A CYTOCHROME-P450 MEDIATED NARINGENIN 3'-HYDROXYLASE FROM SWEET ORANGE CELL-CULTURES

A microsomal flavonoid 3'-hydroxylase (F3'H) catalyzing the metabolism of naringenin to eriodictyol in Citrus sinensis (L.) Osbeck cv. 'Haml in' cell suspension cultures was shown to be a cytochrome P450 enzyme. This reaction required O-2 and NADPH and was inhibited by CO, with pa rtial reversal of CO-inhibition by light at 450 nm. Cytochrome P450 co ntent ranged from 10-20 pmol (mg microsomal protein)(-1). The F3'H rea ction was shown to be linear in regard to protein concentration betwee n 2.5 and 25 mu g of microsomal protein. The optimum pH for the reacti on was 7.4-7.6 and the temperature optimum was between 30 and 37 degre es C. The apparent K-m and V-max for naringenin were 24 mu M +/- 3.2 a nd 81.4 +/- 7.9 pmol eriodictyol min(-1) (mg protein)(-1), respectivel y. The microsomal F3'H was also capable of forming dihydroquercetin fr om dihydrokaempferol (40 pmol min(-1) (mg protein)(-1)) and of quercet in from kaempferol (3.25 pmol min(-1) (mg protein(-1)). Cytochrome c a nd ketoconazole were the best inhibitors of F3'H activity followed by piperonyl butoxide and a-naphthoflavone. Light was shown to be an indu cer of the F3'H almost doubling the specific activity and increasing t he microsomal cytochrome P450 content by 30% over that of dark grown c ells. F3'H activity was also confirmed in microsomal preparations of y oung (new flush) leaves from 'Hamlin' trees and flavedo of 'Hamlin' or anges,'Marsh' grapefruit, and 'Lisbon' lemon. No activity was observed in older, hardened leaves and albedo of all the fruit tested. Initiat ion of embryogenesis in the 'Hamlin' cell suspension cultures by switc hing from a sucrose medium to a glycerol-based medium resulted in the down-regulation of F3'H.