TAILIEUCHUNG - Báo cáo khoa học: Functional expression and mutational analysis of flavonol synthase from Citrus unshiu

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and Fe II /2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. | Eur. J. Biochem. 269 4134-4142 2002 FEBS 2002 doi Functional expression and mutational analysis of flavonol synthase from Citrus unshiu Frank Wellmann Richard Lukacin1 I Takaya Moriguchi2 Lothar Britsch3 Emile Schiltz4 and Ulrich Matern1 1Institut fur Pharmazeutische Biologie Philipps-Universitat Marburg Germany 2 National Institute of Fruit Tree Science Ibaraki Japan 3Merck kgaA Scientific Laboratory Products Darmstadt Germany 4Institut fur Organische Chemie und Biochemie Universitdt Freiburg Germany Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII 2-oxoglutarate for activity and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species but none of the enzymes had been studied in detail. Therefore a cDNA from Citrus unshiu Satsuma mandarin designated as flavonol synthase was expressed in Escherichia coli and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m z 37888 40 as compared to 37899 Da calculated for the translated polypeptide and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37 C. Km values of 272 11 and 36 M were determined for dihydroquercetin Fe11 and 2-oxoglutarate respectively with a sixfold higher affinity to dihydrokaempferol Km 45 M . Flavonol synthase polypeptides share an overall sequence similarity of 85 47 identity whereas only 30-60 similarity were apparent with other dioxygenases. Like the other dioxygenases of this class Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines His221 .

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