The monomeric polypeptide comprises the functional flavanone 3beta-hydroxylase from Petunia hybrida.
Flavanone 3beta-hydroxylase catalyzes the Fe(II)/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 3beta-hydroxylase was cloned and shown to be encoded in a 41655 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 3beta-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 3beta-hydroxylase. The result implies that the monomeric polypeptide comprises the catalytically active flavanone 3beta-hydroxylase of P. hybrida, which may readily associate in vivo with other proteins.
Lukacin
R
R
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, D-35037, Marburg, Germany.
Urbanke
C
C
Gröning
I
I
Matern
U
U
D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005419:Flavonoids / Q000096:biosynthesis; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
D003432:Cross-Linking Reagents; D005419:Flavonoids; D010940:Plant Proteins; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C048797:flavanone 3-dioxygenase