Repertoire of Computationally Designed Peroxygenases for Enantiodivergent C–H Oxyfunctionalization Reactions

Abstract

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.

Peroxygenase‐Catalysed Selective Oxidation of Silanes to Silanols

“…Substrate induced-fit binding simulations provide a good understanding of the selectivity patterns, where steric hindrance was the major contributor. [19] This will put the basis for engineering AaeUPO variants [20] with improved activity and (enantio)selectivity towards sterically more demanding silanes for whose silanol products configurational stability of the enantiomers may be expected. [21]…”

Computational-Aided Engineering of a Selective Unspecific Peroxygenase toward Enantiodivergent β-Ionone Hydroxylation

“…A recent untargeted approach using the FuncLib algorithm led to impressive shifts in enantioselectivity. 18 Our objective was to address the existing limitations and demonstrate the successful engineering of a UPO toward the enantioselective conversion of a selected substrate with low inherent enantioselectivity.…”

“…However, the engineering of UPOs for the oxyfunctionalization of specific substrates with initially poor enantioselectivities in a targeted manner remained elusive. A recent untargeted approach using the FuncLib algorithm led to impressive shifts in enantioselectivity …”

Aromatic hydroxylation of substituted benzenes by an unspecific peroxygenase from Aspergillus brasiliensis

“…3,4 Despite a broad range of catalysed reactions like aliphatic hydroxylation, epoxidation, sulfoxidation, N-oxidation and O-dealkylation, aromatic hydroxylations with UPOs have been rarely reported. [5][6][7] Direct aromatic hydroxylation of substituted benzenes gives access to phenols, versatile synthons in the synthesis of dyes, pharmaceuticals and agrochemicals. Chemical aromatic oxidation remains, however, quite challenging because of low efficiency and poor selectivity.…”