Two-step epoxidation of hyoscyamine to scopolamine is catalyzed by bifunctional hyoscyamine 6 beta-hydroxylase.

In several solanaceous plants, hyoscyamine is first hydroxylated at the 6 beta-position, and then epoxidized to scopolamine. We expressed hyoscyamine 6 beta-hydroxylase (H6H) in Escherichia coli as a fusion protein with maltose-binding protein. The crude cell extract from the bacterium that expressed the soluble fusion protein showed a strong hydroxylase activity and a weak epoxidase activity. When 100 microM of hyoscyamine was fed to the recombinant bacterium, the alkaloid was first converted to 6 beta-hydroxy hyoscyamine, and then to scopolamine, which was almost the only alkaloid found in the culture after one week. Therefore, H6H catalyzes two consecutive reactions that oxidize hyoscyamine to scopolamine.