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

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. Sitedirected 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.

A concerted mechanism for berberine bridge enzyme

“…2% during prolonged incubation times) arise in the conversion of the substrate analog (S)-N-methylcoclaurine (10) 2 , which also lacks the C3'-OH group. This finding is considered supporting evidence for the two-step mechanism 2 . In light of our results with laudanosine and 8, however, it could also be explained by the formation of the intracyclic imine, followed by tautomerisation and hydrolytic demethylation to yield formaldehyde and (S)-coclaurine (11).…”

“…1a) 1 . This cyclization reaction has no known equivalent in organic chemistry and is proposed to proceed via a two step process starting with oxidation of the Nmethyl group to the corresponding iminium ion followed by an ionic ring closure initiated by the deprotonated C3'-OH group of the benzyl moiety 2 . This hydroxyl group apparently plays an important role for the enzymatic reaction, as substrate analogs lacking this group are not processed by the enzyme 2 .…”

“…Berberine bridge enzyme (BBE) plays a central role in benzophenanthridine biosynthesis and channels its substrate (S)-reticuline (1) towards berberine (2) and sanguinarine (3) generation 1 . BBE catalyzes the stereospecific conversion of (S)-reticuline to (S)-scoulerine (4) forming the so called berberine bridge (C8) by linking the isoquinoline ring with the benzyl ring system (Fig.…”

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…Protoberberines, protopines and sanguinarine (S)-Scoulerine, the branch-point intermediate leading to protoberberine alkaloids, is formed from (S)-reticuline by BBe (Dittrich and Kutchan 1991;Facchini et al 1996;Kutchan and Dittrich 1995;winkler et al 2006). (S)-Scoulerine is converted via methylenedioxy bridge formation and O-methylation to a variety of protoberberines including (S)-canadine, (S)-stylopine and (S)-sinactine.…”

Tobacco Nectarin V Is a Flavin-Containing Berberine Bridge Enzyme-Like Protein with Glucose Oxidase Activity

“…Examination of the best characterized of the BBEs (Kutchan and Dittrich, 1995) revealed that these proteins contain a conserved, covalently bound FAD moiety that is required for activity. The translated NEC5 sequence also contains a homologous consen- sus flavin-binding domain (Fig.…”