Isotope effects suggest a stepwise mechanism for berberine bridge enzyme.

Berberine Bridge Enzyme (BBE) is a plant enzyme that participates in an alkaloid biosynthetic pathway that produces a variety of pharmacologically active alkaloids, including berberine, sanguinarine, and palmatine (1). The enzyme, an FAD-containing amine oxidase, catalyzes a unique oxidative cyclization reaction, converting (S)-reticuline to (S)-scoulerine (Scheme 1) (2). This regioselective reaction has not yet been reproduced by synthetic organic methods. Furthermore, only a couple of other enzymes have been identified that catalyze similar reactions: cannabidiolic acid synthase and Δ1-tetrahydrocannabinolic acid synthase from Cannabis sativa (3, 4). Neither of these enzymes has been characterized enzymatically. Scheme 1 Reaction catalyzed by BBE. Stopped-flow analyses of the BBE reductive half-reaction have failed to identify a flavin semiquinone intermediate, suggesting that substrate oxidation does not proceed via single electron transfer from the substrate to the cofactor (5) and instead likely occurs via hydride transfer, as observed with other flavoprotein oxidases (6, 7). Both concerted and stepwise mechanisms have been proposed for BBE (Scheme 2) (8, 9). In the stepwise mechanism, a hydride equivalent is transferred from the substrate to the flavin, forming a methylene iminium ion intermediate; subsequently, an active site base, Glu417, deprotonates a substrate phenol, making the adjacent carbon more nucleophilic and enabling its attack on the N-methylene group, yielding the cyclized product. In the concerted mechanism, deprotonation of the phenol occurs during concomitant attack on the N-methyl group by the C2′ and transfer of a hydride to the flavin. Scheme 2 Proposed mechanisms for the BBE-catalyzed reaction (8, 9) Several studies support each mechanistic proposal. Oxidation by BBE of (S)-N-methylcoclaurine yields a demethylated product, suggesting that the reaction proceeds via formation of a methylene iminium ion intermediate (Scheme 3) (8). BBE oxidizes the tetrahydroprotoberberine alkaloids (S)-coreximine, (S)-scoulerine and (S)-norsteponine, forming a double bond between the nitrogen and C-8 of each (Scheme 3) (8, 10); these observations also suggest a two-step mechanism for the oxidative cyclization of (S)-reticuline (8). However, analyses of the products resulting from a different set of substrate analogs have been taken as support for a concerted mechanism (9). Oxidation of laudanosine is associated with an absorbance increase around 360 nm, suggesting formation of a double bond within the isoquinoline ring system (Scheme 3). BBE oxidizes the C1-N bond of 6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline, as opposed to the N-CH3 bond expected in a two-step mechanism (Scheme 3) (9). Mutation of the proposed active site base Glu417 to glutamine leads to a 1500-fold decrease in the rate constant for flavin reduction, resulting in substrate oxidation becoming the rate-determining step during turnover (9). This has been interpreted as additional evidence for a concerted mechanism in which deprotonation of the substrate phenol is necessary for the concerted attack on the incipient methyl aminium and the transfer of a hydride to the flavin cofactor. In the present study, we performed a series of substrate deuterium and solvent kinetic isotope effect studies to discriminate between these two mechanistic proposals. Scheme 3 BBE-catalyzed oxidation of various substrate analogs (8–10).