Theoretical Evidence for the Nonoccurrence of Tetrahedral Intermediates in the Deacylation Pathway of the Oxacillinase-24/Avibactam Complex

Abstract

Oxacillinases (OXAs) β-lactamases are of special interest because of their capacity to hydrolyze antibacterial drugs such as cephalosporins and carbapenems, which are frequently used as the last option for the treatment of multidrug-resistant bacteria. Although the comprehension of the involved mechanisms at the atomic level is crucial for the rational design of new inhibitors and antibiotics, currently there is no study on the acylation/deacylation mechanisms of the OXA-24/avibactam complex from first principles; therefore, mechanistic details such as activation barriers, characterization of intermediates, and transition states are still uncertain. In this article, we address the deacylation of the OXA-24/avibactam complex by molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics computations. The study supplies mechanistic details not available so far, namely, topology of the potential energy surfaces, characterization of transition states and intermediates, and calculation of the respective activation barriers. The results show that the deacylation occurs via a mechanism of two stages; the first one involves the formation of a dianionic intermediate with a computed activation barrier of 24 kcal/mol. The second stage corresponds to the cleavage of the O_S81-C bond promoted by the protonation of the O_S81 atom by the carboxylated Lys84 and the concomitant formation of the C₇-N₆ bond, allowing the liberation of avibactam and recovery of the enzyme. The calculated activation barrier for the second stage is 13 kcal/mol. The structure of the intermediate, formed in the first stage, does not fulfill the characteristics of a tetrahedral intermediate. These results suggest that the recyclization of avibactam from the OXA-24/avibactam complex may occur without the emergence of tetrahedral intermediates, unlike that observed in the class A CTX-M-15.

Figure 1

(a) rmsd plot of the active site. (b) RMSF plot showing the key residues denoted in blue lines.

Figure 2

(a) Interaction between the sulfate group of avibactam and Arg261. (b) Distribution of the angle Ser128(O–H–N)Lys218 (b).

Figure 3

Orientations of the N₆–H bond along the 100 ns simulation.

Figure 4

Clustering analysis in terms of the dihedral angles α, β, γ. The red dot represents the centroids.

Figure 5

Reduced representation of the centroid taken at 76 ns after cluster analysis.

Scheme 1. Overall Mechanism for the Recyclization of Avibactam from Its Complex with OXA-24

Figure 6

(a) Two-dimensional view of PES-1. (INT: intermediate, MC-2: Michaelis complex, TS-2: transition-state 2, P: products). (b) Optimized Structure of the transition state TS-1, distance in Å. (c) Optimized structure of the dianionic intermediate, INT, angles in degrees.

Figure 7

Avibactam-residue interactions for the 100 ns MD simulation (light green: hydrophobic, light blue: polar).

Figure 8

(a): Two-dimensional view of PES-2. (INT: intermediate, MC-2: Michaelis Complex, TS-2: transition state 2, P: products). (b): Optimized structure of the transition state TS-2, distance in Å.

Scheme 2. Reduced Labeled Scheme of the QM Zone