Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds

• Thermobifida fusca DyP peroxidase is found to show activity for lignin oxidation.
• Oxidation of a β-aryl ether lignin model compound yields an oxidised dimer.
• The crystal structure of this enzyme was determined to 1.8 Å resolution.
• Site-directed mutagenesis of active site residues Asp-203 and Arg-315 was carried out, in order to study catalytic function.

A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at λmax 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a β-aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8 Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS.

Figure optionsDownload high-quality image (296 K)Download as PowerPoint slide