Molecular Cloning and Characterization of CYP80G2, a Cytochrome P450 That Catalyzes an Intramolecular C–C Phenol Coupling of (S)-Reticuline in Magnoflorine Biosynthesis, from Cultured Coptis japonica Cells

Abstract

Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C-C phenol-coupling and intermolecular C-O phenol-coupling reactions. We report here the isolation and characterization of a C-C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C-O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C-C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C-C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.Isoquinoline alkaloids are a large group of alkaloids and include many pharmacologically useful compounds; e.g. the analgesic morphinan alkaloid morphine, the anti-tussive alkaloid codeine, and the anti-microbial alkaloids berberine and sanguinarine. Due to the importance of these pharmaceutically useful alkaloids, their biosynthetic pathways have been well investigated, and several of them have been completely clarified at the enzyme level (1-3). It is now known that many isoquinoline alkaloids share a common biosynthetic pathway from L-tyrosine to the key intermediate (S)-reticuline.(S)-Reticuline is a central precursor of various types of isoquinoline alkaloids such as morphinans, aporphines, pavines, protoberberines, protopines, and benzophenanthridines (1, 4). Although the molecular origin of this chemical diversity has not yet been clarified, recent studies have shown that many of their oxidative steps are catalyzed by cytochrome P450s (P450) 2 ( Fig. 1) (1-3, 5-11). Members of the P450 family are found in a very large number of species, especially in the plant kingdom (246 and 356 species in Arabidopsis thaliana and Oryza sativa in contrast to 57 and 84 species in human and Drosophila melanogaster) (12, 13), and many of them have been shown to be involved in plant secondary metabolism (14, 15).In the biosyntheses of isoquinoline alkaloids, P450-mediated hydroxylation, methylenedioxy bridge formation, and phenolcoupling reactions have been reported. Although members of the CYP80B s...

Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

“…The mechanism of C-C bond formation by SnoK is mechanistically different from intramolecular phenol radical coupling reactions catalyzed by P450 enzymes (29,30). In SnoK, such a reaction would formally require abstraction of a hydrogen atom from the C4 hydroxyl group, generating a phenoxy radical.…”

Unusual Cytochrome P450 Enzymes and Reactions

“…S6) (38), and oxidative coupling in early steps of morphine biosynthesis (supplemental Fig. S7) (39,40). These coupling reactions, generally believed to involve radical chemistry, are not restricted to plants and bacteria.…”

The Roles of a Flavone-6-Hydroxylase and 7-O-Demethylation in the Flavone Biosynthetic Network of Sweet Basil

“…One of the few currently known instances is the N-demethylation of nicotine by CYP450-dependent monooxygenases of the family CYP82E (27). In addition, O-demethylating side activity with the alkaloid codamine was tentatively detected for CYP80G2, primarily a C-C coupling enzyme from Coptis japonica (28). Very recently, two O-demethylases involved in the biosynthesis of morphine from thebaine were spectacularly identified as oxoglutarate-dependent dioxygenases (29).…”