Examination of 2-Oxoglutarate Dependant Dioxygenases Leading to the Production of Flavonols in Arabidopsis thaliana

The flavonols are a varied and abundant sub-class of flavonoids that are associated with a number of essential physiological functions in plants and pharmacological activities in animals. The 2-oxoglutarate-dependant dioxygenases (2-ODDs), flavonol synthase (FLS) and flavanone 3hydroxylase (F3H), are essential for flavonol synthesis. The primary goal of this study has been to gain a deeper understanding of the biochemistry of these enzymes in Arabidopsis. To accomplish this goal, an activity assay employing recombinant protein expression and HPLC as a detection system was developed for F3H and adapted for use with FLS. The assay was employed to establish the biochemical parameters of F3H from Arabidopsis, and to further characterize the F3H mutant allele, tt6(87). Enzymatic activity was demonstrated for F3H enzymes from Ipomoea alba (moonflower), Ipomoea purpurea (common morning glory), Citrus sinensis (sweet orange), and Malus X domestica (newton apple), each of which had previously been identified solely based on sequence homology. Arabidopsis contains six genes with high similarity to FLS from other plant species; however, all other central flavonoid pathway enzymes in Arabidopsis are encoded by single genes. The hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation was tested. Sequence analysis revealed that AtFLS2, 4 and 6 contain premature stop codons that eliminate residues essential for enzyme activity. AtFLS1 was found to have a strong preference for dihydrokaempferol as a substrate. However, no enzyme activity was observed for AtFLS3 or