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J. Biol. Chem., Vol. 280, Issue 2, 899-906, January 14, 2005

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

ENZYMOLOGY AND PHYLOGENETICS OF A NOVEL GLUCURONOSYLTRANSFERASE INVOLVED IN FLOWER PIGMENT BIOSYNTHESIS^*

Shin'ya Sawada, Hirokazu Suzuki, Fumiko Ichimaida, Masa-atsu Yamaguchi, Takashi Iwashita¶, Yuko Fukui||, Hisashi Hemmi, Tokuzo Nishino, and Toru Nakayama**

From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579, the Faculty of Horticulture, Minami-Kyushu University, Miyazaki 884-0003, and the ^¶Suntory Institute for Bioorganic Research and ^||Suntory Research Center, Mishima-gun, Shimamoto-cho, Osaka 618-8503, Japan

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 k_cat value of 34 s^–1 at pH 7.0 and 30 °C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. K_m for cyanidin 3-O-6''-O-malonylglucoside, 19 µM) and UDP-glucuronate (K_m, 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.

Received for publication, September 13, 2004 , and in revised form, October 21, 2004.

^* This work was supported in part by the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1S and 2S.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB190262 (for BpUGAT).

^** To whom correspondence should be addressed. Fax: 81-22-217-7293; E-mail: nakayama{at}seika.che.tohoku.ac.jp.

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