Direct evidence for anthocyanidin synthase as a 2-oxoglutarate-dependent oxygenase: molecular cloning and functional expression of cDNA from a red forma ofPerilla frutescens

Authors

  • Kazuki Saito,

    1. Laboratory of Molecular Biology and Biotechnology, Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan, and
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  • Mii Kobayashi,

    1. Laboratory of Molecular Biology and Biotechnology, Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan, and
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  • Zhizhong Gong,

    1. Laboratory of Molecular Biology and Biotechnology, Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan, and
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  • Yoshikazu Tanaka,

    1. Institute for Fundamental Research, Suntory Ltd, Wakayama-dai 1-1-1, Shimamoto, Mishima, Osaka 618-8503, Japan
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  • Mami Yamazaki

    1. Laboratory of Molecular Biology and Biotechnology, Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan, and
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  • Accession number AB003779 in the GenBank/EMBL/DDBJ databases.

*For correspondence (fax +81 43 290 2905; e-mail ksaito@p.chiba-u.ac.jp).

Summary

Anthocyanidin synthase (ANS), an enzyme of the biosynthetic pathway to anthocyanin, has been postulated to catalyze the reaction(s) from the colorless leucoanthocyanidins to the colored anthocyanidins. Although cDNAs have been isolated that encode putative ANS, which exhibits significant similarities in amino acid sequence with members of a family of 2-oxoglutarate-dependent oxygenases, no biochemical evidence has been presented which identifies the actual reaction that is catalyzed by ANS. Here we show that anthocyanidins are formedin vitrothrough 2-oxoglutarate-dependent oxidation of leucoanthocyanidins catalyzed by the recombinant ANS and subsequent acid treatment. A cDNA encoding ANS was isolated from red and green formas ofPerilla frutescensby differential display of mRNA. Recombinant ANS tagged with maltose-binding-protein (MBP) was purified, and the formation of anthocyanidins from leucoanthocyanidins was detected by the ANS-catalyzed reaction in the presence of ferrous ion, 2-oxoglutarate and ascorbate, being followed by acidification with HCl. Equimolar stoichiometry was confirmed for anthocyanidin formation and liberation of CO2 from 2-oxoglutarate. The presumptive two-copy gene of ANS was expressed in leaves and stems of the red forma ofP. frutescensbut not in the green forma plant. This corresponds to the accumulation pattern of anthocyanin. The mechanism of the reaction catalyzed by ANS is discussed in relation to the molecular evolution of a family of 2-oxoglutarate-dependent oxygenases.

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