©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chimeric Flavonol Sulfotransferases Define a Domain Responsible for Substrate and Position Specificities (*)

Luc Varin (1)(§), Frédéric Marsolais (1), Normand Brisson (2)

From the (1) Département de Biologie, Pavillon Charles-Eugène Marchand, Université Laval, Ste-Foy, Québec G1K 7P4 and the (2) Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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() 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).

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 -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.


EXPERIMENTAL PROCEDURES

Quercetin was purchased from Sigma, and quercetin 3-sulfate was from Extrasynthèse, Bordeaux, France.

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 HO as solvent for the identification of monosulfated flavonols or 1-butanol/acetic acid/HO (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.


RESULTS

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) .

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.


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.


DISCUSSION

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-Ser-Gly) at their N-terminal, the recombinant enzymes retain the same specificities and kinetic parameters as the native enzymes purified from the plant.

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.


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).

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.

  
Table: Substrate specificity of the flavonol STs



FOOTNOTES

*
This work was supported by the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dépt. de Biologie, Pavillon Charles-Eugène Marchand, Université Laval, Ste-Foy, Québec G1K 7P4, Canada. Tel.: 418-656-3708; Fax: 418-656-7176; E mail: lucvarin@rsvs.ulaval.ca.

The abbreviations used are: ST, sulfotransferase; PAPS, 3`-phosphoadenosine 5`-phosphosulfate.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.