A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion

Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl- O -demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl- O -demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion. Catabolizing lignin-derived aromatic compounds requires an aryl- O -demethylation step. Here the authors present the structures of GcoA and GcoB, a cytochrome P450-reductase pair that catalyzes aryl- O -demethylations and show that GcoA displays broad substrate specificity, which is of interest for biotechnology applications.