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

Abstract

(2S)‐Flavanone 3‐hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)‐naringenin to (2R, 3R)‐dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2‐oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200‐fold by a six‐step procedure with low recovery. The Mr 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, Fe2+ and ascorbate. With 2‐oxo[1‐14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)‐(2R,3R)‐dihydrokaempferol. (2S)‐Naringenin, but not the (2R)‐enantiomer is a substrate of the hydroxylase. (2S)‐Eriodictyol is converted to (2R,3R)‐dihydroquercetin. In contrast, 5,7,3′, 4′, 5′‐pentahydroxy‐flavanone is not a substrate. Apparent Michaelis constants for (2S)‐naringenin and 2‐oxoglutarate were determined to be respectively 5.6 μmolx1−1 and 20 μmolx1−1 at pH 8.5. The Km for (2S)‐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 Ki values of 1.2 μmolx1−1 and 40 μmolx1−1, respectively. Copyright © 1986, Wiley Blackwell. All rights reserved