Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase

Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2 R,3 R)-dihydroflavonols, i.e. dihydrokaempferol, to the corresponding flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2 S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3β-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2 R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1H NMR and CD spectroscopies as (−)- trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional non-specific activity trans-hydroxylating the flavanones (2 S)-naringenin as well as the unnatural (2 R)-naringenin at C-3. The recombinant flavonol sythase also catalyzes the 3-hydroxylation of (±) naringenin.