Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana

Article abstract of DOI:10.1093/jb/mvt102

The discovery of sulfated flavonoids in plants suggests that sulfation may play a regulatory role in the physiological functions of flavonoids. Sulfation of flavonoids is mediated by cytosolic sulfotransferases (SULTs), which utilize 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfate donor. A novel SULT from Arabidopsis thaliana, designated AtSULT202B7 (AGI code: At1g13420), was cloned and expressed in Escherichia coli. Using various compounds as potential substrates, we demonstrated, for the first time, that AtSULT202B7 displayed sulfating activity specific for flavonoids. Intriguingly, the recombinant enzyme preferred flavonoid glycosides (e.g. kaempferol-3- glucoside and quercetin-3-glucoside) rather than their aglycone counterparts. Among a series of hydroxyflavones tested, AtSULT202B7 showed the enzymatic activity only for 7-hydroxyflavone. pH-dependency study showed that the optimum pH was relatively low (pH 5.5) compared with those (pH 6.0-8.5) previously reported for other isoforms. Based on the comparison of high performance (pressure) liquid chromatography (HPLC) retention times between sulfated kaempferol and the deglycosylated product of sulfated kaempferol-3-glucoside, the sulfation site in sulfated kaempferol-3-glucoside appeared to be the hydroxyl group of the flavonoid skeleton. In addition, by using direct infusion mass spectrometry, it was found that the sulfated product had one sulfonate group within the molecule. These results indicated that AtSULT202B7 functions as a flavonoid glycoside 7-sulfotransferase.

Full text of DOI:10.1093/jb/mvt102

J. Biochem. 2014;155(2):91–97 doi:10.1093/jb/mvt102
Identification of a novel flavonoid glycoside sulfotransferase
in Arabidopsis thaliana
Received September 5, 2013; accepted October 19, 2013; published online November 6, 2013
Takuyu Hashiguchi1,2, Yoichi Sakakibara1,2,*,
PCR, polymerase chain reaction; TLC, thin-layer
chromatography; SDS—PAGE, sodium dodecyl
sulphate—polyacrylamide gel electrophoresis; SULT,
cytosolic sulfotransferase.
Takehiko Shimohira1,2, Katsuhisa Kurogi1,2
,
Masao Yamasaki1,2, Kazuo Nishiyama1,2
Ryo Akashi2, Ming-Cheh Liu3 and
Masahito Suiko1,2
,
1Department of Biochemistry and Applied Biosciences;
2Interdisciplinary Graduate School of Agriculture and Engineering,
University of Miyazaki, Miyazaki 889-2192, Japan; and
3Department of Pharmacology, College of Pharmacy and
Pharmaceutical Sciences, The University of Toledo, OH 43614, USA
Flavonoids are a group of secondary metabolites dis-
tributed in a wide range of plant species. They share a
common phenyl benzopyrone structure and are
divided into major subclasses (e.g. flavanone, flavonol,
flavone, isoflavone and anthocyanidin) based on the
numbers and positions of the hydroxyl group and the
C-ring structure. Flavonoids have been implicated in a
variety of physiological functions, e.g. provision of
colors attractive to pollinators (1); protection of the
plant body from external stress such as fungal infection
and UV irradiation (2); communication with the sym-
biont Rhizobia (3); and influence in the transport of the
plant hormone, auxin (4). To date, nearly 9000 struc-
tural variants of flavonoids have been reported (5). In
different plants, flavonoids occur as glycosides (e.g.
glucoside, galactoside, rhamnoside and arabinoside),
which, except for flavanols such as catechins and
proanthocyanidins, are generated upon glycosylation
by uridine-diphosphate glucose glycosyltransferases
(6). Glycosylation increases the solubility and the sta-
bility of flavonoids, and reduces their reactivity (7). In
addition to glycosylation, flavonoids are known to
undergo sulfation conjugation in at least 32 families
of plants (8). Sulfation conjugation has been exten-
sively studied in humans, but not in plants. Sulfation
is catalyzed by the so-called cytosolic sulfotransferases
(SULTs). The universal sulfate donor for
SULT-mediated sulfation is 30-phosphoadenosine
50-phosphosulfate (PAPS). During the sulfation reac-
tion, the sulfonate group (SOꢀ3 ) is transferred from
PAPS to an acceptor hydroxyl or amino group of the
substrate compounds with the concomitant formation
of 30-phosphoadenosine 50-phosphate (PAP). In
humans, SULT-mediated sulfation is one of the
major phase II conjugation reactions, which plays an
important role in the metabolism and regulation of key
endogenous compounds and the detoxification of
xenobiotics (9). Interestingly, SULT homologs have
been shown to be present in a variety of plant species
as indicated by a large number of plant SULT protein
sequences deposited in different databases. However,
their physiological functions have remained poorly
characterized. In Arabidopsis thaliana, a popular
*Yoichi Sakakibara, Department of Biochemistry and Applied
Biosciences, University of Miyazaki, 1-1, Gakuenkibanadai-Nishi,
Miyazaki 889-2192, Japan. Tel/Fax: þ81-985-58-7211,
email: ysakaki@cc.miyazaki-u.ac.jp
The discovery of sulfated flavonoids in plants suggests
that sulfation may play a regulatory role in the physio-
logical functions of flavonoids. Sulfation of flavonoids is
mediated by cytosolic sulfotransferases (SULTs), which
utilize 30-phosphoadenosine 50-phosphosulfate (PAPS)
as the sulfate donor. A novel SULT from Arabidopsis
thaliana, designated AtSULT202B7 (AGI code:
At1g13420), was cloned and expressed in Escherichia
coli. Using various compounds as potential sub-
strates, we demonstrated, for the first time, that
AtSULT202B7 displayed sulfating activity specific for
flavonoids. Intriguingly, the recombinant enzyme pre-
ferred flavonoid glycosides (e.g. kaempferol-3-glucoside
and quercetin-3-glucoside) rather than their aglycone
counterparts. Among
a series of hydroxyflavones
tested, AtSULT202B7 showed the enzymatic activity
only for 7-hydroxyflavone. pH-dependency study
showed that the optimum pH was relatively low (pH
5.5) compared with those (pH 6.0—8.5) previously re-
ported for other isoforms. Based on the comparison of
high performance (pressure) liquid chromatography
(HPLC) retention times between sulfated kaempferol
and the deglycosylated product of sulfated kaemp-
ferol-3-glucoside, the sulfation site in sulfated kaemp-
ferol-3-glucoside appeared to be the hydroxyl group of
the flavonoid skeleton. In addition, by using direct in-
fusion mass spectrometry, it was found that the sulfated
product had one sulfonate group within the molecule.
These results indicated that AtSULT202B7 functions
as a flavonoid glycoside 7-sulfotransferase.
Keywords: Arabidopsis thaliana/AtSULT202B7/
flavonoid glycoside/sulfation/sulfotransferase.
Abbreviations: HPLC, high performance (pressure)
liquid chromatography; IPTG, isopropyl b-D-thioga-
lactopyranoside; PAP, 30-phosphoadenosine 50-phos-
phate; PAPS, 30-phosphoadenosine 50-phosphosulfate;
ß The Authors 2013. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
91

T. Hashiguchi et al.
digestion buffer (50 mM Tris—HCl, pH 8.0, 150 mM NaCl and
2.5 mM CaCl2) containing 5 units/mL bovine thrombin. Following
a 2-h incubation period at 4ꢁC with constant agitation, the prepar-
ation was subjected to centrifugation, and the supernatant contain-
ing purified recombinant SULT was collected and used in the
enzymatic assay.
model plant species, there are 17 SULT genes (10). To
date, only a few A. thaliana SULT isoforms have been
isolated and biochemically characterized.
In this communication, we report the identification
and characterization of a novel A. thaliana sulfotrans-
ferase, designated AtSULT202B7 (AGI code:
At1g13420). The enzymatic activity of recombinant
AtSULT202B7 toward a major flavonol and its glyco-
sides was tested. A systematic analysis of the optimum
pH and kinetics parameters toward flavonols and their
glycosides was performed. To the best of our know-
ledge, this is the first report on a sulfotransferase cap-
able of catalyzing the sulfation of flavonoid glycosides
in A. thaliana.
Enzymatic assay
Sulfating activity of AtSULT202B7 was assayed using 35S-PAPS as
the sulfate donor. The standard assay mixture, with a final volume of
25 mL, contained 50 mM sodium phosphate buffer, pH 7.5, 0.2 mM
35S-PAPS (45 Ci/mmol) and 100 mM substrate. The reaction was
started by the addition of the enzyme, allowed to proceed for
20 min at 22ꢁC, and terminated by heating at 98ꢁC for 3 min. The
precipitates formed were removed by centrifugation, and the super-
natant was subjected to the analysis of the 35S-sulfated product using
a previously developed TLC separation procedure (11) with n-buta-
nol/isopropanol/formic acid/water (3:1:1:1 by volume) or n-butanol/
acetic acid/water (3:1:1 by volume) as the solvent system.
Afterwards, the plate was air-dried and analyzed using a Fluoro
Image Analyzer FLA-3000 (Fujifilm). To examine the pH-depend-
ence, different buffers (50 mM sodium acetate buffer at pH 4.0—6.0
and 50 mM sodium phosphate buffer at 6.5—8.0) were used in the
reaction mixtures. For the kinetic studies on the sulfation of flavon-
oids, substrates with different concentrations ranging from 0.01
to 40 mM were used. Data obtained were processed using the Excel
program to generate the best fitting trendline for the
Lineweaver—Burk plots. No enzyme inhibition was observed over
the range of the PAPS and flavonoids concentration used.
Materials and Methods
Materials
Kaempferol, quercetin and glucose were purchased from Wako Pure
Chemical Industries. Quercetin-3-glucoside, 5-hydroxyflavone, arbu-
tin, salicin and 3-hydroxyflavone were products of Sigma-Aldrich
Co. LLC. Quercetin-3-galactoside, quercetin-3-rutinoside, kaemp-
ferol-3-glucoside, kaempferol-7-glucoside and kaempferol-3-robino-
side-7-rhamnoside were from Extrasynthese. 7-Hydroxyflavone was
purchased from Tokyo Kasei Kogyo Co., Ltd. Quercetin-7-gluco-
side was a product of Apin Chemical Ltd. Quercetin-3-rhamnoside,
30-hydroxyflavone and 40-hydroxyflavone were obtained from
Indofine Chemical. pBluescript II SK (þ) vector, XL1-Blue MRF’
and BL21 Escherichia coli host strain were obtained from
Stratagene. pGEX-4 T-1 prokaryotic GST fusion vectors and gluta-
thione sepharose 4B were from GE Healthcare Biosciences. Cellulose
thin-layer chromatography (TLC) plates were products of Merck.
All other chemicals were of the highest grade commercially available.
Identification of sulfated kaempferol-3-glucoside by
reverse phase high performance (pressure) liquid
chromatography followed by direct infusion mass
spectrometry
The sulfation reaction was performed in a reaction mixture (with a
final volume of 250 ml) containing 50 mM sodium acetate buffer, a
pH of 5.5, 100 mM PAPS, 20 mM kaempferol or kaempferol-3-gluco-
side and 25 mg of purified AtSULT202B7. The reaction was started
by the addition of the enzyme, allowed to proceed for 12 h at 22ꢁC,
and terminated by heating at 98ꢁC for 5 min. The precipitates
formed were removed by centrifugation, and the supernatant was
subjected to the analysis using a Shimadzu Prominence HPLC
system consisting of a photodiode array detector and the LC
Solution software was used for all high performance (pressure)
liquid chromatography (HPLC) analysis. 5 mm Capcell PAK C18
MG column (250ꢃ4.6 mm; SHISEIDO), and a gradient elution of
acetonitrile and ultrapure water were used for the HPLC separ-
ations. For all analyses, the injection volume was 40 mL; the flow
rate was 1 mL/min; and the controlled oven temperature was 40ꢁC.
Fraction containing sulfated kaempferol-3-glucoside was lyophilized
and dissolved in dimethylsulfoxide. Deglucosylation reaction was
conducted in a reaction mixture (with a final volume of 250 ml) con-
taining 100 mM sodium acetate buffer, pH 5.0, 8 units/mL of
b-glucosidase from sweet almond and 10 mL of the redissolved sul-
fated kaempferol-3-glucoside. The reaction was started by the add-
ition of the enzyme, allowed to proceed for 24 h at 37ꢁC, and
terminated by heating at 98ꢁC for 5 min. The precipitates formed
were removed by centrifugation, and the supernatant was subjected
to the earlier described HPLC analysis. The sulfated kaempferol-3-
glucoside dissolved in methanol was analyzed by Q Exactive hybrid
quadrupole-Orbitrap mass spectrometer (Thermo Fisher scientific)
with a heated electrospray ionization source through direct infusion
using a syringe pump. These data were acquired using Targeted-MS2
scan event. Typical mass spectrometric conditions were as follows:
polarity, negative ionization mode; spray voltage, 3.5 kV; sheath gas
flow rate, 6; auxillary gas, 0; sweep gas, 0; heated capillary tempera-
ture, 320ꢁC. The resolution was set at 140,000. The AGC target was
2E5. The maximum ion injection time was 100 ms. The normalized
collision energy was 20%. The raw data files were analyzed using
Qual Browser software in Xcalibur (Thermo fisher scientific).
Molecular cloning of AtSULT202B7
Arabidopsis thaliana ecotypes Col-0 were grown with a 16-h photo-
period at 22ꢁC with 50—60% humidity. Two-week-old Arabidopsis
seedlings were frozen in liquid nitrogen and homogenized in a
TRIzol RNA Isolation Reagent (Life Technology), according to
the manufacturer’s instructions. With 1 mg of the isolated total
RNA as the template and oligo (dT) as the primer, first-strand
cDNA was synthesized using the First-Strand cDNA Synthesis Kit
(TOYOBO). Polymerase chain reaction (PCR) was carried out in a
20 ml reaction mixture using AtSULT202B7-sense (50-CGCGGATC
CATGGGTGAGAAAGATATTCCA-30) and AtSULT202B7-anti-
sense (50-CGGAATTCCTACAATTTCAAACCAGAGCCT-30) pri-
mers under the action of KOD-Plus-Neo DNA polymerase
(TOYOBO). The PCR conditions were 94ꢁC for 2 min, followed
by 35 cycles of 10 s at 98ꢁC, 30 s at 55ꢁC, 40 s at 68ꢁC and a final
incubation at 68ꢁC for 7 min. The amplified product was restricted
using BamHI and EcoRI (TOYOBO), subcloned into pBluescript II
SK (þ), and transformed into E. coli XL1-Blue MRF’. To verify its
authenticity, the cDNA insert was subjected to nucleotide sequen-
cing. Upon verification, the insert was subcloned into pGEX-4 T-1
prokaryotic expression vector.
Bacterial expression and purification of recombinant
AtSULT202B7
pGEX-4 T-1 harbouring the cloned AtSULT202B7 cDNA
(GenBank ID: NP_172799) was transformed into competent
E. coli BL21 cells. Transformed BL21 cells were grown to
OD600nm ¼ ꢂ0.3 in 100 mL LB medium supplemented with 100 mg/
mL ampicillin, and induced with 0.1 mM isopropyl b-D-thiogalacto-
pyranoside (IPTG). After a 12-h induction at 24ꢁC, the cells were
collected by centrifugation and homogenized in 15 mL of ice-cold
lysis buffer (50 mM Tris—HCl, pH 8.0, 150 mM NaCl and 1 mM
ethylenediaminetetraacetic acid) using
a French Press (Ohtake
Miscellaneous methods
Works Co. Ltd.). The crude homogenate was subjected to centrifu-
gation at 20,400ꢃg for 15 min at 4ꢁC. The supernatant collected was
fractionated using 0.5 mL of glutathione Sepharose 4B, and the
bound GST fusion protein was treated with 0.2 mL of a thrombin
To prepare the solutions for use in the enzymatic assay, all substrate
compounds were dissolved in dimethylsulfoxide. 35S-PAPS (45 Ci/
mmol) was synthesized from ATP and 35S-sulfate using recombinant
92

Flavonoid glycoside sulfation by AtSULT202B7
human bifunctional ATP sulfurylase/adenosine 50-phosphosulfate
kinase, as previously described (12). Sodium dodecyl sulphate—
polyacrylamide gel electrophoresis (SDS—PAGE) was performed
on 10% polyacrylamidegels using the method of Laemmli (13).
Protein determination was performed based on Lowry’s method,
with bovine serum albumin as the standard (14).
(http://web.expasy.org/protparam/) and was deter-
mined to have an expected molecular mass of
37.7 kDa. In line with the predicted molecular mass,
purified AtSULT202B5, upon SDS—PAGE, migrated
at 40.7 kDa position (data not shown).
Results and Discussion
Determination of pH optimum
In higher plants, flavonoids commonly exist as glyco-
side such as glucosides (Fig. 3). In an initial experi-
ment, kaempferol-3-glucoside and quercetin-3-
glucoside were tested as substrates for AtSULT202B7
at pH 7.5. AtSULT202B7 showed strong activities
(138.6 and 80.9 pmol/min/mg enzyme, respectively)
toward kaempferol-3-glucoside and quercetin-3-gluco-
side. Next, the pH dependence of the activity of
AtSULT202B7 was determined using two substrates,
Cloning of AtSULT202B7
For the A. thaliana genome, the At1g13420 (AGI code)
gene was named AtSULT202B7 according to a previ-
ously reported nomenclature system (15, 16). The
deduced amino acid sequence of AtSULT202B7 was
aligned with AtSULT202B1, a flavonol-7 sulfotrans-
ferase (17), and AtSULT202B5, an uncharacterized
sulfotransferase, using the Clustal W program. Box
shade software (http://www.ch.embnet.org/software/
BOX_form.html) was used to show identical residues
in the alignment (Fig. 1). The four highly conserved
regions containing the PAPS binding and catalytic
sites (18—20) were identified in the AtSULT202B7
sequence. Using GENETYX-MAC version 11.1.0,
AtSULT202B7 was shown to display 66.6% and
1 2 3 4 5 6
79 kDa
73.0%
amino
acid
sequence
identities
to
AtSULT202B1 and AtSULT202B5, respectively. The
open reading frames of AtSULT202B7 sequence was
PCR-amplified using gene-specific primers (cf.
Materials and Methods). The PCR product was
cloned into the pBluescript II SK (þ) vector and
sequenced. Sequence data obtained completely
matched with that shown for the GenBank accession
number NP_172799 (At1g13420). The coding sequence
of AtSULT202B7 was subsequently subcloned into
pGEX-4T-1, a prokaryotic expression vector, for the
expression of recombinant enzymes in E. coli. As
shown in Fig. 2, a GST fusion protein (ꢂ64 kDa)
was expressed upon induction with IPTG, and recom-
binant AtSULT202B7 was purified to near homogen-
eity. The molecular weight of AtSULT202B7 was
calculated using the online program Protparam
42 kDa
30 kDa
Fig. 2 SDS gel electrophoretic pattern of recombinant
AtSULT202B7 at different stages during purification. Samples were
subjected to SDS—PAGE, followed by Coomassie blue staining.
Lane 1, molecular weight markers; lane 2, BL21 Escherichia coli
homogenate prior to IPTG induction; lane 3, BL21 E. coli
homogenate after IPTG induction; lane 4, cytosolic fraction of
IPTG-induced BL21 E. coli homogenate; lane 5, AtSULT202B7-
GST-fusion protein before thrombin cleavage; lane 6, purified
AtSULT202B7 after treatment of thrombin.
AtSULT202B7
AtSULT202B5
AtSULT202B1
1 MGEKDIPRNLKEEEEEEEENQSEETKSLISSLPSDIDCSGTKLYKYQGCWYDKDILQAILNFNKNFQPQETDIIVASFPKSGTTWLKALTFALA
1 MDEKDILRNLREEEEEEEENQSEETKILISSLPWEIDYLGNKLFKYQGYWYYEDVLQSIPNIHSSFQPQETDIVVASFYKSGTTWLKALTFALV
1 -----------MEMNLRIEDLNEETKTLISSLPSDKDFTGKTICKYQGCWYTHNVLQAVLNFQKSFKPQDTDIIVASFPKCGTTWLKALTFALL
5’-PSB
NAAC
AtSULT202B7
AtSULT202B5
AtSULT202B1
95 QRSKHTSDN--HPLLTHNPHELVPYLELDLYLKSSKPDLTKLPSSS-----PRLFSTHMSFDALKVPLKESPCKIVYVCRNVKDVLVSLWCFEN
95 QRSKHSLEDHHHPLLSHNPHEIVPYLELDLYLNSSKPDLTKFLSSSSSSSSPRLFSTHMSLDALKLPLKKSPCKVVYVCRNVKDVLVSLWCFLN
84 HRSKQPSHDDDHPLLSNNPHVLVPYFEIDLYLRSENPDLTKFSSSP------RLFSTHVPSHTLQEGLKGSTCKIVYISRNVKDTLVSYWHFFT
3’-PB
AtSULT202B7 182 SMS--------------GENNLSLEALFESLCSGVNLCGPLWENVLGYWRGSLEDPKHVLFLRYEELKTEPRVQIKRLAEFLDCPFTKEEEDSG
AtSULT202B5 189 ANKGVEWGDFSQNEKIIRAENYSFKAIFESFCNGVTLHGPFWDHAQSYWRGSLEDPKHFLFMRYEELKAEPRTQVKRLAEFLDCPFTKEEEDSG
AtSULT202B1 172 KKQT------------DEKIISSFEDTFEMFCRGVSIFGPFWDHVLSYWRGSLEDPNHVLFMKFEEMKAEPRDQIKKFAEFLGCPFTKEEEESG
AtSULT202B7 262 GVDKILELCSLRNLSGLEINKTGSLSEGVSFKSFFRKGEVGDWKSYMTPEMENKIDMIVEEKLQGSGLKL
AtSULT202B5 283 TVDKILELCSLSNLSSLEINKTGSLGG-VDYKTYFRKGQVGDWKSYMTSEMVNKIDMIVEEKLKGSGLKF
AtSULT202B1 254 SVDEIIDLCSLRNLSSLEINKTGKLNSGRENKMFFRKGEVGDWKNYLTPEMENKIDMIIQEKLQNSGLKF
P-loop related motif
Fig. 1 Amino acid sequence comparison of AtSULT202B7, AtSULT202B5 and AtSULT202B1. Identical residues conserved among at least 2 of
the 3 enzymes are indicated by black background, and similar residues are indicated in grey. The NAAC (the N-terminal acidic amino acid
cluster), which might act as a potencial sorting determinant, is underlined. The 50-PSB loop (including the conserved lysine residue in the
N terminal), which interacts with the 50-phosphate of PAP, and the 30-PB motif, for the binding of the 30-phosphate of PAP, are underlined.
The conserved P-loop related motif (GXXGXXK) is also underlined. The catalytic histidine residue, which is highly conserved in almost all
known SULTs, is indicated by an arrow.
93

T. Hashiguchi et al.
kaempferol-3-glucoside and quercetin-3-glucoside.
AtSULT202B7 displayed the highest sulfating activity
at pH 5.5 for these two substrates, and the activity
started decreasing at higher pHs (Fig. 4). Previous stu-
dies have shown that flavonol sulfotransferases derived
from Flaveria species displayed optimum pH ranging
6.0—8.5 (21). Considering that the vacuolar pH of
plant cells is about 5.0—5.5 (22) and that the flavonol
glycosides accumulates mainly in the vacuole (23), it is
possible that AtSULT202B7 might somehow be trans-
ported into this acidic organelle and sulfate the flavo-
nol glycosides locally as substrates. No reported
vacuole sorting determinant, however, could be dis-
cerned in the amino acid sequence of AtSULT202B7.
Using the computer program PSORT (http://psort.
hgc.jp/), AtSULT202B7 has been reported as a cyto-
plasmic protein (10), while its exact subcellular loca-
tion still remains unknown. Further examination of
the amino acid sequence of AtSULT202B7 revealed
an acidic amino acid cluster located in its N-terminal
region of AtSULT202B7 (Fig. 1). It may be worth-
while mentioning that previous studies have reported
that acidic amino acid cluster might serve as a
basolateral sorting signal in MDCK cell (24). The N-
terminal acidic amino acid cluster in AtSULT202B7,
therefore, could be proposed to function as a novel
sorting determinant for a certain organelle in the
cells of A. thaliana. To this end, immunocytochemical
analysis using specific antibody against AtSULT202B7
will be required to unequivocally identify the subcellu-
lar location of AtSULT202B7.
Substrate specificity of AtSULT202B7 for a variety of
flavonoids and related compounds
Under optimum pH conditions, the enzymatic activity
of AtSULT202B7 toward a variety of flavonoids
and related compounds were examined. As shown in
Table I, AtSULT202B7 displayed considerably higher
activity toward a wide range of flavonol glycosides
(e.g. quercetin-3-glucoside, quercetin-3-galactoside,
quercetin-3-rutinoside, quercetin-3-rhamnoside and
kaempferol-3-glucoside) than their aglycone counter-
parts. These results suggested that AtSULT202B7
has no strict sugar selectivity in the sulfation of flavon-
oid glycosides. Interestingly, no activity was detected
with flavonols having a sugar at position 7 (e.g.
quercetin-7-glucoside, kaempferol-7-glucoside and
3′
4′
B
kaempferol-3-robinoside-7-rhamnoside),
suggesting
that AtSULT202B7 may sulfate the 7-hydroxyl
group of flavonols in a position-specific manner. This
possibility was confirmed in a subsequent experiment
using five kinds of hydroxyflavones that contain only
one hydroxyl group at 3, 5, 7, 30 and 40 as substrates.
Of the five, only 7-hydroxyflavone was sulfated. These
results supported the notion that AtSULT202B7 was
active toward only flavonoids with a hydroxyl group at
position 7. Moreover, no sulfation products of glucose
and phenolic plant glucosides, such as arbutin and sali-
cin, were detected. Collectively, these results suggested
7
A
5
C
3
Compound
C3
C5
C7
C3′
H
OH
H
OH
H
H
C4′
OH
OH
OH
OH
H
Kaempferol-3-glucoside
Quercetin-3-glucoside
Kaempferol
Quercetin
3-Hydroxyflavone
7-Hydroxyflavone
Glucose
Glucose
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
H
H
H
OH
H
Fig. 3 A chemical structure of some representative flavonoids used
as substrates for this study.
Table I. Specific activities of AtSULT202B7 with various flavonoids
as substrates.
Specific activity
(pmol/min/mg)
Substrate compound
180
Kaempferol
63.3 ꢄ 3.4
343.8 ꢄ 8.5
N.D.
160
Kaempferol-3-glucoside
Kaempferol-7-glucoside
Kaempfrol-3-robinoside-7-rhamnoside
Quercetin
Kaempferol-3-glucoside
140
N.D.
47.8 ꢄ 3.2
311.7 ꢄ 4.5
N.D.
235.0 ꢄ 3.6
183.1 ꢄ 4.5
153.8 ꢄ 3.6
N.D.
Quercetin-3-glucoside
120
Quercetin-3-glucoside
Quercetin-7-glucoside
Quercetin-3-galactoside
Quercetin-3-rutinoside
Quercetin-3-rhamnoside
3-Hydroxyflavone
5-Hydroxyflavone
30-Hydroxyflavone
40-Hydroxyflavone
7-Hydroxyflavone
Salicin
100
80
60
40
20
0
N.D.
N.D.
N.D.
16.9 ꢄ 1.5
N.D.
N.D.
Arbutin
Glucose
UDP-glucose
3
4
5
6
7
8
9
N.D.
N.D.
pH
Fig. 4 pH dependency of the sulfating activity of AtSULT202B7 with
flavonols and flavonol glucosides as substrates. The enzymatic assays
with 10 mM of substrate compounds were carried out under standard
assay conditions using different buffer systems.
Specific activity refers to pmol of sulfated product formed/min/mg
enzyme. Data shown represent means ꢄ S.D. from three determin-
ations. N.D. refers to activity not detected (55.0 pmol/min/mg).
94

Flavonoid glycoside sulfation by AtSULT202B7
that sulfation by AtSULT202B7 requires the presence
of a flavone backbone in substrate compounds. It is
interesting to note that despite the high amino acid
sequence similarity between AtSULT202B5 and
AtSULT202B7, AtSULT202B5 showed no activity
toward a variety of flavonoids, including its glycosides.
Other phenolic and steroidal compounds, such as
phytohormones and brassinosteroids, were also tested
as substrates for AtSULT202B7. No activity, however,
was detected.
The Km value for kaempferol-3-glucoside was 14.4 mM,
and the Vmax was 592.1 pmol/min/mg of enzyme.
Based on these results, the Vmax/Km for kaempferol-
3-glucoside was about five times higher than that for
its aglycone counterpart. Similar results were also
found with quercetin and its glucoside as substrates.
These findings suggested that AtSULT202B7 sulfates
flavonol-3-glucosides more efficiently than flavonol
aglycones. The Km values for PAPS, determined
with fixed concentration of kaempferol-3-glucoside,
was 0.4 mM. which is within the range of Km values
(0.2—0.4 mM) previously reported for other flavonoid
sulfotransferases (21).
Determination of kinetic parameters for
AtSULT202B7-mediated sulfation of flavonols and
flavonol-3-glucosides
Kinetic parameters (Vmax, Km and Vmax/Km) for the
sulfation of kaempferol, kaempferol-3-glucoside, quer-
cetin, or quercetin-3-glucoside by AtSULT202B7 were
determined. The results compiled in Table II indicate
that the apparent Km for kaempferol was 27.6 mM, and
that the Vmax value was 209.5 pmol/min/mg of enzyme.
HPLC detection and mass spectrometry of sulfated
kaempferol 3-glucoside
To further determine the structure of sulfated flavonol
glucoside, deglucosylated products of sulfated kaemp-
ferol-3-glucoside, generated upon treatment with
b-glucosidase, were subjected to HPLC analysis. The
retention time and the wavelength of maximum
absorption (lmax) of the deglucosylated products were
found to be almost identical to those of sulfated
kaempferol (Fig. 5). These results suggested that
AtSULT202B7 sulfates the hydroxyl group of the fla-
vone backbone. To date, 3-sulfatoglucoside and
3-sulfatorhamnoside of flavonols have been detected
in some plant species (8). To clarify whether the glu-
cose moiety of kaempferol-3-glucoside can indeed be
sulfated by AtSULT202B7, we performed mass spec-
trometry of sulfated kaempferol-3-glucoside in nega-
tive ion mode. The parent ion, the highest relative
abundance of ion in full scan mode was at m/z
527.05. As shown in Fig. 6, further selected ion moni-
toring of the parent ion and the following high-energy
Table II. Kinetic constants of the sulfation of flavonols and flavonol
glucosides by AtSULT202B7.
Vmax
(pmol/min/mg)
Km (6M)
Vmax/Km
Kaempferol
Kaempferol-3-glucoside
Quercetin
Quercetin-3-glucoside
PAPS
27.6 ꢄ 2.7
14.4 ꢄ 2.7
42.1 ꢄ 8.1
18.1 ꢄ 1.9
0.4 ꢄ 0.1
209.5 ꢄ 16.4
592.1 ꢄ 57.0
224.1 ꢄ 25.1
418.2 ꢄ 31.5
495.4 ꢄ 43.4
7.6
41.1
5.3
23.1
1238.5
The kinetic parameters of PAPS were examined with 10 mM of
kaempferol 3-glucoside. Data shown represent means ꢄ S.D.
from three determinations.
Fig. 5 HPLC detection of sulfated kaempferol-3-glucoside. (A) Kaempferol; (B) sulfated kaempferol generated using AtSULT202B7;
(C) kaempferol-3-glucoside; (D) sulfated kaempferol-3-glucoside generated using AtSULT202B7; (E) sulfated kaempferol-3-glucoside;
(F) deglucosylated products of sulfated kaempferol-3-glucoside generated using b-glucosidase.
95

T. Hashiguchi et al.
447.09
C21H19O11
100
90
80
70
60
50
40
30
20
10
0
m/z 527.05 [M-H]-
527.05
C21H19O14
S
-SO3
100
200
300
400
500
600
700
800
900
1000
m/z
Fig. 6 MS2 spectrum of the sulfated kaempferol-3-glucoside. MS2 data were obtained from the 527.05 m/z ion as the precursor for high energy
collisional dissociation.
collision dissociation fragmentation of the parent ion
produced authentic kaempferol-3-glucoside lacking
one SOꢀ3 group (m/z 79.96) at m/z 447.09. These results
strongly suggests that sulfated kaempferol-3-glucoside
by AtSULT202B7 contains no sulfonate group in
glucose moiety.
growth and development of the root via flavonoid
glycosides sulfation.
Acknowledgements
The authors thank Dr. Takehito Inaba for his support and discus-
sions. They also thank Mr. Yosuke Hara for his assistance.
To summarize, we have cloned, expressed, purified
and characterized a novel A. thaliana sulfotransferase,
AtSULT202B7, in this study. AtSULT202B7 was
shown to exhibit higher affinity toward flavonol gluco-
sides than its aglycone derivative. AtSULT202B7 dis-
played sulfating activity only for 7-hydroxyflavone,
but not for flavonol-7-glucosides. Moreover, the deglu-
cosylated product of sulfated kaempferol-3-glucoside
is more likely to be the sulfated kaempferol, and the
product lost the sulfonate group to become kaemp-
ferol-3-glucoside, suggesting that AtSULT202B7
might be a flavonol glucoside 7-sulfotransferase. The
high content of flavonoid glycosides in plants and the
high specific activity of AtSULT202B7 for these
compounds might imply the presence of sulfated
flavonoid glycosides in plants. Attempting to detect
sulfated flavonoid glycoside in vivo, we performed
an Liquid Chromatography-Mass Spectrometry/Mass
Spectrometry (LC-MS/MS) analysis of the methanol
extracts of mature A. thaliana plants. Repeated experi-
ments, however, failed to detect the presence of
sulfated flavonoid glycoside. Moreover, extensive
metabolomic database search yielded no evidence for
the existence of sulfated flavonoid glycoside in A. thali-
ana. It is therefore likely that the abundance of those
compounds may be extremely low, or that the sulfated
flavonoid glycosides might be produced only under
certain conditions, such as at a particular developmen-
tal stage or under environmental stress. To date, it has
been reported that AtSULT202B7 is expressed pre-
dominantly in the root under a publicly available
microarray expression data bank (http://www.bar.
utoronto.ca/efp/cgi-bin/efpWeb.cgi). Flavonoids are
involved in the polar auxin transport in roots (25),
suggesting that SULT202B7 might regulate the
Funding
This work was supported by Grant-in-Aid for Scientific Research
(C) from JSPS 23580138 (to M.S.), 21580114 (to Y.S.), Grant-in-Aid
for JSPS Fellows 24-1404 (to T.H.) and National Institutes of Health
grant GM08756 (to M.C.L.).
Conflict of interest
None declared.
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