Download Partial Purification and Characterization of Three Flavonol

Received for publication December 13, 1988
and in revised form February 22, 1989
Plant Physiol. (1989) 90, 977-981
0032-0889/89/90/0977/05/$0 1 .00/0
Partial Purification and Characterization of Three
Flavonol-Specific Sulfotransferases from
Flaveria chloraefolia'
Luc Varin and Ragai K. Ibrahim*
Plant Biochemistry Laboratory, Department of Biology, Concordia University, Montreal, Quebec, Canada H3G 1M8
assay for flavonoid ST2 activity. The recent advances in the
chemical synthesis of flavonoid sulfate esters (2) and the
development of a simple, accurate flavonoid ST assay (20)
enabled us to demonstrate the enzymic synthesis of sulfated
flavonols in Flaveria spp. (21). The latter reaction involves
the transfer of the sulfate group from a sulfate donor, PAPS
to the hydroxyl group(s) of flavonols. This study (21) seemed
to indicate that the enzymic synthesis of polysulfated flavonols
is catalyzed by a family of position-specific STs (EC 2.8.2-).
Recent studies (2, 9) indicated that Flaveria chloraefolia is
a rich source of mono- and disulfate esters of quercetin and
patuletin (see Scheme I), with sulfation at positions 3, 3', or
4' on the flavonoid ring (3, 7), as well as several 6-methoxyflavonol 3-monosulfates (8). It was considered of interest,
therefore, to study the ST system of this tissue and to determine the sequence of enzymic sulfation of these metabolites.
In this paper, we describe the partial purification and some
properties of three novel, position-specific STs that are involved in the biosynthesis of flavonol sulfate esters in F.
ABSTRACT
Three distinct flavonol-specific sulfotransferases were partially
purified from the shoot tips of Flaveria chloraefolia A. Gray by
fractional precipitation with ammonium sulfate, followed by gel
filtration on Sephacryl S-200, 3'-phosphoadenosine 5-phosphateAgarose affinity chromatography and chromatofocusing on Mono
P. These enzymes exhibited expressed specificity for positions
3 of various flavonol acceptors and of 3' and 4' of flavonol 3sulfate. The three sulfotransferases had similar molecular
weights (35,000), exhibited no requirement for divalent cations
and were not inhibited by SH group reagents. Their Km values for
both the sulfate donor and the flavonol acceptors were of the
same order of magnitude (ca. 0.2-0.4 micromolar). Except for the
3-sulfotransferase, which exhibited two optima at pH 6.5 and 8.5,
the 3' and the 4'-sulfotransferases had a pH optimum of 7.5. The
three enzymes could be resolved only by chromatofocusing and
were eluted at pH 5.4, pH 6.0, and pH 5.1 for the 3-, 3'- and 4'sulfotransferases, respectively. The substrate specificity of these
three enzymes is discussed in relation to the biosynthesis of
polysulfated flavonols in F. chloraefolia.
chloraefolia.
MATERIALS AND METHODS
Plant Material
Sulfated flavonoids, a recently discovered class of organic
sulfur compounds (12), are not considered to be of common
occurrence in a number of plant families, especially the
Compositae (9). They consist mostly of mono- to tetrasulfate
esters of common hydroxyflavones and hydroxyflavonols.
Whereas the enzymic O-methylation (15, 18) and O-glucosylation (14, 16) of flavonoids have been extensively studied,
little is known of their enzymic sulfation in plants. In contrast,e there have been extensive studies of the enzymic sulfation of endogenous metabolites and xenobiotics in animal
tissues, which led to the purification of several classes of
sulfotransferases (13, 17, 19). This paucity in knowledge of
flavonoid sulfation in plants may have been due to the unavailability of specifically sulfated flavonoid substrates and
reaction products, as well as to the absence of an adequate
Seeds of Flaveria chloraefolia A. Gray (Compositae) were
kindly supplied by Dr. M. Powell, Sul Ross University, Alpine, TX and Dr. S. Holaday, Texas Tech University, Lubbock, TX. These were germinated in a 1-cm layer of vermiculite on top of potting soil and their growth was maintained
under greenhouse conditions. Terminal buds and the first pair
of expanded leaves were used for enzyme attraction.
' This work was supported by the Natural Sciences and Engineering
Research Council of Canada and the Department of Higher Education, Government of Quebec. L. V. was the recipient of an NSERC
post-graduate scholarship and a Concordia University Fellowship.
Presented in part at the 14th International Conference of Groupe
Polyphenols, St. Catherines, Ontario, August 1988.
Scheme I
2 Abbreviations: ST, sulfotransferase; PAP, 3'-phosphoadenosine 5'-phosphate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate;
TBADP, tetrabutylammonium dihydrogen phosphate; APS, adenosine 5'-phosphosulfate.
977
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978
VARIN AND IBRAHIM
Chemicals
3 '-Phosphoadenosine 5 '-phospho-[35S]sulfate was purchased from NEN (Boston, MA) and diluted with unlabeled
PAPS (Sigma, St. Louis, MO) as required. Flavonoid aglycones were purchased from Roth (Karlsruhe, FRG) or Sarsynthese (Bordeaux, France) and were further purified by
TLC or HPLC. All flavonol sulfate esters used in this study
were from our laboratory collection (2). These compounds
were synthesized by previously described methods (4-6) and
their identity was confirmed by spectroscopic methods.
TBADP was obtained from Aldrich (Milwaukee, WI). PAPAgarose was obtained from Sigma (St. Louis, MO). Sephacryl
S-200, Polybuffer 74, as well as the fast protein liquid chromatography system were from Pharmacia (Uppsala, Sweden).
All other chemicals were of analytical grade.
Buffers
Buffer A, 0.2 M Tris-HCl (pH 7.5), contained 14 mM 2mercaptoethanol, 10 mm diethylammonium diethyldithiocarbamate, and 5 mM EDTA; B, 50 mm Tris-HCl (pH 7.5),
contained 14 mM 2-mercaptoethanol; C, 25 mm bis-Tris-HCI
(pH 6.3), contained 14 mM 2-mercaptoethanol; D, 25 mM
bis-Tris-iminodiacetic acid (pH 7.1), contained 14 mm 2mercaptoethanol; E, Polybuffer 74-iminodiacetic acid (1:10
v/v) (pH 4.8), contained 14 mm 2-mercaptoethanol.
Protein Extraction
Unless stated otherwise, all procedures were carried out at
4°C. Shoot tips (ca. 100 g) were frozen in liquid N2, mixed
with Polyclar AT (10%, w/w), and ground to a fine powder.
The latter was homogenized with buffer A (1:4, w/v). The
homogenate was filtered through nylon mesh and the filtrate
centrifuged at 1 5,000g for 15 min. The supernatant was stirred
for 15 min with Dowex 1X2 (5%, w/v) that had previously
been equilibrated with the same buffer, then filtered. The
filtrate was fractionated with solid ammonium sulfate, and
the protein fraction which precipitated between 35 and 75%
saturation was collected by centrifugation.
Enzyme Purification
The protein pellet was suspended in 20 mL buffer B and
chromatographed on a Sephacryl S-200 column (3 x 65 cm)
preequilibrated with the same buffer. The column was developed with buffer B, and 3-mL fractions were collected for ST
assay using quercetin and quercetin 3-sulfate as substrates.
The active fractions were pooled and concentrated by ultrafiltration (PM 10 Amicon), and the buffer was changed by
chromatography on a Sephadex G-25 column that had previously been equilibrated with buffer C. The eluted protein
was applied to a PAP-Agarose affinity column (1.5 x 12 cm)
that was preequilibrated with buffer C and washed with three
column volumes. The bound proteins were eluted with a
linear salt gradient of 0.0 to 1.0 M NaCI in buffer C. Fractions
(1 mL) were collected and assayed for ST activity using
quercetin and quercetin 3-sulfate as substrates. The active
fractions were concentrated by ultrafiltration and then chro-
Plant Physiol. Vol. 90,1989
matographed on a Sephadex G-25 column which was preequilibrated with buffer D. The desalted proteins were finally
chromatographed on a Mono P (HR 5/20) column previously
equilibrated in buffer D. The bound proteins were eluted at a
flow rate of 0.4 mL/min with buffer E, which generated a
gradient between pH 7.0 and 4.8. Fractions (1 mL) were
collected and assayed for ST activity against both quercetin
and quercetin 3-sulfate.
Sulfotransferase Assay and Identification of Reaction
Products
The standard enzyme assay was used as previously described (20). The assay mixture contained 0.1 nmol flavonoid
substrate, 0.1 nmol labeled PAPS (containing 220,000 dpm),
and up to 60 ,ug protein (in 25 mm Tris-HCl buffer [pH 7.5])
in a total volume of 100 1.L. Control incubations containing
boiled instead of active enzyme were routinely used. The
enzyme reaction was started by the addition of protein, was
incubated at 30°C for 15 min, and was stopped by the successive addition of 20 IuL of 2.5% acetic acid, followed by 20 ,uL
of 0. 1 M TBADP, which resulted in the formation of ion-pairs
with the flavonoid sulfate esters. The latter were extracted
with 250 ,uL ethyl acetate, whereas PAPS remained in the
aqueous layer of the assay mixture (20). An aliquot of the
organic phase was counted for radioactivity in a toluene-based
scintillation fluid. The remaining fraction was concentrated
under N2 and used for identification of the reaction products
by cochromatography with reference compounds (21). TLC
was carried out on Avicel cellulose using H20 or n-BuOHHOAc-H20 (3:1:1 or 4:1:5, v/v/v) as solvents. Paper electrophoresis was carried out as previously described (12). Developed chromatograms were visualized in UV light (366 nm)
before and after spraying with 1% diphenylborinate and then
autoradiographed on x-ray film.
Mol Wt Determination
The highly purified enzymes were applied to a Superose 12
(HR 10-30) column which had previously been equilibrated
with ribonuclease A (Mr 13,700), chymotrypsinogen A (Mr
25,000), ovalbumin (Mr 45,000), and BSA (Mr 67,000) as
reference proteins. The apparent mol wt of each enzyme was
estimated by its elution volume from the column.
Protein Estimation
Protein concentration was measured by the method of
Bradford (10) using the Bio-Rad dye reagent and BSA as
standard protein.
RESULTS
Purification of Flavonol Sulfotransferases
Three distinct, position-specific flavonol STs were highly
purified from F. chloraefolia by ammonium sulfate precipitation and successive chromatography on Sephacryl S-200,
PAP-Agarose, and chromatofocusing on Mono P columns.
Figure 1 shows that when the Sephacryl S-200 fractions were
assayed against quercetin and quercetin 3-sulfate, the ST
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FLAVONOL-SPECIFIC STs OF FLAVERIA CHLORAEFOLIA
979
3
a
I~~~~~~~~~~4.
Xt
30
20
U)
~1
10
FRACTION NUMBER
10
Figure 1. Elution profile of the ST-activities after gel filtration on
Sephacryl S-200. The column was preequilibrated and developed
with buffer B and 3-mL fractions were collected and assayed for
enzyme activity against quercetin (A) and quercetin 3-sulfate (A) as
substrates. The absorbance (. ) was monitored at 280 nm.
Ouercetin
1.0
Quercetin-3-S04
)
4
I~
0
30
0.6
~ ~
D-
20
~
~
~
~
I
0.4
1<
02
10
20
30
40
50
FRACTION NUMBER
Figure 2. Elution profile of the ST-activities after affinity chromatography on PAP-Agarose. The column was preequilibrated with buffer
C, and the bound proteins were eluted using a linear salt gradient of
0.0 to 1.0 M NaCI in buffer C (- -). Two-mL fractions were collected
and assayed for enzyme activity against quercetin (A) and quercetin
3-sulfate (A) as substrates. The absorbance (.) was monitored
at 280 nm.
activities accepting both substrates coeluted as a discrete peak.
Analysis of the reaction products revealed that quercetin was
converted to its 3-sulfate ester, whereas quercetin 3-sulfate
gave rise to its 3,3'- and 3,4'-disulfate ester derivatives. These
results indicated the presence in F. chloraefolia of the 3-, 3'and 4'-ST activities.
Chromatography of the active fractions on PAP-Agarose
resulted in partial but incomplete separation of the 3-ST from
the 3'- and 4'-ST activities (Fig. 2). However, this affinity
column was efficient in eliminating approximately 95% of
the contaminating protein. Further chromatography of the
combined active fractions by chromatofocusing on Mono P
revealed four ST activity peaks (Fig. 3), two of which (peaks
and 2) accepted quercetin as substrate, whereas the other
two (peaks 3 and 4) were active against quercetin 3-sulfate.
Analysis of the enzyme reaction products revealed that peak
1 which eluted at pH 5.7 contained both the 3- and 3'-ST
activities. However, the low activity and instability of this
protein fraction precluded its further characterization. Peak 2
20
30
40
50
60
FRACTION NUMBER
Figure 3. Elution profile of the ST-activities after chromatofocusing
on Mono-P. The column was preequilibrated with buffer D, and the
bound proteins were eluted with buffer E at a flow rate of 0.4 mL/
min which generated a gradient between pH 7 and 4.8. Fractions of
1-mL were collected and assayed for enzyme activity against quercetin (A) and quercetin 3-sulfate (A) as substrates. The absorbance
.)
was monitored at 280 nm.
(.
which eluted at pH 5.4 accepted quercetin as substrate and
gave quercetin 3-sulfate as the only reaction product. Substrate specificity of this protein fraction (see below) established
its identity as the 3-ST which was free from other contaminating activity. Both peaks 3 and 4 which eluted at pH 6.0
and pH 5.1 (Fig. 3) accepted quercetin 3-sulfate as substrate
and gave rise to the 3,3'- and 3,4 '-disulfate esters, respectively,
thus representing the 3'- and 4'-ST activities. This purification protocol resulted in 550-, 630-, and 500-fold increase in
specific activity of the 3-, 3'-, and 4'-ST activities, respectively, with recoveries of 9 to 12% as compared with the crude
extract (Table I).
Stability of Sulfotransferases
The highly purified enzymes were very unstable, with a
half-life of 24 h when stored at 4°C. Storing the protein at
-20°C in presence of glycerol or sucrose did not significantly
improve the stability. However, the addition of BSA up to
mg/mL increased the half-life of the three enzymes up to 3 d.
Substrate Specificity of Sulfotransferases
Flavonol Substrate
II summarizes the relative activities of the three
when assayed against different substrates. The 3-ST
exhibited strict specificity for position 3 of several flavonols
in the following order of decreasing activity, with rhamnetin
> isorhamnetin > quercetin > patuletin > kaempferol, as
sulfate acceptors. However, it accepted neither flavonols with
additional hydroxyl groups at position 6 (quercertagetin), 8
(gossypetin), or 5' (myricetin) nor flavonols lacking ring-B
hydroxylation (e.g. galangin). The accumulation of quercetin
3-sulfate and patuletin 3-sulfate in F. chloraefolia (2) as well
as the common occurrence of flavonol 3-sulfates in plants (9)
suggest that 3-sulfation is the first step in the enzymic synthesis
of polysulfated flavonols.
Table
enzymes
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980
VARIN AND IBRAHIM
Plant Physiol. Vol. 90,1989
Table I. Purification of Flaveria Sulfotransferases
The sulfotransferase activities were assayed as described in "Materials and Methods" using quercetin
as subtrate for the 3-ST and quercetin 3-sulfate for the 3'- and the 4'-STs.
Step
Volume
mL
Dowex
3-ST
3' + 4'-ST
S-200
3-ST
3' + 4'-ST
PAP-Agarose
3-ST
3' + 4'-ST
Mono-P
3-ST
3'-ST
4'-ST
170
170
Total
protein
mg
187
187
Rhamnetin
Isorhamnetin
Quercetin
Patuletin
Kaempferol
Ombuin
Punfiation
Recovery
pKatl
mg
pKat
-fold
%
0.079
0.065
14.80
12.16
100
100
15.2
15.2
0.808
0.647
12.29
9.83
10
10
83
81
15
15
0.8
0.8
9.328
6.413
7.46
5.13
118
99
50
42
1.26
1.15
549
633
503
9
10
12
2
2
2
0.029
0.028
0.044
AM.
Relative Activity
3-ST
Total
activity
20
20
Table II. Substrate specificity of Flaveria sulfotransferases
The standard enzyme assay was used as described in "Materials
and Methods," at a substrate concentration of 1 Mm. The order of
specificity was the same when the substrates were assayed at 10
Substrateb
Specific
activity
3'-ST
4'-ST
100
94
58
52
48
37
31
43.47
41.17
32.71
1.44
well as quercetin 3,4'-disulfate (2), and suggests that sulfation
at either of these two positions is the second step in the
biosynthesis of disulfate esters in this tissue. None of the three
enzymes described here exhibited any activity with phenylpropanoids, flavones, or dihydroflavonols, thus indicating
their strict specificity toward flavonol aglycones or their partially sulfated esters.
Cosubstrate
When [35S]APS was used as the sulfate donor with the
standard enzyme assay, no flavonol sulfation was observed
with either of the ST preparations.
100
100
12
58
33
Tamarixetin-3-SO4
0
0
Kaempferol-3-SO4
45
lsorhamnetin-3-SO4
0
38
a
The maximum activities (100%) observed were 65, 39, and 32
b The following
pKat/mg for the 3-, 3'-, and 4'-STs, respectively.
substrates were utilized as sulfate acceptors at <10% of controls:
myricetin, quercetagetin, dihydroquercetin, and galangin (for the 3ST); apigenin, luteolin, caffeic acid, ferulic acid and p-coumaric acid
(for the 3'- and 4'-STs).
Other Properties
The three flavonol STs were found to have similar mol wt
(ca. 35,000) on a calibrated Superose 12 column using an
FPLC system. However, they displayed different pI values
(Table III) which allowed their separation on chromatofocusing. Except for the 3-ST, which exhibited two pH optima at
pH 6.5 in bis-Tris and 8.5 in Tris buffers, the 3'- and 4'-STs
had an optimum pH of 7.5 in Tris buffer. However, it is
interesting to note that the 4'-ST activity was completely
inhibited in the presence of phosphate buffer. The pH behavior of the 3-ST may be explained by its requirement for
catalysis of a specific ionized form of the substrate or of an
amino acid involved in the catalysis.
The 3'-ST accepted the following flavonols in a decreasing
order with quercetin 3-sulfate > patuletin 3-sulfate > tamarixetin 3-sulfate for further sulfation at the 3'-position. It did
not accept kaempferol 3-sulfate, isorhamnetin 3-sulfate, or
any of the flavonol aglycones tested. The 4'-ST accepted
quercetin 3-sulfate > kaempferol 3-sulfate > isorhamnetin 3sulfate > patuletin-3-sulfate (in order of decreasing activity).
It did not react with either tamarixetin 3-sulfate or any
flavonol aglycone (Table II). The specificity of the two latter
enzymes is consistent with the accumulation, in F. chloraefolia, of quercetin 3,3'- and patuletin 3,3'-disulfates (7) as
Table l1l. Properties of Flaveria Sulfotransferases
3-ST
Property
3T-ST
4'-ST
0.2
Km Flavonoid (,M)a
0.29
0.36
Km PAPS (Mm)
0.2
0.35
0.38
7.5
7.5
pH optimum
6.0, 8.5
5.4
5.1
6.0
Apparent pi
Apparent mol wt
35,000
35,000
35,000
a With quercetin as substrate for the 3-ST and quercetin 3-sulfate
for the 3'- and the 4'-STs.
Tamarixetin
Quercetin-3-SO4
Patuletin-3-SO4
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FLAVONOL-SPECIFIC STs OF FLAVERIA CHLORAEFOLIA
The highly purified enzymes exhibited no requirement for
divalent cations and were not inhibited by EDTA up to 10
mM. They were insensitive to the SH group reagents, pchloromercuribenzoate, iodoacetate, or iodoacetamide, when
assayed at 1 to 10 mm. The Km values of the three enzymes
for both the flavonol acceptors and the sulfate donor (Table
III) were of the same order of magnitude (ca 0.2-0.4 AM).
DISCUSSION
Three distinct, flavonol-specific STs were highly purified
from shoot tips of F. chloraefolia and exhibited strict specificity for positions 3 of various flavonol acceptors and of 3' and
4' of flavonol 3-sulfate. These novel STs constitute the enzyme complement involved in the sequential sulfation of
flavonols in this tissue and represent the first report of a
family of position-specific flavonol STs in plants. We propose,
therefore, the following designations for these enzymes:
PAPS:flavonol 3-ST; PAPS:flavonol 3-sulfate 3'-ST; and
PAPS:flavonol 3-sulfate 4'-ST. Flaveria STs are quite distinct
from the much studied sulfating enzymes in mammalian
tissues (13, 17, 19), which exhibited specificity toward major
chemical classes of substrates (e.g. phenols, steroids, and bile
acids), but not for specific groups or positions. In addition,
the involvement of position-specific STs in the formation of
steroid and bile salt disulfates has yet to be demonstrated ( 17,
19). The enzymes reported here mediate substrate-specific
and position-oriented sulfations which follow the orderly sequence: flavonol
--
flavonol 3-sulfate
--
flavonol 3,3'-
or
3,4'-disulfates, which are natural constituents of this tissue.
Furthermore, it is interesting to note that the sulfation reaction by Flaveria STs was catalyzed in the presence of PAPS,
but not APS, which supports earlier proposals (1, 12) that the
former is the physiological sulfate donor in plant systems.
This is in agreement with the recently reported ST of cress
seedlings (1 1), which utilized PAPS in the sulfation of both
desulfobenzylglucosinolate and desulfoallylglucosinolate to
their sulfated derivatives.
Enzymic sulfation seems to be a later step in flavonoid
biosynthesis since a number of methylated flavonols (e.g.
rhamnetin, isorhamnetin, and patuletin) were good sulfate
acceptors. However, the co-occurrence in F. chloraefolia of
patuletin 3-glucoside and patuletin 3-sulfate raises an interesting question as to the competition among the transferase
reactions (glucosylation, sulfation) for similar aglycones (9).
The high affinity of these enzymes for both the sulfate
donor and the flavonol acceptors seem to reflect the low
concentration of sulfated flavonoids inside the cell. This offers
a means for modifying the existing pools of PAPS and the
naturally occurring acceptors toward the formation of flavonol sulfates in other plant species. Such strategy may be
achieved by the insertion of a cDNA gene coding for a specific
ST with the aim of altering the secondary metabolism in the
target plant.
of the flavonoid sulfates used in this study, and J. Seguin for her
technical assistance.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
ACKNOWLEDGMENTS
We wish to thank Dr. M. Powell and Dr. S. Holaday for their
generous gifts of F. chloraefolia seeds, Dr. D. Barron for the synthesis
981
21.
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