A cytochrome P450 mediated naringenin 3'-hydroxylase from sweet orange cell cultures
1995
Doostdar, H. | Shapiro, J.P. | Niedz, R. | Burke, M.D. | McCollum, T.G. | McDonald, R.E. | Mayer, R.T.
A microsomal flavonoid 3'-hydroxylase (F3'H) catalyzing the metabolism of naringenin to eriodictyol in Citrus sinensis (L.) Osbeck cv. 'Hamlin' cell suspension cultures was shown to be a cytochrome P450 enzyme. This reaction required O2 and NADPH and was inhibited by CO, with partial reversal of CO-inhibition by light at 450 nm. Cytochrome P450 content ranged from 10-20 pmol (mg microsomal protein)-1. The F3'H reaction was shown to be linear in regard to protein concentration between 2.5 and 25 micrograms of microsomal protein. The optimum pH for the reaction was 7.4-7.6 and the temperature optimum was between 30 and 37 degrees C. The apparent Km and Vmax for naringenin were 24 micromolar +/- 3.2 and 81.4 +/- 7.9 pmol eriodictyol min-1 (mg protein)-1, respectively. The microsomal F3'H was also capable of forming dihydroquercetin from dihydrokaempferol (40 pmol min-1 (mg protein)-1) and of quercetin from kaempferol (3.25 pmol min-1 (mg protein- 1). Cytochrome c and ketoconazole were the best inhibitors of F3'H activity followed by piperonyl butoxide and alpha-naphthoflavone. Light was shown to be an inducer of the F3'H almost doubling the specific activity and increasing the microsomal cytochrome P450 content by 30% over that of dark grown cells. F3'H activity was also confirmed in microsomal preparations of young (new flush) leaves from 'Hamlin' trees and flavedo of 'Hamlin' oranges, 'Marsh' grapefruit, and 'Lisbon' lemon. No activity was observed in older, hardened leaves and albedo of all the fruit tested. Initiation of embryogenesis in the 'Hamlin' cell suspension cultures by switching from a sucrose medium to a glycerol-based medium resulted in the down-regulation of F3'H.
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