Elicitor induction and characterization of microsomal protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosynthesis

Abstract

The chemical synthesis of [6- 3H]protopine is described. Microsomal preparations from Eschscholtzia californica cell suspension cultures challenged with a crude elicitor preparation from yeast, catalyse the hydroxylation of [6- 3H]protopine with the concomitant formation of [11- 3H]dihydrosanguinarine and HO 3H. This reaction served as an assay for the enzyme. The hydroxylase reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen. Cytochrome c, inhibitors such as prochloraz and ketoconazole and carbon monoxide inhibit the hydroxylation reaction, suggesting that the enzyme is a cytochrome P-450 linked monooxygenase. The hydroxylase was induced by the yeast elicitor ca eight-fold, 20 hr after challenging the plant cell culture with the yeast carbohydrate, having no effect on the cell dry weight of the culture. The hydroxylase is specifically present in different plant species that produce benzo[c]phenanthridine alkaloids in culture and also respond to induction by the elicitor.