Structural Insights into the Substrate Promiscuity of a Laboratory-Evolved Peroxygenase

ACS Chem Biol. 2018 Dec 21;13(12):3259-3268. doi: 10.1021/acschembio.8b00500. Epub 2018 Nov 16.


Because of their minimal requirements, substrate promiscuity and product selectivity, fungal peroxygenases are now considered to be the jewel in the crown of C-H oxyfunctionalization biocatalysts. In this work, the crystal structure of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å. Notable differences were detected between the evolved and native peroxygenase from Agrocybe aegerita, including the presence of a full N-terminus and a broader heme access channel due to the mutations that accumulated through directed evolution. Further mutagenesis and soaking experiments with a palette of peroxygenative and peroxidative substrates suggested dynamic trafficking through the heme channel as the main driving force for the exceptional substrate promiscuity of peroxygenase. Accordingly, this study provides the first structural evidence at an atomic level regarding the mode of substrate binding for this versatile biocatalyst, which is discussed within a biological and chemical context.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrocybe / enzymology
  • Catalytic Domain / genetics
  • Crystallography, X-Ray
  • Directed Molecular Evolution
  • Escherichia coli / genetics
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Ligands
  • Mixed Function Oxygenases / chemistry*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Mutagenesis, Site-Directed
  • Mutation
  • Organic Chemicals / chemistry
  • Organic Chemicals / metabolism
  • Pichia / genetics
  • Protein Binding
  • Protein Structure, Tertiary / genetics
  • Saccharomyces cerevisiae / genetics
  • Substrate Specificity / genetics


  • Fungal Proteins
  • Ligands
  • Organic Chemicals
  • Mixed Function Oxygenases
  • peroxygenase