Lid-loop flexibility (RMSF), THR12 side-chain orientation, and distance analysis for important residues of the active site. (A) The RMSF for L-ASN WT (black) and 5 mutants are depicted in different colors calculated for the residue range 6-38. The lowest RMSF was found for N24T (blue) and N24A (green). N24H (yellow) shows a very high c-terminal RMSF. N24G (red) showed the highest RMSF of all L-ASN mutants. The double mutant N24S D281E (light brown) had a slightly increased RMSF compared with the WT. (B) The fraction of correctly placed hydroxyl groups toward the active site compared with the incorrect conformation involving the methyl group is shown. The highest fraction can be seen for N24A, the lowest for N24T and N24G. (C) Key distances measured in Å. The average and SDs are calculated for all snapshots found in the MD simulations of the WT (black) and the mutants N24G (red), N24A (green), and N24T (blue). Hydrogen bonds can be formed for distances around 2.8Å.