Kinetic Characterization of a Lytic Polysaccharide Monooxygenase Reveals a Unique Specificity for Depolymerization at β-O-4 of Lignin Compounds

Abstract

The intrinsic role of lytic polysaccharide monooxygenases (LPMOs) for oxidative cleavage of a wide range of polysaccharides like cellulose, starch, chitin, and xyloglucan has been studied extensively. Lignin and its compounds are known to act as electron donors for activation of LPMOs. However, the role of LPMOs in the cleavage of lignin compounds and their complete kinetic analysis has not been well understood. Here, an in-depth kinetic characterization was conducted to study the depolymerization of the phenolic β-O-4 lignin compound, guaicyl glycerol β-guaicyl ether (GGE), by LPMO. Ultrahigh-pressure liquid chromatography, thin-layer chromatography, and NMR analysis of the oxidized reaction mixture confirmed the formation of guaiacol from selective cleavage of GGE at β-O-4 linkage by LPMO. Michaelis–Menten and Lineweaver Burk plot kinetics of LPMOs revealed a Km of 3.566 ± 1.002 mM and a catalytic efficiency (kcat/Km) of 43 × 103 M–1 s–1 for GGE. A complete catalytic mechanism involving O2 and H2O2 was proposed and validated for the LPMO-driven cleavage of GGE. The oxidation reactions involving GGE and LPMO in the N2 atmosphere and competitive inhibition studies using catalase confirmed the role of O2 and H2O2 in completing the catalytic cycle of LPMO. Molecular docking and molecular dynamics simulation studies revealed the interaction of amino residues around the copper active site of LPMO with GGE. Per-residue interaction energy of amino acid depicted the maximum interactive energy contribution by MET1 of LPMO. The presence of GGE around the substrate-binding site further confirms the strong interaction of LPMO and GGE. Hence, the interaction between GGE and LPMO confirmed the role of LPMO in cleaving the β-O-4 linkage of lignin, thus making the LPMO an attractive biocatalyst for use in enzymatic cocktails for lignin valorization.