cDNA cloning and characterization of two Dendranthema x morifolium anthocyanin malonyltransferases with different functional activities

In the flowers of chrysanthemum (Dendranthema x morifolium), cyanidin 3-O-3",6"-O-dimalonylglucoside is accumulated as a dominant anthocyanin. In this study, we isolated two full-length cDNAs from chrysanthemum flowers, Dm3MaT1 and Dm3MaT2, coding for enzymes that should be responsible for malonylation steps involved in the biosynthesis of the pigment. Both cDNAs were heterologously expressed in Escherichia coli cells, and the expressed products were functionally characterized. The Dm3MaT1 cDNA coded for a protein of 459 amino acid residues with a calculated molecular weight of 51,224, which showed >70% of sequence identity to anthocyanidin 3-O-glucoside-6"-O-malonyltransferases of Dahlia variabilis and Senecio cruentus. Specificity analyses of Dm3MaT1 further confirmed its identity as an anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, which exclusively catalyzed the malonyl-CoA-dependent malonyl transfer to anthocyanidin 3-O-glucoside to produce anthocyanidin 3-O-6"-O-malonylglucoside. On the other hand, the Dm3MaT2 cDNA coded for a protein of 460 amino acid residues with a calculated molecular weight of 51,7 10, showing identities of 93% (for the nucleotide sequence) and 89% (for the deduced amino acid sequence) to Dm3MaT1. Unlike Dm3MaT1, the recombinant Dm3MaT2 catalyzed the consecutive dimalonyl transfer to anthocyanidin 3-O-glucoside; it produced anthocyanidin 3-O-6"-O-malonylglucoside from anthocyanidin 3-O-glucoside and could subsequently produce anthocyanidin 3-O-3",6"-O-dimalonylglucoside from anthocyanidin 3-O-6"-O-malonylglucoside. Thus, Dm3MaT2 was the anthocyanidin 3-O-glucoside-3",6"-O-dimalonyltransferase and should be responsible for the second malonylation of anthocyanins in the flower, providing the first example of anthocyanin acyltransferase that can catalyze the consecutive malony transfer. (C) 2003 Elsevier Ireland Ltd. All rights reserved.