Publications

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

  Sawada S., Suzuki H., Ichimaida F., Yamaguchi M.A., Iwashita T., Fukui Y., Hemmi H., Nishino T., Nakayama T.

  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 th ... >> More

  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 UDP-glucuronate to the 2''-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6''-O-malonylglucoside with a kcat value of 34 s(-1) at pH 7.0 and 30 degrees C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6''-O-malonylglucoside, 19 microM) and UDP-glucuronate (Km, 476 microM). 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. << Less

  J. Biol. Chem. 280:899-906(2005) [PubMed] [EuropePMC]

• Catalytic key amino acids and UDP-sugar donor specificity of a plant glucuronosyltransferase, UGT94B1: molecular modeling substantiated by site-specific mutagenesis and biochemical analyses.

  Osmani S.A., Bak S., Imberty A., Olsen C.E., Moeller B.L.

  The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidin-derived flavonoid in red daisy (Bellis perennis). The functional properties of BpUGT94B1 were investigated using protein modeling, site-directed mutagenesis, and analysis of the subst ... >> More

  The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidin-derived flavonoid in red daisy (Bellis perennis). The functional properties of BpUGT94B1 were investigated using protein modeling, site-directed mutagenesis, and analysis of the substrate specificity of isolated wild-type and mutated forms of BpUGT94B1. A single unique arginine residue (R25) positioned outside the conserved plant secondary product glycosyltransferase region was identified as crucial for the activity with UDP-glucuronic acid. The mutants R25S, R25G, and R25K all exhibited only 0.5% to 2.5% of wild-type activity with UDP-glucuronic acid, but showed a 3-fold increase in activity with UDP-glucose. The model of BpUGT94B1 also enabled identification of key residues in the acceptor pocket. The mutations N123A and D152A decreased the activity with cyanidin 3-O-glucoside to less than 15% of wild type. The wild-type enzyme activity toward delphinidin-3-O-glucoside was only 5% to 10% of the activity with cyanidin 3-O-glucoside. Independent point mutations of three residues positioned near the acceptor B ring were introduced to increase the activity toward delphinidin-3-O-glucoside. In all three mutant enzymes, the enzymatic activity toward both acceptors was reduced to less than 15% of wild type. The model of BpUGT94B1 allowed for correct identification of catalytically important residues, within as well as outside the plant secondary product glycosyltransferase motif, determining sugar donor and acceptor specificity. << Less

  Plant Physiol. 148:1295-1308(2008) [PubMed] [EuropePMC]