TY - JOUR
T1 - Repertoire of Computationally Designed Peroxygenases for Enantiodivergent C-H Oxyfunctionalization Reactions
AU - De Santos, Patricia Gomez
AU - Mateljak, Ivan
AU - Hoang, Manh Dat
AU - Fleishman, Sarel J.
AU - Hollmann, Frank
AU - Alcalde, Miguel
PY - 2023
Y1 - 2023
N2 - 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 this dramatic functional divergence stems from beneficial epistasis among the mutations and an extensive reorganization of the heme channel. Our work demonstrates that FuncLib can rapidly design highly functional libraries enriched in enantioselective peroxygenases not seen in nature for a range of biotechnological applications.
AB - 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 this dramatic functional divergence stems from beneficial epistasis among the mutations and an extensive reorganization of the heme channel. Our work demonstrates that FuncLib can rapidly design highly functional libraries enriched in enantioselective peroxygenases not seen in nature for a range of biotechnological applications.
UR - http://www.scopus.com/inward/record.url?scp=85147116057&partnerID=8YFLogxK
U2 - 10.1021/jacs.2c11118
DO - 10.1021/jacs.2c11118
M3 - Article
C2 - 36689349
AN - SCOPUS:85147116057
SN - 0002-7863
VL - 145
SP - 3443
EP - 3453
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 6
ER -