Abstract
Saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a fungal laccase previously engineered in the lab. Mutant libraries were screened using sinapic acid as a model substrate, and those mutants presenting increased activity were selected for exploring the oxidation of lignin-derived phenols. The latter comprised a battery of phenolic compounds of interest due to their use as redox mediators or precursors of added-value products and their biological activity. The new laccase variants were investigated in a multi-screening assay and the structural determinants, at both the substrate and the protein level, for the oxidation of the different phenols are discussed. Laccase activity greatly varied only by changing one or two residues of the enzyme pocket. Our results suggest that once the redox potential threshold is surpassed, the contribution of the residues of the enzymatic pocket for substrate recognition and binding strongly influence the overall rate of the catalytic reaction.
Highlights
Laccases are blue-multicopper oxidases that are widely distributed in nature
Laccase mutants studied in this work come from the saturation mutagenesis of six residues of the substrate binding pocket of a high-redox potential laccase (3A4) previously engineered in the lab by recombination of PcL and PM1-L [25]
Mutant libraries were submitted to a high-throughput screening (HTS) assay based on the activity towards sinapic acid (14 in Table 1), a lignin related phenol whose oxidation renders a pinkish colored product [24,30]
Summary
Laccases are blue-multicopper oxidases that are widely distributed in nature. They primarily catalyze the oxidation of substituted phenols and aromatic amines, among other aromatic compounds, coupled to Molecules 2015, 20 the reduction of O2 to water. Wood-rotting fungi are the most important producers of laccases, and white-rot basidiomycetes in particular are the only organisms capable of completely degrading lignin, thanks to the coordinated action of a battery of oxidoreductases (ligninolytic peroxidases and laccases), auxiliary enzymes (H2O2-producing enzymes, cellobiose dehydrogenase, etc.) and oxidized species of low-molecular weight compounds (e.g., Mn3+, Fe3+, ROS, and phenoxyl radicals). White-rot basidiomycetes produce the laccases with the highest redox potential in nature, with up to +0.8 V (vs +0.4 to +0.7 V of laccases from bacteria, plants and other fungi), this is still insufficient to oxidize certain components of lignin or other substrates of higher redox potential. Mediators may act as electron shuttles, diffusing far away from the laccase active site and oxidizing complex compounds that cannot fit into the substrate binding pocket [2]
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