Chimeric flavonol sulfotransferases define a domain responsible for substrate and position specificities.
The pFST3 and pFST4' cDNAs encode flavonol sulfotransferases (ST) that are 69% identical in amino acid sequence yet exhibit strict substrate and position specificities. To determine the domain responsible for the properties of the flavonol STs, several chimeric flavonol STs were constructed by the reciprocal exchange of DNA fragments derived from the plasmids pFST3 and pFST4' and by the expression of the corresponding chimeric proteins in Escherichia coli. The chimeric enzymes were enzymatically active even though their activities were reduced compared to the parent enzymes. The specificity of the resulting hybrid proteins indicates that an interval of the flavonol STs spanning amino acids 92-194 of the flavonol 3-ST sequence contains the determinant of the substrate and position preferences. From the comparison of the amino acid sequences between plant and animal STs, this interval can be subdivided into a highly conserved region corresponding to positions 134-152 of the flavonol 3-ST, flanked by two regions of high divergence from 98 to 110 and 153 to 170. In view of the similarities in length and hydropathic profiles as well as the presence of four conserved regions between plant and animal STs, the results of these experiments suggest that this interval is involved in the recognition of substrates and/or catalysis in all STs.
Varin
L
L
Département de Biologie, Université Laval, Ste-Foy, Québec, Canada.
Marsolais
F
F
Brisson
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005419:Flavonoids / Q000378:metabolism*; D044948:Flavonols; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*
D005419:Flavonoids; D044948:Flavonols; D011993:Recombinant Fusion Proteins; D015238:Sulfotransferases; C072964:flavonol 4'-sulfotransferase; C072963:flavonol 3-sulfotransferase