Anaerobic demethylation of guaiacyl-derived monolignols enabled by a designed artificial cobalamin methyltransferase fusion enzyme.
Christopher GrimmSimona PompeiKristina EggerMichael FuchsWolfgang KroutilPublished in: RSC advances (2023)
Lignin-derived aryl methyl ethers ( e.g. coniferyl alcohol, ferulic acid) are expected to be a future carbon source for chemistry. The well-known P450 dependent biocatalytic O -demethylation of these aryl methyl ethers is prone to side product formation especially for the oxidation sensitive catechol products which get easily oxidized in the presence of O 2 . Alternatively, biocatalytic demethylation using cobalamin dependent enzymes may be used under anaerobic conditions, whereby two proteins, namely a methyltransferase and a carrier protein are required. To make this approach applicable for preparative transformations, fusion proteins were designed connecting the cobalamin-dependent methyltransferase (MT) with the corrinoid-binding protein (CP) from Desulfitobacterium hafniense by variable glycine linkers. From the proteins created, the fusion enzyme MT-L5-CP with the shortest linker performed best of all fusion enzymes investigated showing comparable and, in some aspects, even better performance than the separated proteins. The fusion enzymes provided several advantages like that the cobalamin cofactor loading step required originally for the CP could be skipped enabling a significantly simpler protocol. Consequently, the biocatalytic demethylation was performed using Schlenk conditions allowing the O -demethylation e.g. of the monolignol coniferyl alcohol on a 25 mL scale leading to 75% conversion. The fusion enzyme represents a promising starting point to be evolved for alternative demethylation reactions to diversify natural products and to valorize lignin.