Abstract
A flavonol sulfotransferase (EC 2.8.2.-), which catalyzes the transfer of the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the 3-hydroxyl group of flavonol aglycones, has been purified to apparent homogeneity from Flaveria chloraefolia. The specific activity of flavonol 3-sulfotransferase was enriched 2000-fold, as compared with the homogenate, with a recovery of 9%. The molecular mass of the native and denatured enzyme was found to be 34.5 kDa, suggesting that the active from of the enzyme is a monomer. The enzyme exhibited expressed specificity for position 3 of flavonol aglycones, showed two activity optima at pH 6.0 and 8.5, did not require divalent cations, and was not inhibited by either EDTA or sulfhydryl group reagents. The results of substrate interaction kinetics and product inhibition are consistent with an Ordered Bi Bi mechanism where 3'-phosphoadenosine 5'-phosphosulfate is the first substrate to bind to the enzyme and 3'-phosphoadenosine 5'-phosphate is the final product to be released. The amino acid sequence of two peptides representing 17 and 33 amino acids showed no significant sequence similarity with the amino acid sequences reported for animal sulfotransferases. Antibodies raised against F. chloraefolia 3-sulfotransferase were found to cross-react with the 3'- and 4'-sulfotransferase activities of the same plant, suggesting that the three enzymes are structurally related.
Highlights
From the Plnnt Biochemistry Laboratory, Department of Biology, Concordin University, Montreal, Quebec H3G lM8, C a d a
This paper describes the purification and some properties of the 3-ST from Flaveria chloraefolia
A longitudinal section of the gel was stained for protein visualization, whereas the remaining gel was sliced into 2-mm bands, andthe enzyme protein was eluted and assayedfor 3-ST activity using quercetin as the substrate
Summary
Q 1992 hy The American Societyfor Biochemistryand MolecularBiology, Inc. Vol 267, No 3, Issue of January 25, pp. 1858-1863,1992 Print& in U.S.A. A flavonol sulfotransferase(EC 2.8.2.-), which cat- number of plant families [49]. Investigation of sulfate metabolism in animaltissues led tothe recognition of a number of sulfotransferases (STs)’ (EC 2.2.8.-) with specificity towards a variety of metabolites, including arylamines, phenols, steroids, and bile acids [10,11,12]. A number of these enzymes have been purified to homogeneity and theirproperties studied. A number product inhibition are consistent with an Ordered Bi of position-specific flavonol STs have been characterized from. Anti-flavonol 3-ST antibodies and a partial amino acid sequence of the purified protein were used for comparison with other plant anadnimal. Plants accumulate a variety of natural products These STs. compounds are synthesized in response toenvironmental stimuli and genetically programmed developmental signals
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