Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis.

ABSTRACT: BACKGROUND: Wheat (Triticum aestivum L.) O-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin), 3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous Medicago sativa caffeic acid/5-hydroxyferulic acid O-methyltransferase (MsCOMT) as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat. RESULTS: This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate. CONCLUSIONS: We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.

SUBMITTER: Zhou JM 

PROVIDER: S-EPMC3017781 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature