To further enhance the diastereo-and enantioselectivity of this biocatalytic cyclopropanation, using ApePgb W59S Y60G and RmaNOD Y32K V97L as the template, we performed enzyme engineering through site-saturation mutagenesis (SSM) and screening by targeting active-site residues in proximity to the substrate (Figure ). At the time when we started this project, the structure of ApePgb was unknown. We thus used Al-phaFold2 to generate the structure of ApePgb W59S Y60G and docked the heme cofactor into this apo protein based on the structure of a homologous protoglobin from Methanosarcina acetivorans (MaPgb, Protein Data Bank (PDB) ID: 3ZJI). Guided by this structure and previous engineering efforts with ApePgb, SSM libraries of the ApePgb W59S Y60G variant were created for active-site residues 59, 60, 93, and 145 (Figure , see Table -S4 for details). In a similar vein, SSM libraries of RmaNOD Y32K V97L were generated by randomizing active-site residues 32, 46, 52, and 97 (Figure , see Table -S7 for details).