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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Baker's yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-␤-glucoside revealed considerable ␤-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the S. cerevisiae genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic ␣-or ␤-glucosides identified three efficient ␤-glucosidases (EXG1, SPR1, and YIR007W), which were further assayed with natural flavonoid ␤-glucoside substrates and product verification by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Preferential hydrolysis of 7-or 4-O-glucosides of isoflavones, flavonols, flavones, and flavanones was observed in vitro with all three glucosidases, while anthocyanins were also accepted as substrates. The glucosidase activities of EXG1 and SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue, as reported for other glucosidases. Most importantly, biotransformation experiments with knockout yeast strains revealed that only EXG1 knockout strains lost the capability to hydrolyze flavonoid glucosides.Glycoside hydrolases, in particular glucosidases (EC 3.2.1.-), are widespread in pro-and eukaryotic organisms and play a pivotal role in many biological processes, such as the metabolism of oligosaccharides or the degradation of endogenous and exogenous glycosides. Beta-glucosidases (␤-GHs) are among the oldest classes of enzymes, and microbial ␤-GHs have been identified often as molecular factors indispensable for growth; for example, they enable phytopathogenic fungi to colonize host plant tissues by hydrolyzing plant fungitoxic glucosides to less toxic or less soluble aglyca (5). They are also important for cleaving cellulose and fulfill essential functions in the sporulation of yeast cells (27,38). In plants, ␤-GHs are involved in crucial growth processes, such as the degradation of endosperm cell walls during germination or the formation of intermediates in cell wall lignification, as well as in the activation of defense compounds and the formation of phytohormones (references 49 and 58 and references therein). Moreover, plant ␤-GHs are essential for turnover of flavonoid glucosides, which are exclusively found in ␤ configuration, as had been reported for malonylglucosides of the isoflavones genistein and daidzein in soybean (21) or isoflavone 7-O-␤-glucosides in chickpea (20). Even the endophytic bacterium Pseudomonas strain ZD-8 (61), as well as cell-associated ␤-GHs (bglH and yckE) from Bacillus subtilis natto, used for fermentation of soy products (28), were shown to metabolize apigenin 7-O-␤-glucoside or glucosides and malonylglucosides of genistein and daidzein. The capacity of multiple Bifidobacterium strains of human origin to digest isoflavonoid glucosides (32, 41) is particularly noteworthy because of their releva...

Five glucosyltransferases were cloned by RT-PCR ampliWcation using total RNA from Hieracium pilosella L. (Asteraceae) inXorescences as template. Expression was accomplished in Escherichia coli, and three of the HIS-tagged enzymes, UGT90A7, UGT95A1, and UGT72B11 were partially puriWed and functionally characterised as UDP-glucose:Xavonoid O-glucosyltransferases. Both UGT90A7 and UGT95A1 preferred luteolin as substrate, but possessed diVerent regiospeciWcity proWles. UGT95A1 established a new subgroup within the UGT family showing high regiospeciWcity towards the C-3Ј hydroxyl group of luteolin, while UGT90A7 primarily yielded the 4Ј-O-glucoside, but concomitantly catalysed also the formation of the 7-O-glucoside, which could account for this Xavones glucoside in H. pilosella Xower heads. Semi quantitative expression proWles revealed that UGT95A1 was expressed at all stages of inXorescence development as well as in leaf and stem tissue, whereas UGT90A7 transcript abundance was nearly limited to Xower tissue and started to develop with the pigmentation of closed buds. Other than these enzymes, UGT72B11 showed rather broad substrate acceptance, with highest activity towards Xavones and Xavonols which have not been reported from H. pilosella. As umbelliferone was also readily accepted, this enzyme could be involved in the glucosylation of coumarins and other metabolites.

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