The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6؆-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3؆, 6؆-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N-and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acylacceptor specificity of BAHD family to be understood.Plants produce more than 2 ϫ 10 5 types of secondary metabolites, many of which are of biomedical, pharmaceutical, and agricultural importance. A class of acyl-CoA-dependent acyltransferases plays versatile roles in the biosyntheses of these metabolites, making a very important contribution to the establishment of the structural and functional diversities of the metabolites (1). Although these acyltransferases show only low sequence similarities (15-30% identity) with each other, they share two highly conserved sequences, suggesting that they evolved from a common ancestor. Thus, a single protein family has been proposed for these diversified acyltransferases of biological and industrial significance and is referred to as the BAHD 4 family (1-4). Anthocyanin acyltransferases (AATs) form an important class of this family, catalyzing the transfer of an acyl group from acyl-CoA to a sugar moiety of an anthocyanin (2), a class of flavonoids that is the origin of most of the orange to blue colors of flowers and that plays important roles in plant reproduction and survival (5). The AAT-catalyzed acylation of anthocyanins is important for the stabilization, accumulation in vacuoles, and modulating the coloration of the pigments (2). It has been proposed that AAT catalysis proceeds through the formation of a ternary complex consisting of acyl-CoA, an anthocyanin, and the enzyme, where a general-base amino acid residue deprotonates a hydroxy group of the anthocyanin substrate, thereby promoting its nucleophilic attack on the carbonyl of the thioester of acyl-CoA (6).The red color of the chrysanthemum (Dendranthema ϫ morifolium) mainly arises from cyanidin 3-O-3Љ,6Љ-O-d...

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.

No abstract

No abstract

No abstract