Role of Serine 286 in cosubstrate binding and catalysis of a flavonol O-methyltransferase.
O-Methyltransferases catalyze the transfer of the methyl groups of S-adenosyl-L-methionine to specific hydroxyl groups of several classes of flavonoid compounds. Of the several cDNA clones isolated from a Chrysosplenium americanum library, FOMT3' encodes the 3'/5'-O-methylation of partially methylated flavonols. The recombinant protein of another clone, FOMTx which differs from FOMT3' by a single amino acid residue (Ser286Arg) exhibits no enzymatic activity towards any of the flavonoid substrates tested. Replacement of Ser 286 in FOMT3' with either Ala, Leu, Lys or Thr, almost abolished O-methyltransferase activity. In contrast with FOMT3', no photoaffinity labeling could be achieved using [(14)CH(3)]AdoMet with the mutant recombinant proteins indicating that Ser 286 is also required for cosubstrate binding. These results are corroborated by isothermal titration microcalorimetry measurements. Circular dichroism spectra ruled out any significant conformational differences in the secondary structures of both FOMT3' and Ser286Arg. Modeling FOMT3' on the structure of chalcone methyltransferase indicates that serine 286 is greater than 10 A from any of the residues of the active site or the AdoMet binding site of FOMT3'. At the same time, residues 282 to 290 are conserved in most of the Chrysosplenium americanum OMTs. These residues form a large part of the subunit interface, and at least five of these residues are within 4 A of the opposing subunit. It would appear, therefore, that mutations in Ser286 exert their influence by altering the contacts between the subunits and that these contacts are necessary for maintaining the integrety of the AdoMet binding site and active site of this group of enzymes.
Kornblatt
Jack
J
Biology Department's Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, QC H4B 1R6, Canada.
Muzac
Ingrid
I
Lim
Yoongho
Y
Ahn
Joong Hoon
JH
Ibrahim
Ragai K
RK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D002384:Catalysis; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D044948:Flavonols / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D044948:Flavonols; D010940:Plant Proteins; D012694:Serine; D008780:Methyltransferases; C114844:trimethylflavonol 3'-O-methyltransferase, Chrysosplenium americanum