CHARACTERIZATION AND MECHANISM OF THE BERBERINE BRIDGE ENZYME, A COVALENTLY FLAVINYLATED OXIDASE OF BENZOPHENANTHRIDINE ALKALOID BIOSYNTHESIS IN PLANTS

The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (met hylene-bridge-forming), EC 1.5.3.9) catalyzes the oxidative cyclizatio n of the N-methyl moiety of (S)-reticuline into the berberine bridge c arbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniquene ss of this catalytic reaction prompted an in depth study that began wi th the isolation of the cDNA encoding the berberine bridge enzyme foll owed by the overexpression of this cDNA in insect cell culture. The he terologously expressed enzyme has herein been shown to contain covalen tly attached FAD in a molar ratio of cofactor to protein of 1:1.03. Si te-directed mutagenesis and laser desorption time of-flight mass spect rometry suggest that the site of covalent attachment is at His-104. Th e holoenzyme exhibited absorbance maxima at 380 and 442 nm and a fluor escence emission maximum at 628 nm (310 nn excitation). Enzymic transf ormation of a series of (S)-reticuline derivatives modified with respe ct to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the met hylene iminium ion and subsequent ring closure via an ionic mechanism.