Intrigued by how mutation M45F enhances HheG's activity and enantioselectivity towards the epoxide-ring opening reaction of cyclohexene oxide (1) with azide, we decided to computationally evaluate HheG wild type and mutant M45F by means of Molecular Dynamics (MD) simulations in the presence and absence of substrates. M45F is contained in loop3947, whose conformational dynamics regulate the formation of the available tunnels for substrate binding and product release. Our study therefore first focused on the comparison of the conformational dynamics of HheG wild type and variant M45F (Figure ). We performed 5 replicas of 250 ns MD simulations (1.25 µs per system in tetrameric conformation) for both systems in (i) the absence of any ligand, and (ii) with both azide and epoxide 1 bound in the active site (see Experimental section for computational details). We performed Principal Component Analysis (PCA) considering the pairwise distances between the heavy atoms of residues included in loop3947 and the rest of the residues of the protein. , the closed conformation of loop3947 favors the formation of tunnels named T1, previously found to be present in most HHDHs, and T2, which is mostly found in HheG and HheC. The open conformation of loop3947 with the sidechain of M/F45 in the active site blocks the formation of tunnel 2 (T2), but instead opens tunnel 3 (T3) that is mostly observed in G-type HHDHs (Figures 4B, S3 and Table ). The analysis of the conformational landscape and available tunnels for HheG wild type and variant M45F suggests that the introduced mutation favors the exploration of both open and closed conformations of loop3947, which affects T2/T3 formation potentially impacting the productive binding of both substrates in the active site pocket.