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

Flavanone 3 beta-hydroxylase catalyzes the Fe-II/oxoglutarate-dependent hyd roxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the biosynthes is of flavonoids, catechins and anthocyanidins, The enzyme had been partial ly purified from Petunia hybrida and proposed to be active as a dimer of ro ughly 75 kDa in size. More recently, the Petunia 3 beta-hydroxylase was clo ned and shown to be encoded in a 41 655 Da polypeptide, In order to charact erize the molecular composition, the enzyme was expressed in a highly activ e state in Escherichia coli and purified to apparent homogeneity. Size excl usion chromatographies of the pure, recombinant enzyme revealed that this f lavanone 3 beta-hydroxylase exists in functional monomeric and oligomeric f orms. Protein cross-linking experiments employing a specific homobifunction al sulfhydryl group reagent or the photochemical activation of tryptophan r esidues confirmed the tendency of the enzyme to aggregate to oligomeric com plexes in solution. Thorough equilibrium sedimentation analyses, however, r evealed a molecular mass of 39.2 +/- 12 kDa for the recombinant flavanone 3 beta-hydroxylase. The result implies that the monomeric polypeptide compri ses the catalytically active flavanone 3 beta-hydroxylase of P, hybrida, wh ich may readily associate in vivo with other proteins. (C) 2000 Federation of European Biochemical Societies.