Structural and mechanistic studies on anthocyanidin synthase catalysed oxidation of flavanone substrates: the effect of C-2 stereochemistry on product selectivity and mechanism

During the biosynthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C-ring are catalysed by Fe(ii) and 2-oxoglutarate dependent oxygenases. The reactions catalysed by three of these enzymes; flavone synthase I, flavonol synthase and anthocyanidin synthase (ANS), are formally desaturations. In comparison, flavanone 3beta-hydroxylase catalyses hydroxylation at the C-3 pro-R position of 2S-naringenin. Incubation of ANS with the unnatural substrate (+/-)-naringenin results in predominantly C-3 hydroxylation to give cis-dihydrokaempferol as the major product; trans-dihydrokaempferol and the desaturation product, apigenin are also observed. Labelling studies have demonstrated that some of the formal desaturation reactions catalysed by ANS proceed via initial C-3 hydroxylation followed by dehydration at the active site. We describe analyses of the reaction of ANS with 2S- and 2R-naringenin substrates, including the anaerobic crystal structure of an ANS-Fe-2-oxoglutarate-naringenin complex. Together the results reveal that for the 'natural' C-2 stereochemistry of 2S-naringenin, C-3 hydroxylation predominates (>9 : 1) over desaturation, probably due to the inaccessibility of the C-2 hydrogen to the iron centre. For the 2R-naringenin substrate, desaturation is significantly increased relative to C-3 hydroxylation (ca. 1 : 1); this is probably a result of both the C-3 pro-S and C-2 hydrogen atoms being accessible to the reactive oxidising intermediate in this substrate. In contrast to the hydroxylation-elimination desaturation mechanism for some ANS substrates, the results imply that the ANS catalysed desaturation of 2R-naringenin to form apigenin proceeds with a syn-arrangement of eliminated hydrogen atoms and not via an oxygenated (gem-diol) flavonoid intermediate. Thus, by utilising flavonoid substrates with different C-2 stereochemistries, the balance between C-3 hydroxylation or C-2, C-3 desaturation mechanisms can be altered.