The monomeric polypeptide comprises the functional flavanone 3β-hydroxylase from Petunia hybrida

Abstract

Flavanone 3β-hydroxylase catalyzes the FeII/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the biosynthesis of flavonoids, catechins and anthocyanidins. The enzyme had been partially purified from Petunia hybrida and proposed to be active as a dimer of roughly 75 kDa in size. More recently, the Petunia 3β-hydroxylase was cloned and shown to be encoded in a 41 655 Da polypeptide. In order to characterize the molecular composition, the enzyme was expressed in a highly active state in Escherichia coli and purified to apparent homogeneity. Size exclusion chromatographies of the pure, recombinant enzyme revealed that this flavanone 3β-hydroxylase exists in functional monomeric and oligomeric forms. Protein cross-linking experiments employing a specific homobifunctional sulfhydryl group reagent or the photochemical activation of tryptophan residues confirmed the tendency of the enzyme to aggregate to oligomeric complexes in solution. Thorough equilibrium sedimentation analyses, however, revealed a molecular mass of 39.2±12 kDa for the recombinant flavanone 3β-hydroxylase. The result implies that the monomeric polypeptide comprises the catalytically active flavanone 3β-hydroxylase of P. hybrida, which may readily associate in vivo with other proteins.