F. Marsolais et L. Varin, MUTATIONAL ANALYSIS OF DOMAIN-II OF FLAVONOL 3-SULFOTRANSFERASE, European journal of biochemistry, 247(3), 1997, pp. 1056-1062
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.