Mechanistic study on the oxidation of anthocyanidin synthase by quantum mechanical calculation

Anthocyanidin synthase (ANS), a member of the 2-oxoglutarate-dependent dioxygenase family in flavonoid biosynthesis, catalyzes the conversion of leucoanthocyanidins (e.g. 2R,3S,4S-cis-leucocyanidin, LCD) to flav-2-en-3,4-diols, a direct precursor of colored anthocyanidins via flavan-3,3,4-triols. The detailed oxygenation mechanism of 2R,3S,4S-cis-LCD to flav-2-en-3,4-diols was investigated using the density functional theory method. An initial model for the calculation was constructed from a structure obtained by a 100-ps molecular dynamics simulation of Arabidopsis ANS under physiological conditions. This model consisted of an LCD molecule as the substrate together with an iron atom, two histidine residues, an aspartic acid residue, a succinate, and an oxygen atom as ligands of the iron atom. The results of the calculation indicated that both the C-3 and C-4 positions of LCD can be oxidized, although C-4 oxidation is preferable. The C-3 oxidation required several steps to form flavan-3,3,4-triol: 1) formation of Fe(III)-OH and a substrate C-3 radical via hydrogen atom abstraction by Fe(IV)=O, 2) formation of a C-3 ketone and a water molecule, 3) addition of OH(-) into the C-3 position of the ketone, and 4) addition of H(+) to form flavan-3,3,4-triol. On the other hand, C-4 oxidation of 2R,3S,4S-cis-LCD resulted in the direct formation of 2R,3R-trans-dihydroquercetin. These results suggest that the oxidation at C-3 of LCD, a key reaction for coloring in anthocyanin biosynthesis, can be regarded as a "side reaction" from the viewpoint of quantum mechanics of enzymatic reactions. Molecular evolutional implications of ANS and related proteins are discussed in terms of reaction dynamics.