Structural insights into S-lignin O-demethylation via a rare class of heme peroxygenase enzymes

Abstract

Abstract The O -demethylation of lignin aromatics is a rate-limiting step in their bioconversion to high-value compounds. A recently discovered cytochrome P450 enzyme SyoA was found to demethylate the sinapyl alcohol-derived (S-lignin) aromatic syringol. In this work, we solved high-resolution X-ray crystal structures of SyoA in the substrate-free and substrate-bound states and evaluate the demethylation of para -substituted S-lignin aromatics via the monooxygenase pathway and peroxide shunt pathway. We found that SyoA demethylates S-lignin aromatics with the following activity: 4-methylsyringol > syringaldehyde > syringol exclusively using the peroxide shunt pathway. The atomic-resolution structure of SyoA reveals the position of the non-canonical residues in the I-helix (Gln252 and Glu253). Site-directed mutagenesis of this amide-acid pair of a homologous CYP255 enzyme GcoA, which can catalyze the O-demethylation of guaiacol using both monooxygenase and peroxygenase activity, showed the amide-acid pair is critical for both pathways. This work expands the enzymatic toolkit for improving the capacity to funnel lignin towards high-value compounds, and defines the new chemistry within the active site of the enzyme that enables efficient peroxygenase activity. These insights provide a framework for engineered peroxygenase activity in other cytochrome P450 enzymes, with the potential for more facile catalysis, relative to traditional P450 monooxygenases which require difficult to handle redox partners and expensive nicotinamide cofactors.