Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana

Abstract

Formation of the methylenedioxy bridge is an integral step in the biosynthesis of benzo[ c]phenanthridine and protoberberine alkaloids in the Papaveraceae family of plants. This reaction in plants is catalyzed by cytochrome P450-dependent enzymes. Two cDNAs that encode cytochrome P450 enzymes belonging to the CYP719 family were identified upon interrogation of an EST dataset prepared from 2-month-old plantlets of the Mexican prickly poppy Argemone mexicana that accumulated the benzo[ c]phenanthridine alkaloid sanguinarine and the protoberberine alkaloid berberine. CYP719A13 and CYP719A14 are 58% identical to each other and 77% and 60% identical, respectively, to stylopine synthase CYP719A2 of benzo[ c]phenanthridine biosynthesis in Eschscholzia californica. Functional heterologous expression of CYP719A14 and CYP719A13 in Spodoptera frugiperda Sf9 cells produced recombinant enzymes that catalyzed the formation of the methylenedioxy bridge of ( S)-cheilanthifoline from ( S)-scoulerine and of ( S)-stylopine from ( S)-cheilanthifoline, respectively. Twenty-seven potential substrates were tested with each enzyme. Whereas CYP719A14 transformed only ( S)-scoulerine to ( S)-cheilanthifoline ( K m 1.9 ± 0.3; k cat / K m 1.7), CYP719A13 converted ( S)-tetrahydrocolumbamine to ( S)-canadine ( K m 2.7 ± 1.3; k cat / K m 12.8), ( S)-cheilanthifoline to ( S)-stylopine ( K m 5.2 ± 3.0; k cat / K m 2.6) and ( S)-scoulerine to ( S)-nandinine ( K m 8.1 ± 1.9; k cat / K m 0.7). These results indicate that although CYP719A14 participates in only sanguinarine biosynthesis, CYP719A13 can be involved in both sanguinarine and berberine formation in A. mexicana.