To estimate the differences in enantioselectivity, we performed Quantum Mechanics (QM) theozyme calculations to determine the ideal distances and angles for the azide-mediated cyclohexene oxide ring-opening reaction and evaluated the number of MD frames displaying a pro-S/pro-R conformation. QM calculations indicate that at the transition state the nucleophilic attack distance is 2.2 Å with an Nazide-Nazide-C(1) angle of ca. 109º, whereas in the reactant complex the distance is 3.2 Å and the angle 85º (Figure ). We filtered the number of MD frames presenting catalytically competent poses by considering distances < 4 Å between the epoxide oxygen of 1 and the catalytic Tyr165, as well as the distance (< 4 Å) and angle (range of 80 to 120º) between azide and either C1/C2 of cyclohexene oxide corresponding to pro-S/pro-R attacks, respectively (see Figure ). Using this filtering scheme, we could get an estimation of the enantiomeric excess, which our simulations predict to be 13.7 % and 40.7 % for wild type and variant M45F, respectively. These simulations therefore suggest that mutation M45F restricts the flexibility of position 45 by establishing non-covalent C-H••π interactions with the epoxide. This interaction observed in M45F helps retaining the substrate and azide in the active site and promotes the enantioselective epoxide-ring opening reaction by positioning C1 closer to the azide for the selective formation of the (1S,2S)-product. and especially azide can adopt multiple conformations in the active site pocket of HheG wild type, in line with its inferior catalytic activity. Nucleophile binding site residues are shown using gray sticks, catalytic residues in teal, and loop3947 and position 45 in light pink. Cyclohexene oxide (1) and azide are represented using spheres and black sticks.