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 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, 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 K_m 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 K_i values of 1.2 μmolx1⁻¹ and 40 μmolx1⁻¹, respectively.