Structural basis for imipenem inhibition of class C beta-lactamases

Abstract

To determine how imipenem inhibits the class C beta-lactamase AmpC, the X-ray crystal structure of the acyl-enzyme complex was determined to a resolution of 1.80 A. In the complex, the lactam carbonyl oxygen of imipenem has flipped by approximately 180 degrees compared to its expected position; the electrophilic acyl center is thus displaced from the point of hydrolytic attack. This conformation resembles that of imipenem bound to the class A enzyme TEM-1 but is different from that of moxalactam bound to AmpC.

FIG. 1.

Structures of β-lactams that contain large 6(7)α substituents. (A) Imipenem; (B) moxalactam; (C) cefoxitin.

FIG. 2.

The active site for molecule 1 of the AmpC-imipenem complexed structure. (A) Stereoview of the electron density of the active site. The 2F_o-F_c electron density of the refined protein model is shown in blue, contoured at 1 σ, and the simulated annealing omit electron density of the refined ligand covalently bound to Ser64 is shown in green, contoured at 3.3 σ. Carbon atoms are colored gray, oxygen atoms are colored red, and nitrogen atoms are colored blue. Cyan spheres indicate water molecules. The figure was generated using SETOR software (4). (B) Stereoview of the interactions observed in the AmpC-imipenem complexed structure. Atoms are colored as described for panel A, except the carbon atoms of the ligand are colored green. Dashed yellow lines indicate hydrogen bonds. Figures 2B and 3 were generated using MidasPlus software (5).

FIG. 3.

Comparison of the acyl adducts of AmpC and imipenem (PDB entry 1LL5) (A), AmpC and moxalactam (PDB code 1FCO) (14) (B), and TEM-1 and imipenem (PDB code 1BT5) (10) (C), showing that AmpC binds imipenem differently than it does moxalactam but similarly to TEM-1. Atoms are colored as described for Fig. 2A, except the carbon atoms of imipenem in panel A are colored green, the carbon atoms of moxalactam in panel B are colored orange, and carbon atoms of imipenem in panel C are colored purple. Dashed yellow lines indicate hydrogen bonds of the oxyanion, or electrophilic, hole.