Induction and characterization of a microsomal flavonoid 3'-hydroxylase from parsley cell cultures.
A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4'-trihydroxyflavanone] to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction. In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid. 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase. Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide. Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase. The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases.
D002384:Catalysis; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System; D003580:Cytochromes / Q000378:metabolism; D004790:Enzyme Induction / Q000528:radiation effects; D008027:Light; D008861:Microsomes / Q000201:enzymology* / Q000528:radiation effects; D006899:Mixed Function Oxygenases / Q000378:metabolism* / Q000528:radiation effects; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity
D003580:Cytochromes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C038888:flavonoid 3'-hydroxylase