UDP-glucuronic Acid:Anthocyanin Glucuronosyltransferase from Red Daisy (Bellis perennis) Flowers

Abstract

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°C. BpUGAT was highly specific for cyanidin 3-O-glucosides (e.g. K m for cyanidin 3-O-6؆-O-malonylglucoside, 19 M) and UDP-glucuronate (K m , 476 M). 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.

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

“…Each of these Vv GT genes was predicted to have a single intron. Phylogenetic analysis revealed that each of these genes is related to the GT1 family phylogenetic cluster (cluster I) of flavonoid 3-O-glycosyltransferases, as expected (Vogt and Jones, 2000;Sawada et al, 2005;Noguchi et al, 2007). Moreover, these genes formed a subcluster that was distinct from the Vv GT1-related subcluster ( Figure 1B).…”

“…Previous studies of two GATs, Bp UGAT (UGT94B1) of the red daisy flower (Bellis perennis) (Sawada et al, 2005;Osmani et al, 2008) and Pf F7GAT (UGT88D7) of Perilla frutescens (Noguchi et al, 2009a), showed that Arg-25 in Bp UGAT and Arg-350 in Pf F7GAT play an important role in recognition of UDP-GA. These results suggest plasticity of the mechanisms that alter the sugar donor specificity of UGTs.…”

“…UDP-rhamnose was a generous gift from Keiko YonekuraSakakibara, RIKEN, Yokohama, Japan. Pelargonidin, cyanidin, delphinidin, apigenin, luteolin, (+)-catechin, genistein, naringenin, ferulic acid, caffeic acid, salicylic acid, capsaicin, trans-resveratrol, and esculetin were obtained as described previously (Sawada et al, 2005;Noguchi et al, 2007Noguchi et al, , 2008Noguchi et al, , 2009b.…”

“…The branches shown in red and black indicate the foreground and background lineages, respectively, of the UGT gene clusters on the basis of a branch-specific model with the dN/dS ratios of v 1 = 0.34 and v 0 = 0.22, as suggested by the maximum likelihood analysis (Yang, 2007 (Vogt and Jones, 2000) and a catalytic mechanism that inverts the anomeric configuration of a transferred sugar (Campbell et al, 1997;Wang, 2009). Phylogenetic analyses of plant family 1 UGTs show several phylogenetic clusters, which appear to be characterized by the regiospecificity of their glycosyl transfer to flavonoids (Vogt and Jones, 2000;Sawada et al, 2005;Noguchi et al, 2007). Moreover, the differentiation of the sugar donor specificity of UGTs appears to have occurred in a lineage-specific manner after establishment of general regiospecificity for the glycosyl acceptor (Yonekura-Sakakibara et al, 2007;Noguchi et al, 2009a;Ono et al, 2010).…”

Functional analysis of a cotton glucuronosyltransferase promoter in transgenic tobaccos

“…Furthermore, the a-D-glucuronic acid units can be attached to the O-2 position of the xylan backbone as side groups [2]. Additionally, Sawada et al [3] demonstrated that BpUGAT (Bellis perennis glucurowww.cell-research.com | Cell Research Ai-Min Wu et al 175 npg nosyltransferase) catalyzes the regiospecific transfer of GlcA to the 2-hydroxyl group of the 3-glucosyl moiety of the substrate anthocyanidin. These results suggest that the UGTs may be important in regulation of differentiation and secondary metabolism [4][5][6].…”