biostudies-literatureUnknown12(9)Brissos VLaccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and <i>in silico</i> analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (<i>k</i> _cat 273 s^-1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutaACS catalysis5022-5035https://www.ebi.ac.uk/biostudies/studies/S-EPMC9775220biostudies-literatureDistal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase.PMC9775220Borges PTLucas MFMasgrau LFrazao CCordeiro TNMartins LOMonza EBrissos VNunez-Franco RfalseDistal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase.Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and <i>in silico</i> analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (<i>k</i> _cat 273 s^-1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal muta2022-01-01T00:00:00Z2022 May2025-04-05T14:57:40.434Z2025-04-05T14:57:40.434ZS-EPMC97752203656777210.1021/acscatal.2c00336