Partial Purification and Characterization of Three Flavonol-Specific Sulfotransferases from Flaveria chloraefolia

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 3-sulfate. 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. assay for flavonoid ST2 activity. The -).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.

Partial Purification and Some Properties of Flavonol 7-Sulfotransferase from Flaveria bidentis

“…Their activity was inhibited by the flavonol substrate above 1.5 ,uM. These properties are similar to those previously reported for the 3, 3', and 4'-STs of F. chloraefolia (14). Although the 7-ST isozymes catalysed further sulfation of quercetin 3,3'-and -3,4'-disulfates with equal efficiency (Table II), the physiological significance oftwo isoforms in the 7-sulfation of flavonols remains to be elucidated.…”

“…Due to instability of the 7-ST, a purification protocol was developed to allow the recovery of this enzyme free from other contaminating activities, namely the 3, 3' or 4'-STs (14). Partial purification of the 7-ST was achieved within 8 h after protein extraction by passing the ammonium sulfate pellet through a PD-10 column, followed by chromatography on PAP-agarose.…”

“…The sulfated products were transformed to their ion pairs by the addition of TBADP before extraction with ethyl acetate. The radioactivity in the reaction products was determined by liquid scintillation, and their identity was verified by cochromatography with reference compounds as previously de-scribed (14,16). …”

“…The 7-sulfotransferase isoforms I and 11 had a pH optimum of 7.5 in phosphate buffer, apparent pi values of 6.5 and 6.3, and an M, of 35,000. They had no requirement for divalent cations and were not inhibited by (14). These reactions were shown to be catalyzed by three distinct, flavonol-specific and position-oriented STs2.…”

“…Because of their accumulation in plants growing in saline and marshy habitats, sulfated flavonoids are believed to play a role in sequestering sulfate ions (3). Their 3, 3', and 4' of flavonols in Flaveria chloraefolia (14). These reactions were shown to be catalyzed by three distinct, flavonol-specific and position-oriented STs2.…”