- Mallinson, Sam JB;
- Machovina, Melodie M;
- Silveira, Rodrigo L;
- Garcia-Borràs, Marc;
- Gallup, Nathan;
- Johnson, Christopher W;
- Allen, Mark D;
- Skaf, Munir S;
- Crowley, Michael F;
- Neidle, Ellen L;
- Houk, Kendall N;
- Beckham, Gregg T;
- DuBois, Jennifer L;
- McGeehan, John E
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.