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-h ydroxylation of partially methylated flavonols has been purified to near ho mogeneity 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 immunoaf finity 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 affini ty chromatography steps resulted in the elimination of most contaminating p roteins, but not without loss of enzyme activity and stability. The molecul ar mass of both the native and denatured enzyme was found to be 42 and 45 k Da, respectively, suggesting a monomeric protein. The enzyme exhibits stric t specificity for position 6 of partially methylated flavonols possessing a 7-methoxyl group, indicating its involvement in the biosynthesis of polyme thylated 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 dio xygenases. The results of substrate interaction kinetics and product inhibi tion. studies suggest an ordered, sequential reaction mechanism (TerTer), w here 2-oxoglutarate is the first substrate to bind, followed by O-2 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 succ inate. To our knowledge, this is the first reported 2-oxoglutarate-dependen t dioxygenase that catalyzes the aromatic hydroxylation of a flavonoid comp ound. (C) 2000 Academic Press.