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 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.
Anzellotti
D
D
Department of Biology, Concordia University, Montreal, Québec, Canada.
Ibrahim
R K
RK
D000818:Animals; D002412:Cations / Q000494:pharmacology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism; D044948:Flavonols; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007526:Isoelectric Point; D007700:Kinetics; D019684:Magnoliopsida / Q000201:enzymology*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D011817:Rabbits; D013379:Substrate Specificity
D002412:Cations; D005419:Flavonoids; D044948:Flavonols; D006899:Mixed Function Oxygenases; C416048:flavonol 6-hydroxylase