Novel Flavonol 2-Oxoglutarate Dependent Dioxygenase: Affinity Purification, Characterization, and Kinetic Properties

A 2-oxoglutarate-dependent dioxygenase [EC 1.14.11–] that catalyzes the 6-hydroxylation of partially methylated flavonols has been purified to near homogeneity from Chrysosplenium americanum. Enzyme purification was achieved by fast protein liquid chromatography on Superose 12 and Mono Q columns as...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 2000-10, Vol.382 (2), p.161-172
Main Authors: Anzellotti, Dominique, Ibrahim, Ragai K
Format: Article
Language:English
Subjects:
Animals
Cations - pharmacology
Chemical Phenomena
Chemistry, Physical
Chromatography, Affinity
Chromatography, High Pressure Liquid
Chrysosplenium americanum
Flavonoids - chemistry
Flavonoids - metabolism
flavonol
Flavonols
Hydrogen-Ion Concentration
Immunochemistry
Isoelectric Point
Kinetics
Magnoliopsida - enzymology
Mass Spectrometry
Mixed Function Oxygenases - chemistry
Mixed Function Oxygenases - isolation & purification
Mixed Function Oxygenases - metabolism
Molecular Weight
partially methylated quercetin
Rabbits
Substrate Specificity
α-ketoglutarate-dependent dioxygenase
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Summary:A 2-oxoglutarate-dependent dioxygenase [EC 1.14.11–] that catalyzes the 6-hydroxylation of partially methylated flavonols has been purified to near homogeneity from Chrysosplenium americanum. Enzyme purification was achieved by fast protein liquid chromatography on Superose 12 and Mono Q columns as well as by affinity chromatography on 2-oxoglutarate–Sepharose and immunoaffinity columns. The specific activity of the 6-hydroxylase eluted from Mono Q (97.1 pkat/mg) was enriched 538-fold, with a 0.63% recovery. Both affinity chromatography steps resulted in the elimination of most contaminating proteins, but not without loss of enzyme activity and stability. The molecular mass of both the native and denatured enzyme was found to be 42 and 45 kDa, respectively, suggesting a monomeric protein. The enzyme exhibits strict specificity for position 6 of partially methylated flavonols possessing a 7-methoxyl group, indicating its involvement in the biosynthesis of polymethylated flavonols in this plant. The cofactor dependence of the enzyme is similar to that of other plant dioxygenases, particularly its dependence on ferrous ions for catalytic activity and reactivation. Internal amino acid sequence information indicated its relatedness to other plant flavonoid dioxygenases. The results of substrate interaction kinetics and product inhibition studies suggest an ordered, sequential reaction mechanism (TerTer), where 2-oxoglutarate is the first substrate to bind, followed by O2 and the flavonol substrate. Product release occurs in the reverse order where the hydroxylated flavonol is the first to be released, followed by CO2 and succinate. To our knowledge, this is the first reported 2-oxoglutarate-dependent dioxygenase that catalyzes the aromatic hydroxylation of a flavonoid compound.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.2000.2002