Masa-atsu Yamaguchi

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 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 °C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6″-O-malonylglucoside, 19 μm) and UDP-glucuronate (Km, 476 μm). 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.

Anthocyanin-flavone copigmentation in bluish purple flowers of Japanese garden iris (Iris ensata Thunb.)

The in vitro identification of copigmentation was carried out using anthocyanins, such as malvidin 3RGac5G, petunidin 3RGac5G and delphinidin 3RGac5G and the flavone isovitexin. These are major pigments of Iris ensata. These anthocyanins brought about copigmentation, i.e., the bathochromic shift (bluing effect) of visible λ max due to increased concentrations of isovitexin, and 32 to 35 nm were estimated as the magnitude (Δλ max) of each shift. In addition, the absorption spectrum of 0.1 mM malvidin 3RGac5G, 0.07 mM petunidin 3RGac5G and 0.7 mM isovitexin mixture closely matched those of the fresh outer perianths of the bluish purple cultivars, ‘Suiten-isshoku’, ‘Hekikai’ and ‘Yakonotama’ which belong to the malvidin 3RGac5G - petunidin 3RGac5G type of I. ensata. Therefore, these results indicated that the bluing effect on the flower color of the bluish purple cultivars of this species was caused at least in part by the copigmentation between these anthocyanins and the flavone isovitexin. For the copigment effects of isovitexin among malvidin 3RGac5G, petunidin 3RGac5G and delphinidin 3RGac5G, λ max and Δλ max of delphinidin 3RGac5G was slightly higher than those of malvidin 3RGac5G or petunidin 3RGac5G, and the copigmentation of delphinidin 3RGac5G was characterized by a higher concentration of isovitexin. Finally, the breeding for blue flowers due to copigmentation of delphinidin 3RGac5G with isovitexin in I. ensata was discussed.

Anthocyanins acylated with malic acid in Dianthus caryophyllus and D. deltoides

Abstract The major anthocyanin in pink and red forms of Dianthus caryophyllus has been identified as pelargonidin 3-malylglucoside. The corresponding cyanidin 3-malylglucoside has been found in red flowers of D. deltoides. This is the first complete characterization in plants of anthocyanins substituted with malic acid.

p-Hydroxybenzoyl-Glucose Is a Zwitter Donor for the Biosynthesis of 7-Polyacylated Anthocyanin in Delphinium

Polyacylation of anthocyanin at the 7-position is crucial for the blue pigmentation of delphinium flowers. 7-Polyacylated anthocyanin is synthesized via a step-by-step reaction mediated by vacuolar type acyltransferases and glucosyltransferases in delphiniums. In this reaction sequence, p-hydroxybenzoyl-Glc plays a crucial role as a bifunctional donor for glucosylation and acylation. The blue color of delphinium (Delphinium grandiflorum) flowers is produced by two 7-polyacylated anthocyanins, violdelphin and cyanodelphin. Violdelphin is derived from the chromophore delphinidin that has been modified at the 7-position by Glc and p-hydroxybenzoic acid (pHBA) molecules. Modification of violdelphin by linear conjugation of Glc and pHBA molecules to a Glc moiety at the 7-position produces cyanodelphin. We recently showed that anthocyanin 7-O-glucosylation in delphinium is catalyzed by the acyl-Glc–dependent anthocyanin glucosyltransferase (AAGT). Here, we sought to answer the question of which enzyme activities are necessary for catalyzing the transfer of Glc and pHBA moieties to 7-glucosylated anthocyanin. We found that these transfers were catalyzed by enzymes that use p-hydroxybenzoyl-Glc (pHBG) as a bifunctional acyl and glucosyl donor. In addition, we determined that violdelphin is synthesized via step-by-step enzymatic reactions catalyzed by two enzymes that use pHBG as an acyl or glucosyl donor. We also isolated a cDNA encoding a protein that has the potential for p-hydroxybenzoylation activity and two AAGT cDNAs that encode a protein capable of adding Glc to delphinidin 3-O-rutinoside-7-O-(6-O-[p-hydroxybenzoyl]-glucoside) to form violdelphin.

Characterization of anthocyanin p-coumaroyltransferase in flowers of Iris ensata

Malvidin and petunidin 3-(p-coumaroyl)rhamnosylglucoside-5-glucosides as well as nonacylated 3-rhamnosylglucoside-5-glucoside of these anthocyanidins were detected as major anthocyanins in cyanic flowers of Iris ensata. Enzyme extracts from flower buds of this plant catalyzed the transfer of the p-coumaroyl moiety from p-coumaroyl-CoA to both the anthocyanidin 3-rhamnosylglucoside and 3-rhamnosylglucoside-5-glucoside to form the anthocyanidin 3-(p-coumaroyl) rhamnosylglucoside and 3-(p-coumaroyl) rhamnosylglucoside-5-glucoside, at a ratio of ca. 1 to 4, respectively. The activities of this enzyme were also examined for various cyanic and acyanic cultivars, in addition to the characterization of the p-coumaroyltransferase. The sequence of acylation and 5-glucosylation in the anthocyanin biosynthesis of this plant is discussed.

Purification and characterization of anthocyanin 3-aromatic acyltransferase from Perilla frutescens

Abstract Hydroxycinnamoyl-CoA:anthocyanin 3- O- glucoside-6′′- O -hydroxycinnamoyltransferase (3AT; EC 2.3.1.-) was identified and highly purified from red leaves of Perilla frutescens , which accumulate cyanidin 3-(6- O - p -coumaroyl-β- d -glucoside)-5-(6- O -malonyl-β- d -glucoside). 3AT was a 50 kDa monomer of protein with a pI of 5.3. It catalyzed the transfer of the p -coumaric and caffeic acids from their CoA esters to the 3-glucosyl moiety of delphinidin, cyanidin and pelargonidin 3-glucoside. Anthocyanidin 3,5-diglucosides were also good substrates, while cyanidin 3-rutinoside was a poor substrate. Malonyl-CoA was not used by the enzyme as an acyl-donor. Aromatic acylation of anthocyanin by 3AT caused a bathochromic shift. Lower K m values for anthocyanidin 3-glucoside than 3,5-diglucoside may indicate that acylation of cyanidin 3-glucoside precedes 5- O -glucosylation in vivo.

1H NMR spectral analysis of the malylated anthocyanins from Dianthus

Abstract The structures of malylated anthocyanins from carnation Dianthus caryophyllus flowers were confirmed as the 3- O -(6- O -malyl-β- D -glucopyranosides) of pelargonidin and cyanidin by 400 MHz FT-NMR.

Cyanidin 3-malonylglucoside and malonyl-coenzyme a: Anthocyanidin malonyltransferase in Lactuca sativa leaves

Abstract A pigment isolated from Lactuca sativa leaves was identified as cyanidin 3- O -(6″-malonylglucoside) by chromatographic and spectral methods. The acyltransferase extracted from the leaves catalysed the malonylation of anthocyanidin 3-glucosides with malonyl-CoA as an acyl donor. This enzyme was characterized in more detail.

Anthocyanidin 3-glucoside malonyltransferase from Dahlia variabilis

Abstract Cyanic flowers of Dahlia variabilis contained monomalonylated anthocyanins, 3-(6″-malonylglucoside)-5-glucosides of pelargonidin and cyanidin, and dimalonylated anthocyanins, 3,5-di(malonylglucoside)s of pelargonidin and cyanidin, in addition to nonmalonylated 3,5-diglucosides of these anthocyanidins. Enzyme extracts from this plant catalyzed the malonylation of anthocyanidin 3-glucoside to anthocyanidin 3-(6″-malonylglucoside), but not the 3,5-diglucoside to 3-(6″-malonylglucoside)-5-glucoside or 3,5-di(malonylglucoside). The amounts of this enzyme were also examined for cyanic and acyanic cultivars.

Anthocyanins in The Dark Purple Anthers of Tulipa gesneriana: Identification of Two Novel Delphinidin 3-O-(6-O-(Acetyl-α-Rhamnopyranosyl)-β-Glucopyranosides)

Two novel anthocyanins, delphinidin 3-O-(6-O-(2-O-acetyl-α-rhamnopyranosyl)-β-glucopyranoside) and delphinidin 3-O-(6-O-(3-O-acetyl-α-rhamnopyranosyl)-β-glucopyranoside), were identified from the anthers of Tulipa gesneriana. These and delphinidin 3-O-(6-O-(α-rhamnopyranosyl)-β-glucopyranoside) made up over 80% of the anthocyanin content in the dark purple anthers and could be responsible for the intense color of the anthers.