Figure 1.
Catalytic mechanism of Ntn-hydrolases.
Panel (A). The reaction begins when the nucleophilic oxygen/sulfur of Thr/Ser/Cys donates its proton to its own alpha-amino group and attacks the carbonyl carbon of the substrate [1], leading to a negatively charged tetrahedral intermediate (X represents oxygen or sulfur). The acylation step is completed when the alpha-amino group of the catalytic residue protonates the nitrogen of the scissile amide bond leading to the expulsion of the leaving group. Panel (B) First reaction of the catalytic mechanism of CBAH. A, B, and C are key steps for the cleavage of TAU amide bond.
Figure 2.
CBAH residues interacting with Cys2 as found in the crystal structure.
Secondary structure elements of CBAH are displayed with gray cartoons, while carbon atoms are in black.
Figure 3.
Free energy profile for Cys2 internal proton transfer.
Free energy profile along S, H1 distance as obtained from umbrella sampling calculations and WHAM.
Figure 4.
CBAH in its Michaelis complex with TAU.
CBAH carbon atoms are in black, while TAU carbon atoms are in gray. The CBAH active site is represented as a solid van der Waals surface (white), which is occupied by TAU substrate. Relevant CBAH residues are displayed and labeled. Relevant H-bonds between the substrate and CBAH are shown as dotted green lines.
Figure 5.
Evolution of the reaction path from steered-MD/PCVs simulations.
The initial pulling showing the nucleophilic attack (described by the S, C distance), occurring before the proton transfer reaction (N, H1 distance), is displayed as a black line. The first steered-MD round is featured by a similar reaction path. Conversely, the last three steered-MD runs show a different mechanism, where protonation of the nitrogen (N) occurs at the same time (run three) or even before the nucleophilic attack. The steered-MD rounds third and fourth displayed a very similar distributions of the S-C and N-H1 distances, indicating the reaction path is converged in terms of explored configurations of the system.
Figure 6.
Free energy surface (FES) for TAU hydrolysis catalyzed by CBAH.
Left panel. Bi-dimensional FES in the S and Z space from US simulations. The minimum free energy path is displayed with a continuous black line. Configurations 1–5 are crucial structures identified along the reaction path, S. Right panel, subpanel (A). Projection of the FES of TAU hydrolysis on S. Right panel, subpanel B. Relevant interatomic distances (reported as average over US with error bars representing the standard deviations) are plotted as function of S. Right panel, subpanel C. Improper torsion of the amide nitrogen (N) of TAU as function of S. Right panel, subpanel D. Nucleophile attacking angle as function of S.
Figure 7.
Transition state (TS) and tetrahedral adduct (TA) geometries identified along the path.
Left panel (A). TS structure of TAU (gray carbons) hydrolysis catalyzed by CBAH (black carbons). H1 is nearly equidistant between N and N1 favoring the formation of a pseudo chair structure. Right panel (B) Zwitterionic TI. In both panels, H-bonds are shown as dotted green lines, while secondary structure elements of CBAH are omitted for clarity.
Figure 8.
Activation barriers for TAU hydrolysis as obtained from steered-MD/PCVs simulations.
Barriers are estimated from work profiles and are refereed to CBAH wild type (wt) and to zero-point charge mutants.