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.

SummaryAnthocyanin acyltransferases (AATs) catalyze a regiospeci®c acyl transfer from acyl-CoA to the glycosyl moiety of anthocyanins, thus playing an important role in¯ower coloration. The known AATs are subfamily members of an acyltransferase family, the BAHD family, which play important roles in secondary metabolism in plants. Here, we describe the puri®cation, characterization, and cDNA cloning of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens)¯owers. The puri®ed enzyme (hereafter referred to as Ss5MaT2) is a monomeric 46-kDa protein that catalyzes the transfer of the malonyl group from malonyl-CoA to the 4 HHH -hydroxyl group of the 5-glucosyl moiety of anthocyanins. Thus, it is a malonylCoA:anthocyanin 5-glucoside 4HHH -O-malonyltransferase. On the basis of the partial amino acid sequences of the puri®ed enzyme, we isolated a cDNA that encodes an acyltransferase protein. The steady-state transcript level of the gene was the highest in recently opened, fully pigmented¯owers and was also correlated with the trend observed for an AAT gene responsible for the ®rst malonylation step during salvianin biosynthesis. Immunoprecipitation studies using antibodies against the recombinant acyltransferase protein corroborated the identity of this cDNA as that encoding Ss5MaT2. The deduced amino acid sequence of Ss5MaT2 showed a low similarity (22±24% identity) to those of AATs and lacked the AAT-speci®c signature sequence. A phylogenetic analysis suggested that Ss5MaT2 is more related to acetyl-CoA:benzylalcohol acetyltransferase (BEAT) rather than to AAT. This is another example in which enzymes with similar, although not identical, substrate evolved from different branches of the BAHD family.

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