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 .