The MD simulations performed in the presence of both substrates indicate that in wild-type HheG the side chain of M45 is highly flexible, which affects the positioning of the epoxide and azide in the active site. As shown in Figure , azide can be either retained in the nucleophile binding site (grey region in Figure ) or get displaced close to the catalytic serine (teal region in Figure ), which hampers the epoxide-ring opening reaction. The introduction of mutation M45F favors the produtive binding of cyclohexene oxide (1) in the active site by establishing non-covalent C-H ••π interactions with F45 (Figure ). At the same time, azide is preferentially bound at the nucleophile binding pocket (grey region in Figure ). These simulations therefore indicate that mutation M45F helps retaining both the epoxide and azide in a catalytically competent pose in the active site pocket, which is in line with the lower K50 for azide and the higher kobs,max observed experimentally (Table ).