Catalyzing New Hydroxylations

This enzyme was first reported in the microsomal fractions of Haplopappus gracilis cell cultures,33 studied in more detail in parsley,34 and later shown to occur in the flowers of several species.7 F3 H catalyzes the hydroxylation of naringenin and dihydrokaempferol (DHK), as well as of apigenin or kaempferol to their respective 3 -hydroxy derivatives, eriodictyol, dihydroquercetin (DHQ), luteolin, and quercetin, but does not accept the flavan 3,4-diols or anthocyanidins as substrates,34 indicating that B-ring hydroxylation of the latter is determined at the dihydroflavonol level. It was more than two decades later that the first cDNA clone encoding F3 H was isolated and characterized from the flowers of Petunia hybridal Arabidopsis thaliana,36 and Perilla frutescens.37 Their recombinant proteins were shown to 

It has recently been reported that cytochrome bj is required for full activity of F3 /5 H. The gene encoding this protein, difF, was cloned from petunia42 and shown to be exclusively expressed in flowers generating purple or blue petals. Inactivation of the gene reduced both F3 /5 H activity and 5 -substituted pigments, thus resulting in altered flower color.42 

Microsomal preparations from yeast-elicited cell cultures of chickpea catalyzed the hydroxylation of formononetin and biochanin A to their 2 - and 3 -hydroxy derivatives.43 44 Neither daidzein nor genistein were accepted as substrates. Both hydroxylation reactions seem to be catalyzed by two distinct enzymes, since they exhibited different physicochemical properties and induction kinetics in cell cultures and roots of chickpeas.45 Both 2 - and 3 -hydroxylations of formononetin are prerequisite reactions in the pathway for biosynthesis of the phytoalexins, medicarpin and maackiain in alfalfa and chickpea, respectively8. 

Microsomal preparations of Dahlia variabilis flowers have been reported to catalyze the 3-hydroxylation of the 6 -deoxychalcone, isoliquiritigenin, to butein.46 This enzyme activity was shown to be a typical cytochrome P450 monooxygenase that appears to be different from the F3 H, although its occurrence in other species remains to be investigated. 

A feature of some pterocarpan phytoalexins (e.g., pisatin and glyceollin of pea and soybean, respectively) is their hydroxylation at position 6a, a reaction catalyzed by a microsomal cytochrome P450 monooxygenase.49 50 A cDNA encoding this enzyme was recently characterized from elicited soybean cell cultures.51 The microsomal protein, expressed in yeast cells, catalyzed the stereoselective hydroxylation of (6a/ , lla/ )-3,9-dihydroxypterocarpan to its 6a-hydroxy derivative. It was also demonstrated that the enzyme expression is regulated at the transcriptional level.51 

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