Close-up of the active site and high-occupancy hydrogen bonds during MD simulations. (A) Overall view of the L-ASN tetramer with focus on a single monomer (green). In the close-up, the active site (orange sticks) and lid-loop (blue) are shown, as well as important residues from the interacting second monomer (light pink). An aspartic acid molecule, in space fill, is shown within the active site. (B) Residues of the active site are shown in different conformations and con-figurations found throughout the MD simulation. Simulations I through IV are ordered in a time-dependent fashion denoted by arrows. (I) A more closed conformation for the active site lid-loop is shown, where a hydrogen bond (red) between N24 and E283 is found. (II) One of the main hydrogen bonds in the active site of the WT enzyme is found between D281 and N24. Furthermore, the very important stabilization of T12 by Y25 can be seen. (III) The hydrogen bond between D281 and N24 fluctuates and can be found in different conformations. (IV) Y25 can also form a stabilizing hydrogen bond to residue E283. (C) The active site and surrounding is shown for mutant N24A. The lid-loop is colored blue. D281 and E283 are colored light pink. Y25 (orange) makes a hydrogen bond (red dashed line) to E283 and the nucleophile T12 (orange). This conformation is found more often for the mutant N24A.