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.