Abstract
In the conversion from colorless leucoanthocyanidin to colored anthocyanidin 3-glucoside, at least two enzymes, anthocyanidin synthase (ANS) and UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT), are postulated to be involved. Despite the importance of this reaction sequence for coloring in anthocyanin biosynthesis, the biochemical reaction mechanism has not been clarified, and the possible involvement of a dehydratase has not been excluded. Here we show that recombinant ANSs from several model plant species, snapdragon, petunia, torenia, and maize, catalyze the formation of anthocyanidin in vitro through a 2-oxoglutarate-dependent oxidation of leucoanthocyanidin. Crude extracts of Escherichia coli, expressing recombinant ANSs from these plant species, and purified recombinant enzymes of petunia and maize catalyzed the formation of anthocyanidin in the presence of ferrous ion, 2-oxoglutarate, and ascorbate. The in vitro formation of colored cyanidin 3-glucoside from leucocyanidin, via a cyanidin intermediate, was demonstrated using petunia ANS and 3-GT. The entire reaction sequence did not require any additional dehydratase but was dependent on moderate acidic pH conditions following the enzymatic steps. The present study indicated that the in vivo cytosolic reaction sequence involves an ANS-catalyzed 2-oxoglutarate-dependent conversion of leucoanthocyanidin (flavan-3,4-cis-diol) to 3-flaven-2,3-diol (pseudobase), most probably through 2,3-desaturation and isomerization, followed by glucosylation at the C-3 position by 3-GT.
Highlights
In the conversion from colorless leucoanthocyanidin to colored anthocyanidin 3-glucoside, at least two enzymes, anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (3-GT), are postulated to be involved
The reaction leading from colorless leucoanthocyanidin to anthocyanidin and its 3-O-glucoside is the critical step in the formation of colored metabolites in anthocyanin biosynthesis [1,2,3] (Fig. 1)
Heterologous Expression of ANS and 3-GT in E. coli—The open reading frames of cDNAs encoding ANS from snapdragon, torenia, and maize and the cDNA encoding 3-GT from petunia were amplified by PCR and subcloned into pMAL-c2 expression vectors under the control of the tac promoter
Summary
No in vitro biochemical experimental evidence has been provided to confirm the reaction sequence from leucoanthocyanidin to anthocyanidin 3-glucoside, 3-GT activity has been detected in crude enzyme preparations of various plant species (19 –22) and in preparations containing recombinant 3-GT [23, 24]. It is necessary to examine whether anthocyanidin 3-glucoside can be formed from leucoanthocyanidin using both ANS and 3-GT recombinant proteins under physiological conditions, where enzyme reactions take place under cytosolic conditions (pH 7) followed by a shift to vacuolar conditions (pH 5) [26]. We show that the combination of recombinant ANS and 3-GT was sufficient and essential to yield cyanidin 3-glucoside from leucocyanidin under physiological conditions, by mimicking cytosolic enzyme reactions and transport into vacuoles
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