Characterization and Mechanism of the Berberine Bridge Enzyme, a Covalently Flavinylated Oxidase of Benzophenanthridine Alkaloid Biosynthesis in Plants

Toni M Kutchan,Heinz Dittrich

doi:10.1074/jbc.270.41.24475

Abstract

The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9) catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniqueness of this catalytic reaction prompted an in depth study that began with the isolation of the cDNA encoding the berberine bridge enzyme followed by the overexpression of this cDNA in insect cell culture. The heterologously expressed enzyme has herein been shown to contain covalently attached FAD in a molar ratio of cofactor to protein of 1:1.03. Site-directed mutagenesis and laser desorption time-of-flight mass spectrometry suggest that the site of covalent attachment is at His-104. The holoenzyme exhibited absorbance maxima at 380 and 442 nm and a fluorescence emission maximum at 628 nm (310 nm excitation). Enzymic transformation of a series of (S)-reticuline derivatives modified with respect to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the methylene iminium ion and subsequent ring closure via an ionic mechanism.

Highlights

The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase, EC 1.5.3.9) catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniqueness of this catalytic reaction prompted an in depth study that began with the isolation of the cDNA encoding the berberine bridge enzyme followed by the overexpression of this cDNA in insect cell culture
Enzymic transformation of a series of (S)-reticuline derivatives modified with respect to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the methylene iminium ion and subsequent ring closure via an ionic mechanism
The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase, EC 1.5.3.9) transforms the N-methyl group of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine, thereby forming the protoberberine carbon skeleton [1, 2]. (S)-Scoulerine serves as the biosynthetic precursor to a multitude of species-specific protopine, protoberberine and benzophenanthridine alkaloids (Fig. 1)

Summary

Berberine Bridge Enzyme Mechanism

700 nmol of substrate were dissolved in 200 ␮l of 100 mM potassium phosphate buffer, pH 9.3 This solution was divided into two parts and to one part was added 0.9 nanokatal of enzyme. When a dehydrogenation reaction was suspected, the eluted product was first reduced with NaB2H4 in methanol, made to 500 ␮l with 20 mM potassium phosphate buffer, pH 9.3, and extracted 5 times with 300 ␮l of ethyl acetate. Formaldehyde Trapping—The tritiated formaldehyde released upon demethylation of (S)-N-[N-methyl-3H]methylcoclaurine (1 ␮Ci, 85 Ci/ mmol) to (S)-coclaurine by the berberine bridge enzyme (0.9 nanokatal) in a total volume of 160 ␮l of 20 mM potassium phosphate buffer, pH 9.3, incubated for 30 min at 37 °C was diluted by the addition of 80 ␮l of 37% formaldehyde and reacted with dimedone (5,5-dimethyl-1,3-cyclohexanedione) (300 mg in 3 ml of 50% methanol) at room temperature overnight.

RESULTS

TABLE I Substrate specificity for berberine bridge formation

DISCUSSION