Induction and Characterization of a Microsomal Flavonoid 3′‐Hydroxylase from Parsley Cell Cultures

Abstract

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‐β‐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.

Induction of phytoalexin synthesis in soybean. Stereospecific 3,9‐dihydroxypterocarpan 6a‐hydroxylase from elicitor‐induced soybean cell cultures

“…The fact that this typical monooxygenase reaction is inhibited by low concentrations of cytochrome c indicates that it probably belongs to the cytochrome P-450-dependent plant molnooxygenases [21]. Its properties are similar to those of the recently described flavonoid 3'-hydroxylase [14]. Further investigations are necessary to prove the participation of cytochrome P-450.…”

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

“…In plants, different isoforms of Cyt P-450 are implicated in essential biosynthetic pathways such as the production of lignin, at the level of cinnamate hydroxylation (1) and ferulate hydroxylation (11); the biosynthesis of cutin and suberin, at the level of fatty acid hydroxylation (15); and the biosynthesis of hormones (13), pigments (12), and phytoalexins (14). Cyt P-450s are also implicated in the metabolism of various herbicides in several plant species (8).…”

Flavonoids as Flower Pigments: The Formation of the Natural Spectrum and its Extension by Genetic Engineering

“…One is the oxidative rearrangement including a 2,3-aryl shift to isoflavones (genistein/daidzein), which are involved in phytoalexin synthesis. This reaction is catalized by isoflavone synthase, a cytochrome P-450-dependent mono-oxygenase with NADPH as cofactor (HAGMANN andGRISEBACH 1984, KOCHS and.…”

“…Flavonoid 3'-hydroxylase was first demonstrated in microsomal preparations from Haplopappus cell cultures (FRITSCH and GRISEBACH 1975). Later, the enzyme was characterized as a cytochrome P-450-dependent mono-oxygenase which requires NADPH as cofactor and molecular oxygen (HAGMANN et al 1983). Favourable substrates are the flavonoid intermediates naringenin and dihydrokaempferol (DHK) which are hydroxylated in the 3'-position to eriodictyol and dihydroquercetin (DHQ), respectively (Fig.…”