Flavanone 3b-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/ FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by sitedirected mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the b-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by synelimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).Flavones and flavonols are the predominant flavonoids found in tissues of Apiaceae species (Harborne, 1971;Harborne and Williams, 1972;Harborne and Baxter, 1999). Significant functions were ascribed to these metabolites for growth and propagation of plants, as well as for adaptation to ecological niches. Flavonoids have been shown to protect from UV radiation, provide pigmentation, mediate the plant's interaction with insects or microbes, and act as feeding deterrents and phytoalexins (Harborne and Williams, 2000;Martens and Mithö fer, 2005). Flavones (i.e. apigenin) are formed by direct 2,3-desaturation of natural flavanones such as (2S)-naringenin (Fig.

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