We have applied QM/MM well-tempered metadynamics simulations to compare the feasibility of the multiple proposed reaction mechanisms shown in Figure . In case of the AMDase ICPLLG mutant, the starting structures for both hypothesis 1, as well as hypothesis 3 were prepared based on the available information regarding the constitution of the original hydrophobic pocket, the new hydrophobic pocket emerging due to the mutations, and the dioxyanion hole. In total, four substrate orientations were considered, reacting either through retention (hypothesis 1) or inversion (hypothesis 3) of the stereoconfiguration, with either pro-R or pro-S carboxylate cleavage (Figure 3C and 3D).