cDNA cloning and functional characterization of flavonol 3-O-glucoside-6″-O-malonyltransferases from flowers of Verbena hybrida and Lamium purpureum

Complementary DNAs coding for two flavonol 3-O-glucoside malonyltransferases, Vh3MaT1 and Lp3MaT1, were cloned from flowers of Verbena hybrida and Lamium purpureum, respectively, expressed in Escherichia coli cells, and functionally characterized. The cloning strategy took full advantage of the specific conservation of a sequence (–Tyr–Phe–Gly–Asn–Cys–, termed motif 2) in the anthocyanin-specific members of the versatile acyltransferase (VAT) family. Both of the expressed proteins, Vh3MaT1 and Lp3MaT1, effectively catalyzed the regiospecific transfer of the malonyl group from malonyl-CoA to the 6″-hydroxyl group of quercetin 3-O-glucoside and were highly specific for these acyl donor and acceptor. Therefore, these enzymes are malonyl-CoA:flavonol 3-O-glucoside-6″-O-malonyltransferases. Kinetic parameters were determined at pH 7.0 and 30 °C as follows: for Vh3MaT1, kcat, 2.9 s−1; kcat/Km for quercetin 3-O-glucoside, 17 s−1 mM−1; and kcat/Km for malonyl-CoA, 930 s−1 mM−1; for Lp3MaT1, kcat, 9.0 s−1; kcat/Km for quercetin 3-O-glucoside, 28 s−1 mM−1; and kcat/Km for malonyl-CoA, 360 s−1 mM−1. These results suggested that Vh3MaT1 and Lp3MaT1 should serve as useful biocatalysts for the malonylation of quercetin 3-O-glucoside to control the bioactivities and pharmacokinetics of the flavonoid. The results also show that VAT members having motif 2 are not restricted to “anthocyanin” acyltransferases but should include an extended class of enzymes, i.e. “flavonoid glucoside” acyltransferases.