Reaction mechanism of ANS In general, the first colored compound in the biosynthetic pathway of anthocyanin is anthocyanidin (pelargonidin, cyanidin and delphinidin), which is derived from colorless leucoanthocyanidin. In P. frutescens, leucocyanidin is converted into cyanidin (Fig. 3). From leucoanthocyanidin (flavan-3,4-cis-diol) to anthocyanidin (flavylium ion), dehydrogenation from C-2 and dehydration from C-3,4 takes place formally (Fig. 4). However, actual reactions involved in this critical step for coloring of anthocyanin have not been clarified in vitro, because attempts to demonstrate the cell-free activity of conversion from leucoanthocyanidin to anthocyanidin or its 3-glucoside have been unsuccessful (Heller & Forkmann, 1993).
It has been suggested that ANS catalyzes the reaction(s) from leucoanthocyanidin to anthocyanidin. The cDNA encoding putative ANS was isolated from maize mutant (a2) by transposon tagging; and the involvement of the A2 gene in the coloring of anthocyanin was confirmed by the complementation of the a2 mutant by transient expression of the intact A2 gene (Menssen et al., 1990). Although the deduced amino acid sequence of A2 cDNA exhibited significant similarities with those of a family of 2-oxoglutarate-dependent oxygenases, no biochemical investigation was carried out until the first study using the recombinant enzyme from P. frutescens (Saito et al., 1999). This has been ‘a missing link’ where molecular biology failed to meet biochemistry.
A cDNA encoding ANS was isolated from red and green forms of P. frutescens by differential display of mRNA (Yamazaki et al., 1997). As well as recombinant perilla ANS (Saito et al., 1999), ANS proteins of petunia, maize, snapdragon and torenia were produced as fusion proteins with maltose-binding-protein (Nakajima et al., 2001). By using these recombinant proteins, the formation of anthocyanidins from leucoanthocyanidins was detected after the incubation in the presence of Fe2+, 2-oxoglutarate, molecular oxygen and ascorbate, being followed by acidification (pH 1–5). As an important feature of the reaction catalyzed by ANS, no additional enzyme(s) such as dehydratase was required in spite of the involvement of a formal dehydration step.
These results suggest that the reaction mechanism from leucoanthocyanidin to anthocyanidin was catalyzed by ANS as illustrated in Fig. 4(a). The reaction catalyzed by 2-oxoglutarate-dependent oxygenases includes two steps. In the first step, ANS binds with ferrous ion, which acts as a catalytic center of the reaction, and composes a complex with molecular oxygen and 2-oxoglutarate followed by the formation of an oxoferryl enzyme complex, succinate and CO2 (reaction (i) in Fig. 4). This first step is common to all 2-oxoglutarate-dependent oxygenases. In the second step, the oxoferryl species is used for the hydrogen radical abstraction from C-2 and C-3, or for hydroxylation at C-2 or C-3 followed by spontaneous dehydration that yields 2-flaven-3,4-diol and H2O (reaction (ii)). Subsequently, isomerization of the hydroxyl group and double bond of 2-flaven-3,4-diol occurs spontaneously to yield the thermodynamically more stable 3-flaven-2,3-diol (pseudobase) at cytosolic pH (reaction (iii)). The enzyme 3-GT catalyzes 3-O-glucosylation of the pseudobase form under cytosolic neutral conditions (reaction (iv)) (see below).
In the flavonoid biosynthetic pathway, there are four reactions which are catalyzed by 2-oxoglutarate-dependent oxygenases, ANS, F3H (Britsch, 1990b; Britsch et al., 1992), flavonol synthase (Holton et al., 1993) and flavone synthase I (Britsch, 1990a). All these reactions are concerned with oxidation at the C-2 and/or C-3 position of the flavonoid skeleton. Since no stable hydroxylated intermediates were detected in the reactions of ANS, flavonol synthase and flavone synthase I, the mechanism involving direct 2,3-desaturation seems to be the most likely route for the formation of 2-flaven-3,4-diol (Fig. 4a). Regarding the reaction mechanism of flavone synthase I, a radical mechanism was proposed for the direct 2,3-dehydrogenation of flavanone (Britsch, 1990a).