Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase

Vânia Brissos, Patrícia T. Borges, Reyes Núñez-Franco, Maria Fátima Lucas, Carlos Frazão, Emanuele Monza, Laura Masgrau, Tiago N. Cordeiro, Lígia O. Martins

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)


Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (kcat273 s-1for 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 mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.

Original languageEnglish
Pages (from-to)5022-5035
Number of pages14
JournalACS Catalysis
Issue number9
Publication statusPublished - 6 May 2022


  • allosteric regulation
  • Aquifex aeolicus
  • enzyme dynamics
  • enzyme specificity
  • epistasis
  • hyperthermophiles
  • multicopper oxidases


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