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Purification and characterization of (2S)‐flavanone 3‐hydroxylase from Petunia hybrida

European Journal of Biochemistry, ISSN: 1432-1033, Vol: 156, Issue: 3, Page: 569-577
1986
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(2S)‐Flavanone 3‐hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)‐naringenin to (2R, 3R)‐dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2‐oxoglutarate and Fe had to be added to the buffers. The hydroxylase was purified about 200‐fold by a six‐step procedure with low recovery. The M of the enzyme was estimated by gel filtration to be about 74000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2‐oxoglutarate, Fe and ascorbate. With 2‐oxo[1‐C]glutarate in the enzyme assay dihydrokaempferol and CO are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)‐(2R,3R)‐dihydrokaempferol. (2S)‐Naringenin, but not the (2R)‐enantiomer is a substrate of the hydroxylase. (2S)‐Eriodictyol is converted to (2R,3R)‐dihydroquercetin. In contrast, 5,7,3′, 4′, 5′‐pentahydroxy‐flavanone is not a substrate. Apparent Michaelis constants for (2S)‐naringenin and 2‐oxoglutarate were determined to be respectively 5.6 μmolx1 and 20 μmolx1 at pH 8.5. The K for (2S)‐eriodictyol is 12 μmolx1 at pH 8.0. Pyridine 2,4‐dicarboxylate and 2,5‐dicarboxylate are strong competitive inhibitors with respect to 2‐oxoglutarate with K values of 1.2 μmolx1 and 40 μmolx1, respectively. Copyright © 1986, Wiley Blackwell. All rights reserved

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