Fad-dependent epoxidase as a key enzyme in fungal metabolism of prenylated flavonoids

Crude protein extracts from Botrytis cinerea preincubated with 6-prenylnaringenin (6-PN) for 20 hr catalysed the prenyl epoxidation of 7-O-methyl-luteone. The resulting epoxide was non-enzymatically and slowly converted into the corresponding dihydrofurano derivative in a buffer solution at pH 7.5. Preparation of cell-free extracts in the presence of 6-PN from the mycelia without preincubation with 6-PN hardly showed the epoxidizing activity. These facts revealed that the substrate analogue 6-PN has a role as an enzyme inducer rather than stabilizer. The enzyme reaction depends on molecular oxygen and NADPH. Low amounts of FAD were necessary for maximal enzyme activity. The enzymatic activity was not inhibited by various inhibitors of cytochrome P-450 tested, in addition to carbon monoxide and cytochrome c. The results indicated that this enzyme does not belong to the monooxygenases dependent on cytochrome P-450, but to those dependent on FAD. About half of. the total enzyme activity was found in the 125 000 g supernatant, but the specific activity for the epoxidation reaction in the 125 000 g pellet was 3.7-fold higher than in the soluble fraction. The enzyme showed high specificity to monoprenyl isoflavones. Finally, a preliminary experiment using a cell-free system from white lupin hypocotyls resulted in formation of small amounts of an epoxide corresponding to 7-O-methyl-luteone used as the substrate. (C) 1997 Elsevier Science Ltd.