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

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.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.This work was supported by the European Union (FP7-KBBE-2013-7-613549-INDOX and H2020-BBI-PPP-2015-2-720297-ENZOX2), the CSIC (Project PIE-201580E042), and the Spanish Ministry of Economy, Industry and Competitiveness (Projects LIGNOLUTION, BIO2013-48779-C4-2-R, and BIO2016-76601-C3-3-R).Peer reviewe