UDP-glucuronic acid:anthocyanin glucuronosyltransferase from red daisy (Bellis perennis) flowers -: Enzymology and phylogenetics of a novel glucuronosyltransferase involved in flower pigment biosynthesis

In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDPglucuronate to the 2-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6-O-malonylglucoside with a k(cat) value of 34 s(-1) at pH 7.0 and 30 degreesC. BpUGAT was highly specific for cyanidin 3-O-glucosides (e.g. K-m for cyanidin 3-O-6-O-malonylglucoside, 19 muM) and UDP-glucuronate (K-m, 476 muM). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily ( according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28 - 40% identity). Although the biological role ( pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.