MUTATIONAL ANALYSIS OF DOMAIN-II OF FLAVONOL 3-SULFOTRANSFERASE

The flavonol 3- and 4'-sulfotransferases (ST) from Flaveria chloraefol ia catalyze the transfer of the sulfonate group from 3'-phosphoadenosi ne 5'-phosphosulfate (PAdoPS) to position 3 of flavonol aglycones and position 4' of flavonol 3-sulfates. We identified previously a protein segment, designated domain II, that contains all the determinants res ponsible for the specificity of these enzymes. Within domain II, at le ast five amino acids specific to the 4'-ST that could bind the sulfate group of quercetin 3-sulfate were identified. In this study, these am ino acid residues were introduced at equivalent positions in the flavo nol 3-ST sequence by site-directed mutagenesis of the cloned cDNA. No reversal of tile substrate specificity was observed after the individu al mutations. However, mutation of Leu95 to Tyr had different effects on the kinetic constants depending on the substitution pattern of the flavonoid B ring, suggesting that the tyrosine side chain may be in di rect contact with this part of the molecule. The function of conserved amino acids present in domain II was also investigated. Unconservativ e mutation?, at Lys134, Tyr137 and Tyr150 resulted in protein instabil ity in solution, suggesting that these residues might be important for tile structural stability of the enzyme. Replacement of Arg140 with L ys or Ser had no effect on protein stability, but resulted in a strong reduction in specific activity. The results of photoaffinity-labeling experiments with PAdoP[S-35]S suggest that this residue is required t o bind the cosubstrate. In addition, the reduced affinity of [Ser140]S T for 3'-phosphoadenosine 5'-phosphate (PAdoP)-agarose indicates that Arg140 is also involved in binding the coproduct. Replacement of His11 8 with Glu or Ala resulted in a strong reduction in catalytic activity . However, [Lys118]ST retained a significant amount of catalytic activ ity. The result of photoaffinity-labeling experiments with PAdoP[S-35] S and affinity chromatography on PAdoP-agarose suggest that His118 mig ht be involved in catalysis in the flavonol 3-ST.