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

FEBS Letters

Gröning, Inga²  Urbanke, Claus¹  Lukačin, Richard²  Matern, Ulrich² 

[1] Medizinische Hochschule Hannover, Betriebseinheit Biophysikalisch-Biochemische Verfahren, Carl Neuberg Str. 1, D-30623 Hannover, Germany;Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, D-35037 Marburg, Germany

关键词: Flavonoid biosynthesis;    Monomeric structure;    Flavanone 3β-hydroxylase;    2-Oxoglutarate-dependent dioxygenase;    Petunia hybrida;    ACC oxidase;    1-aminocyclopropane-1-carboxylic acid oxidase;    FHT;    flavanone 3β-hydroxylase;    DAOCS;    deacetoxycephalosporin C synthase;    DTT;    dithiothreitol;    IPNS;    isopenicillin N synthase;    SDS–PAGE;    polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate;   

DOI  :  10.1016/S0014-5793(00)01116-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Flavanone 3β-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 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.

【 授权许可】

Unknown