Mutational analysis of domain II of flavonol 3-sulfotransferase.

The flavonol 3- and 4'-sulfotransferases (ST) from Flaveria chloraefolia catalyze the transfer of the sulfonate group from 3'-phosphoadenosine 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 responsible for the specificity of these enzymes. Within domain II, at least five amino acids specific to the 4'-ST that could bind the sulfate group of quercetin 3-sulfate were identified. In this study, these amino acid residues were introduced at equivalent positions in the flavonol 3-ST sequence by site-directed mutagenesis of the cloned cDNA. No reversal of the substrate specificity was observed after the individual 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 direct contact with this part of the molecule. The function of conserved amino acids present in domain II was also investigated. Unconservative mutations at Lys134, Tyr137 and Tyr150 resulted in protein instability in solution, suggesting that these residues might be important for the structural stability of the enzyme. Replacement of Arg140 with Lys 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[35S]S suggest that this residue is required to bind the cosubstrate. In addition, the reduced affinity of [Ser140]ST for 3'-phosphoadenosine 5'-phosphate (PAdoP)-agarose indicates that Arg140 is also involved in binding the coproduct. Replacement of His118 with Glu or Ala resulted in a strong reduction in catalytic activity. However, [Lys118]ST retained a significant amount of catalytic activity. The results of photoaffinity-labeling experiments with PAdoP[35S]S and affinity chromatography on PAdoP-agarose suggest that His118 might be involved in catalysis in the flavonol 3-ST.