Functional Analysis of a Methyltransferase Involved in Anthocyanin Biosynthesis from Blueberries (Vaccinium corymbosum).

ABSTRACT

Anthocyanins are natural water-soluble pigments that widely exist in plants, with various biological activities, including antioxidant, anti-obesity, and anti-diabetic activities. Currently, monomeric anthocyanins are mainly obtained through natural sources, which limits their availability. In the biosynthesis of anthocyanins, anthocyanin methyltransferases are recognized to play important roles in the water solubility and structural stability of anthocyanins. Blueberries are a rich source of anthocyanins with more than 30 chemical structures. However, the enzymes that were responsible for the methylation of anthocyanidin cores in blueberries had not been reported. Here, blueberries (Vaccinium corymbosum) have been selected as the candidate for characterization of the key enzyme. Phylogenic analysis, enzymatic activity assay, homology modeling, molecular simulation, protein expression and purification assay, site-directed mutation, isothermal titration calorimetry assay, and enzyme kinetic assay were used to identify the enzymatic function and molecular mechanism of VcOMT, which was responsible for the methylation of anthocyanidin cores. VcOMT could use delphinidin as a substrate but not cyanidin, petunidin, anthocyanins, flavonols, and flavonol glycosides. Ile191 and Glu198 were both identified as important amino acid residues for the binding interactions of anthocyanidins with VcOMT.