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 agly‐cones and position 4′ of flavonol 3‐sulfates. We identified previously a protein segment, designated domain 11, that contains all the determinants responsible for the specificity of these enzymes. Within domain 11, 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 solu