Laccase-catalysed cleavage of malvidin-3-O-galactoside to 2,6-dimethoxy-1,4-benzoquinone and a coumarin galactoside

Abstract

Malvidin-3-O-galactoside, a widely occurring anthocyanin, was incubated with laccases (LCCs) from the basidiomycetes Pleurotus pulmonarius (Ppu) and Funalia trogii (Ftr), and with bilirubin oxidases (BODs) from Bacillus pumilus (Bpu) and Magnaporthe oryzae (Mor). LC-MS and LC-MS2 analyses identified 2,6-dimethoxy-1,4-benzoquinone (DMBQ) and tentatively identified the corresponding coumarin galactoside fragment (m/z 355) resulting from the cleavage of the substrate. The enzymes cleaved malvidin-3-O-galactoside within a few minutes under acidic and neutral conditions and in the absence of a mediator. The fungal LCCs were more active at pH 4 and 5.5, while the BODs worked best in the neutral range. The initial cleavage product coumarin galactoside was further oxidised, as indicated by a continuous decline of concentration and concurrent formation of a brown precipitate. Based on the structure of the products and the general LCC mechanism, a reaction sequence was proposed starting with a stable tertiary carbocation, attack of molecular oxygen and formation of a 1,2-dioxetane intermediate between B and C rings which is finally split into the two carbonyl products. Similar reactions may be catalysed by plant oxidases in anthocyanin-rich tissues resulting in the formation of the strong antibacterial and cytotoxic DMBQ.