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Direct evidence for anthocyanidin synthase as a 2‐oxoglutarate‐dependent oxygenase: molecular cloning and functional expression of cDNA from a red forma of Perilla frutescens
Author(s) -
Saito Kazuki,
Kobayashi Mii,
Gong Zhizhong,
Tanaka Yoshikazu,
Yamazaki Mami
Publication year - 1999
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1999.00365.x
Subject(s) - anthocyanidin , anthocyanidins , biochemistry , chemistry , perilla frutescens , biology , complementary dna , anthocyanin , stereochemistry , gene , botany , flavonoid , antioxidant
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 CO 2 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.