Role of Serine 286 in cosubstrate binding and catalysis of a flavonolO-methyltransferase

Abstract

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 Å 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 Å 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.Key words: flavonoids, O-methyltransferase, photoaffinity labeling. Résumé : Les O-méthyltransférases (OMTs) catalysent le transfert de groupes méthyles de la S-adénosyl-méthionine sur des groupes hydroxyles spécifiques de plusieurs classes de flavonoïdes. Parmi plusieurs clones d'ADNc isolés d'une banque deChrysosplenium amiricanum, le FOMT3′ code une enzyme catalysant la 3′-5′-O-méthylation de flavonols partiellement méthylés. La protéine recombinante issue d'un autre clone, FOMTx, qui ne diffère de FOMT3′ que par un seul acide aminé (Ser286Arg), ne démontre aucune activité enzymatique envers les substrats flavonoïdes testés. Le remplacement de la Ser286 de FOMT3′ par une Ala, Leu, Lys ou Thr abolit presque totalement l'activité O-méthyltransférase. Contrairement à la FOMT3′, aucun marquage par photoaffinité n'est obtenu avec le [ 14 CH 3 ]AdoMet sur les protéines recombinantes mutantes, indiquant que la Ser286 est aussi requise pour la liaison du co-substrat. Ces résultats sont corroborés par des mesures de titration microcalorimétrique isothermique. Le spectre en dichroïsme circulaire exclut toute différence de conformation significative dans la structure secondaire de FOMT3′ et le mutant Ser286Arg. La modélisation de FOMT3′ sur la structure de la chalcone méthyltransférase indique que la sérine 286 est é...

Structure–activity relationships of wheat flavone O‐methyltransferase – a homodimer of convenience

“…The situation is more complex when looking at residues that are not at the active site of the protein and are a few angstroms from the interface. In the case of flavonol 3′,5′‐ O ‐methyltransferase, mutation S286R which definitely does not involve an interfacial residue, results in almost complete loss of activity [24]. It would appear that Ser286 communicates with the active site of the opposing subunit across the interface.…”

Photoaffinity Labeling and Mutational Analysis of 24‐C‐Sterol Methyltransferase Defines the AdoMet Binding Site

“…In several instances, radiolabeled AdoMet and 8-azido AdoMet were shown to specifically label the MTase using UV radiation [6][7][8][9][10][11]. In addition, trypsin digestion of AdoMet-bound proteins followed by chromatography to establish the peptide/amino acid-AdoMet adduct and site-directed mutagenesis experiments of amino acid residues predicted to be associated with the AdoMet binding site demonstrated the requirement for one or more conserved residues (i.e., Tyr, Cys or Pro) or motifs in MTase specific activity [12][13][14].…”

Functional analysis of members of the isoflavone and isoflavanone O-methyltransferase enzyme families from the model legume Medicago truncatula

“…Substrate specificities of OMTs can not be accurately predicted on the basis of sequence similarity alone (Schroder et al 2002), and in some cases a single amino acid change can alter substrate specificity (Frick and Kutchan 1999;Gang et al 2002). Elucidation of the crystal structures of caffeoyl CoA 3-OMT and three different group I OMTs from alfalfa (Zubieta et al 2001Ferrer et al 2005) has now made it possible to explore the structural basis of OMT substrate specificity by homology-based modeling (Hoffmann et al 2001;Gang et al 2002;Kornblatt et al 2004;Yang et al 2004).…”