Purification and characterization of (2 S )‐flavanone 3‐hydroxylase from Petunia hybrida

(2 S )‐Flavanone 3‐hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2 S )‐naringenin to (2 R , 3 R )‐dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2‐oxoglutarate and Fe 2+ had to be added to the buffers. The hydroxylase was purified about 200‐fold by a six‐step procedure with low recovery. The M r of the enzyme was estimated by gel filtration to be about 74000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2‐oxoglutarate, Fe 2+ and ascorbate. With 2‐oxo[1‐ 14 C]glutarate in the enzyme assay dihydrokaempferol and 14 CO 2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)‐(2 R ,3 R )‐dihydrokaempferol. (2 S )‐Naringenin, but not the (2 R )‐enantiomer is a substrate of the hydroxylase. (2 S )‐Eriodictyol is converted to (2 R ,3 R )‐dihydroquercetin. In contrast, 5,7,3′, 4′, 5′‐pentahydroxy‐flavanone is not a substrate. Apparent Michaelis constants for (2 S )‐naringenin and 2‐oxoglutarate were determined to be respectively 5.6 μmolx1 −1 and 20 μmolx1 −1 at pH 8.5. The K m for (2 S )‐eriodictyol is 12 μmolx1 −1 at pH 8.0. Pyridine 2,4‐dicarboxylate and 2,5‐dicarboxylate are strong competitive inhibitors with respect to 2‐oxoglutarate with K i values of 1.2 μmolx1 −1 and 40 μmolx1 −1 , respectively.