The anthocyanin biosynthesis pathway is now well known and most of the enzymes are characterised. In the formation of blue pigments a few enzymes have particularly important roles, in particular flavonoid 35-hydroxylase (F35H) and dihydroflavonol 4-reductase (DFR).
The flavonoid 35H-hydroxylase is responsible for the introduction of the second and third hydroxyl group in the B-ring of dihydrokaempferol (DHK) or naringenin which are regarded as the main substrates of the reaction. Product of the reaction with DHK is dihydromyricetin (DHM), precursor for synthesis of all delphinidin type anthocyanin. Enzyme is a member of cytochrome P450 protein family (P450s). It is a very diverse group of heme-containing oxidases, which catalyse NADPH- or NADH-dependent oxidation. F35H was classified into CYP75A subfamily. This enzyme this is regarded as necessary for the blue pigment formation.
Dihydroflavonol 4-reductase is the oxidoreductase that catalyses in the presence of NADPH the stereospecific reduction of the keto group in position 4 of dihydroflavonols producing colourless leucoanthocyanidins as a precursor for anthocyanin formation. Enzyme can show substrate specificity with respect to the B-ring hydroxylation pattern of the dihydroflavonol and can therefore have an influence on the type of formed anthocyanin. For the blue pigment formation, necessary is enzyme, which accept dihydromyricetin (DHM) as a substrate. Product of DFR reaction with DHM in the following steps of the pathway is converted to delphinidin type blue pigments.