
O
O
O
1''
2
7
3'
HO
OH
HO
OH
66.3
55.4
OH
OH
O
Eriodictyol-7-O-glucoside
(authentic sample)
Position
Reaction product
36.5
2
3
5.44 (tt;3.4,12.6)
2.71 (m)
3.26 (m)
5.43 (m)
2.73 (m)
3.26 (m)
6.13 (t;2.2)
6.14 (t;2.2)
6.88 (s)
Fig. 2. Expression and Purification of AtGT-2 Recombinant Protein.
M, molecular weight marker; 1, induced E. coli lysate; 2, purified
recombinant AtGT-2.
6
6.13 (t;2.2)
6.14 (t;2.2)
6.88 (s)
8
20
500
600
1
6.75 (s)
6.75 (s)
6.75 (s)
6.75 (s)
4.97 (dd;7.5,9.1)
3.17 (m)
3.22 (m)
4.96 (dd;7.3,9.3)
3.17 (m)
3.22 (m)
3.11 (m)
3.38 (m)
3.40 (m)
3.67 (m)
200
300
400
500
3.11 (m)
3.38 (m)
mAU
A
1,000
3.40 (m)
3.67 (m)
S2
600
S1
750
500
250
0
hydroxyl group of C-7.
6
8
10
12
14
min
The relative conversion rates of a variety of flavonoid
substrates catalyzed by AtGT-2 were also determined.
Of the substrates analyzed, eriodictyol was identified as
the best substrate, followed by luteolin, kaempferol,
naringenin, and quercetin (Table 2). AtGT-2 preferred
flavanone to flavone or flavonol, both of which contain a
double bond in the C ring. In addition, the Km and Vmax
values of AtGT-2 for eriodictyol, kaempferol, and
quercetin were determined using Lineweaver-Burk plots
(Table 3). AtGT-2 had a lower Km value and higher
Vmax for eriodictyol than for kaempferol or quercetin.
These data are consistent with the relative conversion
rates for the three flavonoids.
A. thalina contains kaempferol and quercetin 7-O-
rhamnosides,15,23) and while there has been no report to
date that A. thaliana contains a flavonoid 7-O-glucoside,
our data indicate that the in vivo function of AtGT-2 is
indeed to transfer a rhamnose to kaempferol and/or
quercetin. We were unable to determine whether AtGT-
2 uses UDP-rhamnose, since the substrate is not
commercially available; however, even though AtGT-2
might prefer UDP-rhamnose to UDP-glucose, the
regioselectivity of AtGT-2, regardless of the sugar
donor, would not change.
mAU
300
150
100
50
1,000
S1
P1
P1
750
500
250
0
S3
0
300
350
400
250
Wave length (nm)
6
8
10
12
14
min
mAU
C
D
S5
S4
1,000
750
500
250
0
6
8
10
12
14
min
mAU
P2 300
1,000
S4
P2
150
100
50
750
500
250
0
S6
0
300
Wave length (nm)
350
400
250
6
8
10
12
14
min
Flavonoid 7-O-glucosyltransferases from Scutellaria
bacicalensis (UBGT), Glycyrrhiza echinata (UGT73-
F1), and Allium cepa (UGT73J1) have been also cloned
and characterized. UBGT showed regiospecificity for
baicalein, the major flavonoid found in S. bacicalen-
sis,18) despite an in vitro preference for kaempferol. The
substrate of UGT73F1, an isoflavonoid formonetin, was
Fig. 3. HPLC Chromatogram of AtGT-2 Reaction Products.
A, Authentic apigenin 7-O-glucoside (S1) and apigenin (S2). B,
Reaction product of apigenin (P1) with AtGT-2. S3 is not a
metabolized substrate (apigenin). C, Authentic eriodictyol 7-O-
glucoside (S4) and eriodictyol (S5). D, Reaction product of
eriodictyol (P2) with AtGT-2. S6 is not a metabolized substrate
(eriodictyol). Insets in B and D are comparisons of UV-spectra
between authentic compounds and reaction products.

Flavonoid 7-O-Glycosyltransferase from Arabidopsis thaliana
Table 2. Relative Substrate Preference of AtGT-2
1475
Substrate
Structural formula
Relative conversion rate (%)
OH
OH
HO
O
O
Eriodictyol
100
OH
OH
OH
Flavanone
OH
HO
O
O
Naringenin
71.6
29.6
OH
HO
O
O
Apigenin
Flavone
OH
OH
HO
O
O
Luteolin
92.1
78.9
64.1
OH
OH
HO
O
O
Kaempferol
OH
OH
Flavonol
OH
OH
HO
O
O
Quercetin
OH
OH
ꢂ40 mM of flavonoid was added.
Table 3. Substrate Specificities of the Purified AtGT-2 Recombinant
Protein
genistein, is capable of using quercetin 3-O-glucoside
to produce quercetin 3, 7-O-diglucoside,22) and while
genistein is not found in A. cepa, mono- or diglycosides
of quercetin are commonly found. Thus it is not
surprising that the in vitro substrate specificity of UGTs
can differ from the in vivo specificity. The substrate
discrepancy of flavonoid 7-O-glucosyltransfereases
might be due to the tagging system used to express it
in E. coli and a lack of posttranslational modification.
Differences of kinetic parameter and substrate between
the recombinant protein and the native protein were
found in flavonoid O-methyltransferase.25)
Vmax
Kcat=Km
(mMꢃ1/sꢃ1
)
Substrate
Km (mM)
Vmax=Km
(pkat/mg)
Eriodictyol
Kaempferol
Quercetin
18.0
26.3
35.3
333.3
333.3
250.0
18.5
12.7
7.1
2.66
1.32
0.41
Enzyme assays were carried out using purified AtGT-2 (9 to 23 mg),
substrate (10 to 100 mM), and UDP-glucose (500 mM). Reaction mixtures
were incubated at 37 ꢀC for 30 min.
the major flavonoid constituent of G. echinata, and
UGT73F1 has only minimal activity toward flavonoids
such as quercetin and kaempferol.24) Recombinant
UGT73J1, whose primary substrate is the isoflavon
A more effective method to examine the properties of
UGTs would be to purify the UGT of interest, but some
UGTs involved in secondary metabolism are known to
be unstable enzymes existing in plants in limited

1476
J. H. KIM et al.
200
160
Control
Product
Substrate
120
80
40
0
0 0.5 1
2
3
4
5
6
7
8
9
10
Time (h)
Fig. 4. Biotransformation of Eriodictyol Using E. coli Expressing AtGT-2.
quantities.26) While purification is often difficult and
time-consuming, the use of a heterologous expression
system to study the function of UGTs provides at least a
guide as to which metabolites should studied in further
in vivo analyses.
Last, R. L., Arabidopsis flavonoid mutants are hyper-
sensitive to UV-B irradiation. Plant Cell, 5, 171–179
(1993).
3) Dixon, R. A., and Paiva, N. L., Stress-induced phenyl-
propanoid metabolism. Plant Cell, 7, 1085–1097 (1997).
4) Hain, R., Reif, H. J., Krause, E., Langebartels, R., Kindl,
H., Vornam, B., Wiese, W., Schmelzer, E., Schreier, P.
H., Stocker, R. H., and Stenzel, K., Disease resistance
results from foreign phytoalexin expression in a novel
plant. Nature, 361, 153–156 (1993).
Biotransformation of eriodictyol using E. coli ex-
pressing AtGT-2
Regioselective glycosylation of flavonoids with whole
cells expressing UGT is also possible.27) Such a system
is capable of providing a diverse range of glucosides
using UGTs from various sources without the need for
addition of the expensive cofactor UDP-glucose, which
is endogenously available. In the present study, the
conversion of eriodictyol using the whole E. coli cells
expressing AtGT-2 was investigated. Two hundred mM
of eriodictyol was added to culture media containing
E. coli expressing AtGT-2. Analysis of the substrate and
product indicated that the concentration of eriodictyol
continued to decrease while the amount of reaction
product increased. All of the added eridictyol was
converted into eridicyol 7-O-glucoside within 9 h, while
E. coli containing the empty vector was unable to
metabolize eriodictyol (Fig. 4). Thus glycosylation of a
flavonoid using E. coli expressing UGT was achieved.
With a myriad of UGTs from several organisms, it might
be possible to randomize the glycosylation position of a
small chemical for pharmaceutical purposes.
5) Springob, K., Nakajima, J., Yamazaki, M., and Saito, K.,
Recent advances in the biosynthesis and accumulation of
anthocyanins. Nat. Prod. Rep., 20, 288–303 (2003).
6) Cos, P., De Bruyne, T., Hermans, N., Apers, S., Berghe,
D. V., and Vlietinck, A. J., Proanthocyanidins in health
care: current and new trends. Curr. Med. Chem., 11,
1345–1359 (2004).
7) Hou, D. X., Potential mechanisms of cancer chemo-
prevention by anthocyanins. Curr. Mol. Med., 3, 149–
159 (2003).
8) Harborne, J. B., Nature, distribution, and function of
plant flavonoids. Prog. Clin. Biol. Res., 213, 15–24
(1986).
9) Brugliera, F., Holton, T. A., Stevenson, T. W., Farcy, E.,
Lu, C., and Cornish, E. C., Isolation and characterization
of a cDNA clone corresponding to the Rt locus of
Petunia hybrida. Plant J., 5, 81–92 (1994).
10) Hu, Y., and Walker, S., Remarkable structural similar-
ities between diverse glycosyltransferases. Chem. Biol.,
9, 1287–1296 (2002).
11) Campbell, J. A., Davies, G. J., Bulone, V., and Henrissat,
B., A classification of nucleotide-diphospho-sugar gly-
cosyltransferases based on amino acid sequence sim-
ilarities. Biochem. J., 326, 929–939 (1997).
Acknowledgments
This work was supported from a grant from the
Biogreen 21 Program, Rural Development Admini-
stration, Republic of Korea, R01-2004-000-10688-0
(KOSEF), KRF2004-F00019 (KRF), and the second
Brain Korea 21 (Korea Minister of Education).
12) Mackenzie, P. I., Owens, I. S., Burchell, B., Bock, K.
W., Bairoch, A., Belanger, A., Fournel-Gigleux, S.,
Green, M., Hum, D. W., Iyanagi, T., Lancet, D., Louisor,
P., Magdalow, J., Chowdhury, J. R., Ritter, J. K.,
Schachter, H., Tephly, T. R., Tipton, K. F., and Nebert,
D. W., The UDP glycosyltransferase gene superfamily:
recommended nomenclature update based on evolution-
ary divergence. Pharmacogenetics, 7, 255–269 (1997).
13) Paquette, S., Møller, B. L., and Bak, S., On the origin of
family 1 plant glycosyltransferases. Phytochemistry, 62,
399–413 (2003).
References
1) Bieza, K., and Lois, R., An Arabidopsis mutant tolerant
to lethal ultraviolet-B levels shows constitutively ele-
vated accumulation of flavonoids and other phenolics.
Plant Physiol., 126, 1105–1115 (2001).
14) Summer, L. W., Mendes, P., and Dixon, R. A., Plant
metabolomics: large-scale phytochemistry in the func-
tional genomics era. Phytochem., 62, 817–836 (2003).
2) Li, J., Ou-Lee, T. M., Raba, R., Amundson, R. G., and

Flavonoid 7-O-Glycosyltransferase from Arabidopsis thaliana
1477
15) Jones, P., Messner, B., Nakajima, J.-I., Schaffner, A. R.,
¨
nin biosynthesis from gentia. Plant Physiol., 132, 1652–
1663 (2003).
and Saito, K., UGT73C6 and UGT78D1, glycosyltrans-
ferases involved in flavonol glycoside biosynthesis in
Arabidopsis thaliana. J. Biol. Chem., 278, 43910–43918
(2003).
22) Kramer, C. M., Prata, R. T. N., Willits, M. G., De Luca,
V., Steffens, J. C., and Graser, G., Cloning and
regiospecificity studies of two flavonoid glucosyltrans-
ferases from Allium cepa. Phytochemistry, 64, 1069–
1076 (2003).
23) Tohge, P., Nishiyama, Y., Hirai, M. Y., Yano, M.,
Nakajima, J., Awazuhara, M., Inoue, E., Takahashi, H.,
Goodenowe, D. B., Kitayama, M., Noji, M., Yamazake,
M., and Sait, K., Functional genomics by integrated
analysis of metabolome and transcriptome of Arabidop-
sis plants over-expressing an MYB transcription factor.
Plant J., 42, 218–235 (2005).
24) Nagashima, S., Inagaki, R., Kubo, A., Hirotani, M., and
Yoshikawa, T., cDNA cloning and expression of
isoflavonoid-specific glucosyltransferase from Glycyr-
rhiza echinata cell-suspension cultures. Planta, 218,
456–459 (2004).
25) Ibdah, M., Zhang, X.-H., Schnot, J., and Vogt, T., A
novel Mg2þ-dependent O-methyltransferase in the phen-
ylpropanoid metabolism of Mesembryanthemum crystal-
linum. J. Biol. Chem., 278, 43961–43972 (2003).
26) Vogt, T., and Jones, P., Glycosyltransferases in plant
natural product synthesis: characterization of a super-
gene family. Trends Plant Sci., 5, 380–386 (2000).
27) Lim, E.-K., Ashford, D. A., Hou, B., Jackson, R. G., and
Bowles, D. J., Arabidopsis glycosyltransferases as
biocatalysts in fermentation for regioselective synthesis
of diverse quercetin glucosides. Biotechnol. Bioeng., 87,
623–631 (2004).
16) Kim, D. H., Kim, B. G., Lee, Y., Ryu, J. Y., Lim, Y.,
Hur, H.-G., and Ahn, J.-H., Regiospecific methylation of
naringenin to ponciretin by soybean O-methyltransferase
expressed in Escherichia coli. J. Biotechnol., 19, 155–
162 (2005).
17) Yoon, Y., Yi, Y. S., Lee, Y., Kim, S., Kim, B. G., Ahn,
J.-H., and Lim, Y., Characterization of O-methyltrans-
ferase ScOMT1 cloned from Streptomyces coelicolor
A3(2). Biochim. Biophys. Acta, 1730, 85–95 (2005).
18) Hirotani, M., Kuroda, R., Suzuki, H., and Yoshikawa, T.,
Cloning and expression of UDP-glucose:flavonoid 7-O-
glucosyltransferase from hairy root cultures of Scutel-
laria baicalensis. Planta, 210, 1006–1013 (2000).
19) Ross, J., Li, Y., Lim, E.-K., and Bowles, D., Higher plant
glycosyltransferases. Genome Biol., 2, 3004.1–3004.6
(2001).
20) Vogt, T., Zimmermann, E., Grimm, R., Meyer, M., and
Stack, D., Are the characteristics of betanidin glucosyl-
transferases from cell-suspension cultures of Dorothean-
thus bellidiformis indicative of their phylogenetic rela-
tionship with flavonoid glucosyltransferases? Planta,
203, 349–361 (1997).
21) Fukuchi-Mizutani, M., Okuhara, H., Fukui, Y., Nakao,
M., Katsumoto, Y., Yonekura-Sakakibara, K., Kusumi,
T., Hase, T., and Tanaka, Y., Biochemical and molecular
characterization of a novel UDP-glucose:anthocyanin 30-
O-glucosyltransferase, a key enzyme for blue anthocya-