From the
The pFST3 and pFST4` cDNAs encode flavonol sulfotransferases
(ST) that are 69% identical in amino acid sequence yet exhibit strict
substrate and position specificities. To determine the domain
responsible for the properties of the flavonol STs, several chimeric
flavonol STs were constructed by the reciprocal exchange of DNA
fragments derived from the plasmids pFST3 and pFST4` and by the
expression of the corresponding chimeric proteins in Escherichia
coli. The chimeric enzymes were enzymatically active even though
their activities were reduced compared to the parent enzymes. The
specificity of the resulting hybrid proteins indicates that an interval
of the flavonol STs spanning amino acids 92-194 of the flavonol
3-ST sequence contains the determinant of the substrate and position
preferences. From the comparison of the amino acid sequences between
plant and animal STs, this interval can be subdivided into a highly
conserved region corresponding to positions 134-152 of the
flavonol 3-ST, flanked by two regions of high divergence from 98 to 110
and 153 to 170. In view of the similarities in length and hydropathic
profiles as well as the presence of four conserved regions between
plant and animal STs, the results of these experiments suggest that
this interval is involved in the recognition of substrates and/or
catalysis in all STs.
Plants accumulate a variety of natural products that are
synthesized in response to environmental stimuli and genetically
programmed developmental signals. Of these metabolites, flavonoids are
probably the most ubiquitous. A new class of flavonoids esterified with
sulfate groups have recently been reported to be of common occurrence
in a number of plant families
(1, 2) . Most of these
compounds are mono- to tetrasulfate esters of common flavones and
flavonols or their methyl ethers. The functional significance of
flavonoid sulfates in plant tissue is not clear. Apart from their
possible involvement in the detoxification of reactive hydroxyl groups,
their accumulation in plants growing in saline or marshy habitats
suggests a role in sequestering sulfate ions
(1) .
In animal
tissues enzymatic sulfation is considered an important reaction in the
detoxification of endogenous metabolites and xenobiotics.
Investigations of sulfate metabolism in these tissues led to the
recognition of a number of STs
Research to elucidate the
role of flavonoid sulfation in plant tissues has resulted in the
isolation and biochemical characterization of four position-specific
flavonol STs from Flaveria species
(7, 8) .
These enzymes exhibit strict specificity for position 3 of flavonol
aglycones, positions 3` and 4` of flavonol 3-sulfate and position 7 of
flavonol 3,3`- or 3,4`-disulfates, thus establishing a sequence for the
enzymatic sulfation of flavonol polysulfates. The cDNAs that encode the
flavonol 3- and 4`STs have been isolated from a Flaveria
chloraefolia expression library
(9) . These enzymes catalyze
the stepwise formation of quercetin 3,4`-disulfate (Fig. SI).
Expression of these clones in Escherichia coli led to the
synthesis of
Quercetin was purchased from Sigma, and quercetin 3-sulfate
was from Extrasynthèse, Bordeaux, France.
Eight chimeric
flavonol STs were constructed by taking advantage of the presence of
common restriction endonuclease sites in pFST3 and pFST4`
(Fig. 1). All restriction sites used are located at homologous
positions within the predicted amino acid sequences of the parental
cDNAs. The chimeric constructs were initially characterized by
sequencing to confirm the homologous exchange of protein domains
between the two enzymes. The synthesis in E. coli of the
corresponding parental or chimeric protein was monitored by
SDS-polyacrylamide gel electrophoresis following purification on
Ni-agarose (Fig. 2). The presence of 10 extra amino acids at the
N-terminal of the flavonol 4`-ST compared to the 3-ST is responsible
for the differences in migration observed between the different
enzymes.
Very little is known about the structure and function
relationships of STs despite the fact that a large number of these
enzymes have been purified and characterized and that they play an
important role in animal metabolism. This lack of information is partly
due to the absence of an appropriate experimental system to facilitate
this analysis. The availability of cDNA clones coding for two flavonol
STs of different specificities and sharing a high level of sequence
identity provides a unique experimental system to initiate studies
about the structure and function relationships of STs. Although these
cDNAs encode fusion proteins having additional amino acids
(His
One possible approach to identify
functional regions that are involved in the recognition of substrates
and catalysis in flavonol STs is to modify specific residues in the
protein sequence. However, the choice of target residues for
site-directed mutagenesis was found to be difficult because of the
absence of a known ST tridimensional structure as well as the limited
information obtained from sequence comparison of the flavonol 3- and
4`-STs. Therefore, to determine the basis for the distinct substrate
and position specificities exhibited by the flavonol 3- and 4`-STs, a
number of chimeric flavonol STs were constructed by the homologous
exchange of four protein domains between these enzymes (Fig. 1).
All the chimeric enzymes exhibited ST activity indicating that the
tertiary structure required for activity is preserved in the different
constructs. However, ChA, B, G, and H produced levels of activity lower
than the parent enzymes. In these four constructs, domains I and II are
of different parental origin, suggesting that the N-terminal and
central portions cooperate through complex interactions to give rise to
a tertiary structure optimal for catalysis. Analysis of the catalytic
properties of ChG and H indicates that domain II from amino acids 92 to
194 (numbering refers to the flavonol 3-ST sequence) is sufficient to
determine both substrate preference and the position of sulfation
(Fig. 1). ChG and H can be viewed as mutant forms in which 34
amino acids have been changed in this interval compared with pFST4` and
pFST3, respectively. Even with this number of amino acid changes, ChG
and H are still 89 and 90% identical in amino acid sequence when
compared with the parent enzymes. The alignment of predicted amino acid
sequences of a number of plant and animal STs for domain II allows the
identification of the following subdomains: a highly conserved region
from position 137-152 of the flavonol 3-ST sequence which
corresponds to the previously described region II of sequence
conservation
(6, 9) , flanked by two subdomains of high
divergence from positions 98 to 110 and 153 to 170
(Fig. 3B). The conserved subdomain is likely to
participate in cosubstrate binding or in specifying the correct folding
to form the active site. On the other hand, the flanking subdomains of
high divergence most probably participate in the functions that are not
conserved between STs, such as the binding to the appropriate sulfate
acceptor. In view of the similarity in the organization of domain II in
plant and animal STs, one can predict that substrate specificity is
determined by this domain in all STs.
The results presented here do not
imply that the domain(s) participating in substrate interaction and
catalysis is or are restricted to domain II. Comparison of the
predicted amino acid sequences of plant and animal STs indicates that
they share a high degree of amino acid sequence identity
(Fig. 3A). The identical amino acid residues are not
distributed evenly but are clustered in four distinct regions. The
presence in plant and animal STs of conserved regions lying outside of
the interval defined in this work suggests that protein determinants
located in domains I, III, and IV can participate in ligands binding
and catalysis but can be interchanged without affecting the enzyme
specificity.
This study represents an important step toward the
elucidation of the structure and function relationships of STs. The
identification of the domain II of flavonol STs as the determinant of
substrate and position specificities will allow the choice of specific
residues in the domain II as targets for site-directed mutagenesis. The
construction and analysis of mutant flavonol STs at both conserved and
unconserved residues are presently under progress.
(
)
with specificity
toward a variety of metabolites including arylamines, phenols,
steroids, and bile acids
(3, 4, 5) . cDNA clones
coding for a number of these enzymes have been isolated and
characterized (for a review, see Ref. 6).
-galactosidase-ST fusion proteins having the same
substrate and position specificities as those observed with the 3- and
4`-flavonol ST enzymes isolated from the plant
(7) . Sequence
comparison of the predicted amino acid sequences of the two proteins
revealed an overall identity of 69%. Comparison of the deduced amino
acid sequences of flavonol ST cDNA clones with animal steroid- and aryl
STs revealed the presence of identical amino acid residues distributed
throughout the sequences with four well conserved
regions
(6, 9) . Although the primary structure of
several STs is known, neither the location of the particular amino
acids nor the protein domains involved in the interaction with the
ligands are known. Previous chemical modification studies of STs showed
that arginine, cysteine, and possibly lysine residues are involved in
ST-substrate interactions
(10, 11) . However, the
location of the amino acids involved in substrate binding could not be
determined in these experiments. More recently, affinity labeling
experiments with ATP dialdehyde, a competitive inhibitor of the sulfate
donor 3`-phosphoadenosine 5`-phosphosulfate (PAPS), resulted in the
labeling of lysine 65 and cysteine 66 of the rat hepatic ST
IV
(12) . These residues are not conserved among STs, but their
presence in close proximity with the conserved region I suggests that
the latter participates in the binding of PAPS.
Figure SI:
Scheme IProposed pathway for the
enzymatic synthesis of quercetin 3,4`-disulfate in F.
chloraefolia.
The availability of
cDNA clones coding for flavonol STs with defined substrate and position
specificities opens a way to study the structure and function
relationships of this class of enzymes by recombinant DNA techniques.
In this investigation, we constructed a series of hybrid enzymes by
substituting protein segments between the flavonol 3- and 4`-STs by
in vitro manipulation of the cloned cDNAs. Analysis of the
substrate preference of the chimeric proteins allowed for the
identification of a defined domain that is responsible for both
substrate and position specificities.
Chimeric Constructs
Plasmids pFST3 and pFST4`,
containing full-length cDNAs coding for the flavonol 3- and 4`-STs,
respectively, were previously described
(9) . cDNA subfragments
to be exchanged between pFST3 and pFST4` were produced by restriction
enzyme digestion of sites conserved between the two clones. The
fragments were purified from agarose gels (0.8%) by extraction with
Geneclean II (Bio 101, La Jolla, CA). The appropriate fragments were
religated to the deleted parental cDNA clones and transformed into the
E. coli strain XL1-Blue (Stratagene, CA). Chimera A and B
cDNAs were constructed by the homologous exchange of the
XbaI-BsmI fragments of pFST3 and pFST4`. Chimera C
and D were constructed by the homologous exchange of the 5`
BamHI fragments of pFST3 and pFST4`. Chimera E and F were
constructed by the homologous exchange of the 3` HindIII
fragments of pFST3 and pFST4`. Finally, for the construction of chimera
G and H, pFST3 and pFST4` cDNAs were subcloned in pBluescript lacking a
HindIII site. These vectors were used for the homologous
exchange of the BsmI-HindIII fragments.
Chimeric constructs were screened for proper religation by restriction
mapping and DNA sequencing. For expression studies, the parental and
hybrid cDNAs were polymerase chain reaction-amplified with Vent DNA
polymerase (New England Biolabs, Beverly, MA) to introduce a
BglII restriction site adjacent to the ATG of the respective
constructs. The oligonucleotides used were:
5`-GAAGATCTATGGAAGATATTATCAAAACAC for cDNAs having the 5`-coding
portion of pFST3 and 5`-GAAGATCTATGGAAACTACAAAAACCCAG for cDNAs having
the 5`-coding portion of pFST4`. The 3` primer in all reactions was the
M13-20 oligonucleotide, which allowed the amplification of
full-length cDNAs. The amplified fragments were digested with
BglII and SalI and ligated into the BamHI
and SalI polylinker sites of the bacterial expression vector
pQE30. The constructs were transformed into E. coli XL1-blue
competent cells, and individual clones were isolated and checked for
proper religation by restriction mapping. The chimeric cDNAs were named
ChA to ChH. All enzymes used for cloning were from New England Biolabs
and were used under the conditions recommended.
Sulfotransferase Assay
A culture (10 ml) of the
E. coli strain XL1-blue containing pHFST3, pHFST4`, or the
different chimeric constructs was incubated in LB medium at 37 °C
for 3 h before the addition of the inducer
isopropyl-1-thio--D-galactopyranoside at a final
concentration of 1 mM. Incubation was continued for an
additional 3 h. Cells were harvested by centrifugation, washed in 50
mM sodium phophate, pH 8.0, 0.3 M NaCl, 14
mM
-mercaptoethanol, resuspended, and lysed by sonication
in 1 ml of the same buffer. Cell debris were removed by centrifugation,
and the supernatant was applied to 50 µl of Ni-NTA resin (Qiagen,
Chatsworth, CA) pre-equilibrated with the same buffer. The resin was
then washed three times with 1 ml of sodium phosphate 50 mM,
pH 6.0, 0.3 M NaCl, 14 mM
-mercaptoethanol, and
the proteins were eluted with 250 µl of the same buffer containing
imidazole at a concentration of 150 mM. Proteins were measured
by the method of Bradford
(13) using the dye and the assay
procedure from Bio-Rad. Bovine serum albumin was used as the standard
protein. ST activity in the eluate was determined immediately after
purification by monitoring the incorporation of label from
3`-phosphoadenosine 5`-phospho[
S]sulfate
([
S]PAPS) (New England Nuclear; specific
activity = 1.5-3.0 Ci/mmol) to the flavonol substrate by
using a previously described assay
(14) . An aliquot of the
enzyme reaction products was assayed for radioactivity in a liquid
scintillation counter, and the remaining fraction was used for the
identification of reaction products by cochromatography with reference
compounds. Thin layer chromatography was carried out on Avicel
cellulose using H
O as solvent for the identification of
monosulfated flavonols or 1-butanol/acetic acid/H
O (3:1:1,
v/v) for the identification of disulfated flavonols. Developed
chromatograms were visualized in UV light (360 nm) and then
autoradiographed on x-ray film.
SDS-Polyacrylamide Gel Electrophoresis
An aliquot
of purified parental and chimeric STs was mixed with an equal volume of
2 SDS sample buffer and subjected to 12% polyacrylamide gel
electrophoresis according to the method of Laemmli
(15) . The
proteins were visualized by Coomassie Blue staining.
DNA Sequencing and Sequence Analysis
The
nucleotide sequences of the different constructs were determined on
both strands by the dideoxy chain-termination method
(16) using
oligonucleotide primers. Sequence comparison and alignment was
performed using the Genetics Computer Group software package.
Expression and Characterization of Recombinant Parental
and Chimeric Flavonol STs
The isolation of cDNA clones coding
for the flavonol 3- and 4`-STs had previously been
described
(9) . The proteins encoded by the different constructs
in pQE30 contain His-Gly-Ser before the putative first
methionines of the original proteins FST3 and FST4`. Despite this
difference both recombinant enzymes exhibit the same substrate
preference and position specificities as the enzymes purified from the
plant. In addition, the recombinant flavonol 3- and 4`-STs were shown
to have affinity constants for the flavonol substrates and for the
cosubstrate (PAPS) similar to the values obtained with the homogeneous
native proteins
(7, 9, 17) .
Figure 1:
Schematic representation of pFST3
(open bars), pFST4` (shaded bars), and the various
chimera. The parental origin of each fragment is illustrated for the
chimeric constructs. The sequences were divided into four domains
represented by Roman numbers. The specificity of the parent
and hybrid enzymes is indicated in the right-hand column. ST activities
of ChA, D, F, and G were compared with the one of pFST3 (100%) using
quercetin as substrate. ST activities of ChB, C, E, and H were compared
with the one of pFST4` (100%) using quercetin 3-sulfate as
substrate.
Figure 2:
SDS-PAGE of the purified parental
(pFST3 and pFST4`) and chimeric (ChA to H)
STs.
Enzymatic Properties
In order to compare the level
of activities of the chimeric proteins with the parent enzymes, the
amount of purified STs was measured by the method of Bradford prior to
enzyme assays. Except for ChA, B, G, and H, all of the hybrid STs
exhibited levels of activity comparable to the parental recombinant
enzymes, and the substrate specificity of these enzymes was readily
determined in vitro using the purified proteins. An analysis
of the specificity of the resulting hybrid STs reveals that the
position of sulfation is determined by the interval of the cDNA that
encodes the amino acid 92-194 of the flavonol 3-ST (designated
domain II) (Fig. 1). The substrate preference of the
ChA, D, F, and G was almost identical with that of pFST3 while the one
observed for ChB, C, E, and H was found to match the substrate
preference of pFST4` (). Due to the presence of several
hydroxyl groups that can potentially be sulfated on both quercetin and
quercetin 3-sulfate, other flavonol substrates were tested to confirm
the position specificity of the individual chimeric STs. ChA, D, F, and
G accepted several flavonol aglycones, but did not accept flavonols
substituted at position 3 (). When assayed with quercetin,
the product of the reactions cochromatographed with authentic quercetin
3-sulfate. ChB, C, E, and H accepted quercetin 3-sulfate (3` and 4`
= OH), kaempferol 3-sulfate (3` = H, 4` = OH) and
isorhamnetin 3-sulfate (3` = OMe, 4` = OH) but did not
accept tamarixetin 3-sulfate (3` = OH, 4` = OMe) or
flavonol aglycones (). This corresponds to the same
substrate specificity as the one observed with pFST4`. When assayed
with quercetin 3-sulfate, the product of the reactions
cochromatographed with authentic quercetin 3,4`-disulfate.
-Ser-Gly) at their N-terminal, the recombinant enzymes
retain the same specificities and kinetic parameters as the native
enzymes purified from the plant.
Figure 3:
A,
schematic representation of the conserved regions among STs. The
backbone corresponds to a protein with 312 amino acids (pFST3). The
boxes represent conserved residues. Roman numbers refer to the conserved regions described in Refs. 6 and 9.
Numbers refer to the sequence of the flavonol 3-ST.
B, amino acid sequence alignment of pFST3 and pFST4` with
Flaveria bidentis ST-like cDNA (GeneBank, U10277)), human
liver hydroxysteroid ST (HSST) (18), rat hydroxysteroid ST
(RHST) (19), rat minoxidil ST (RMST) (20), human aryl
ST (HAST) (21), human dehydroepiandrosterone ST
(HDST) (22) and human estrogen ST (HEST) (23). The
alignment and the consensus sequence were obtained using LINEUP in the
GCG software package. Hyphens replace amino acids present in
the consensus sequence. Boxes indicate residues common to the
nine sequences.
An interesting feature of the
catalytic properties of the flavonol 4`-ST is the strict requirement
for a substrate which is already sulfated at position 3
(Fig. SI). This requirement implies that the flavonol 4`-ST
active site is able to discriminate between a flavonol and a flavonol
3-monosulfate. Studies on the interactions between sulfated molecules
and proteins have implicated two types of interactions: hydrogen
bonding with uncharged polar side chains as in the case of the
Salmonella typhimurium periplasmic sulfate binding
protein
(24, 25) , and ionic interactions with basic
amino acids, as in the binding of heparin to antithrombin
(26) .
We have identified at least 5 residues in the 4`-ST sequence which are
different in the 3-ST and which could be involved in the formation of
hydrogen bonds or salt bridges with the sulfate group of quercetin
3-sulfate. These are Tyr, Lys
,
Cys
, Lys
, and His
(Fig. 3B).
Table:
Substrate specificity of the flavonol STs
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.