Structural basis for carbapenemase activity of the OXA-23 β-lactamase from Acinetobacter baumannii

Abstract

Dissemination of Acinetobacter baumannii strains harboring class D β-lactamases producing resistance to carbapenem antibiotics severely limits our ability to treat deadly Acinetobacter infections. Susceptibility determination in the A. baumannii background and kinetic studies with a homogeneous preparation of OXA-23 β-lactamase, the major carbapenemase present in A. baumannii, document the ability of this enzyme to manifest resistance to last-resort carbapenem antibiotics. We also report three X-ray structures of OXA-23: apo OXA-23 at two different pH values, and wild-type OXA-23 in complex with meropenem, a carbapenem substrate. The structures and dynamics simulations reveal an important role for Leu166, whose motion regulates the access of a hydrolytic water molecule to the acyl-enzyme species in imparting carbapenemase activity.

Figure 1

Ribbon representation of the superimposition of OXA-23 at pH 4.1 (green) and pH 7.0 (magenta). The loops that show large-scale structural rearrangements are shown at the lower right, in the “open” conformation at pH 4.1 and in the “closed” conformation at pH 7.0. The loop which corresponds topologically with the Ω-loop in the class A β-lactamases is also indicated. Meropenem (yellow ball-and-stick) is shown in the active site of OXA-23. See also Figure S1, Figure S4, Table S2 and Table S3)

Figure 2

Meropenem binding to OXA-23. (A) Residual F_o-F_c electron density (pink) in the OXA-23 active site modeled with the final coordinates of meropenem (cyan ball-and-stick). Final 2F_o-F_c electron density for Ser79 is indicated. (B) Meropenem bound in the active site in the final model showing the hydrogen-bonding interactions (dashed black lines). See also Figure S2.

Figure 3

Stereoview of the superposition of the acyl-enzyme intermediate in OXA-23 (green), OXA-24 (magenta) and OXA-1 (white). The two hydrophobic residues which interact with the 6a-hydroxyethyl moiety of the intermediate are shown at upper right (Leu166 for OXA-23 and Leu161 for OXA-1) and lower right (Val128 for OXA-23). The difference in conformation of the leucine residue can be seen. The residues which constitute the tunnel-like structure in the OXA enzymes are indicated for OXA-23 (Phe110 and Met221). See also Figure S3 and Table S1.

Figure 4

Molecular surface representation of (A) OXA-1 and (B) OXA-23. In both cases the bound substrate (doripenem and meropenem, respectively) are shown as cyan ball-and-stick. The motion of the Leu166 side chain in response to meropenem binding in OXA-23 opens a channel to the external solvent and allows access of a water molecule to the N-carboxylated Lys82.

Figure 5

Schematic of the active site of the acyl-enzyme species of meropenem in (A) Δ²- and (B) Δ¹-tautomeric forms. Distance distribution of the d₁ and d₂ during the 20 ns simulation of OXA-23 (black) and OXA-1 (red) acylated with meropenem in (C) Δ²- and (D) Δ¹-tautomeric forms. See also Figure S5.