biostudies-literatureGomez de Santos PComunidad de MadridH2020 FoodMinisterio de Ciencia e Innovación3443-3453https://www.ebi.ac.uk/biostudies/studies/S-EPMC9936548biostudies-literatureUnknown145(6)The generation of enantiodivergent biocatalysts for C-H oxyfunctionalizations is ever more important in modern synthetic chemistry. Here, we have applied the FuncLib algorithm based on phylogenetic and Rosetta calculations to design a diverse repertoire of active, stable, and enantiodivergent fungal peroxygenases. 24 designs, each carrying 4-5 mutations in the catalytic core, were expressed functionally in yeast and benchmarked against characteristic model compounds. Several designs were active and stable in a range of temperature and pH, displaying unprecedented enantiodivergence, changing regioselectivity from alkyl to aromatic hydroxylation, and increasing catalytic efficiencies up to 10-fold, with 15-fold improvements in total turnover numbers over the parental enzyme. We find that thiJournal of the American Chemical SocietyRepertoire of Computationally Designed Peroxygenases for Enantiodivergent C-H Oxyfunctionalization Reactions.PMC9936548Y2018/BIO-4738-EVOCHIMERA-CMBES-2017-080040RIS3-S2019\L1-274PTQ2019-010467PTQ2020-011037PID 2019-106166RB-100-OXYWAVE886567Gomez de Santos PMateljak IFleishman SJHoang MDHollmann FAlcalde MfalseRepertoire of Computationally Designed Peroxygenases for Enantiodivergent C-H Oxyfunctionalization Reactions.The generation of enantiodivergent biocatalysts for C-H oxyfunctionalizations is ever more important in modern synthetic chemistry. Here, we have applied the FuncLib algorithm based on phylogenetic and Rosetta calculations to design a diverse repertoire of active, stable, and enantiodivergent fungal peroxygenases. 24 designs, each carrying 4-5 mutations in the catalytic core, were expressed functionally in yeast and benchmarked against characteristic model compounds. Several designs were active and stable in a range of temperature and pH, displaying unprecedented enantiodivergence, changing regioselectivity from alkyl to aromatic hydroxylation, and increasing catalytic efficiencies up to 10-fold, with 15-fold improvements in total turnover numbers over the parental enzyme. We find that thi2023-01-01T00:00:00Z2023 Feb2026-05-28T18:33:40.635Z2024-11-20T17:34:34.248ZS-EPMC99365483668934910.1021/jacs.2c11118