Kinetic properties of four plasmid-mediated AmpC beta-lactamases

Abstract

The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C beta-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC beta-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the K(m) values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward beta-lactam antibiotics are mainly due to the overproduction of the beta-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded beta-lactamases.

FIG. 1.

Titration curve of the ACT-1 β-lactamase by aztreonam. The residual activity of ACT-1 versus the [aztreonam]/[ACT-1] ratio is shown. The linear regression allows the determination of the actual concentrations of active enzyme. E_th, theoretical values of enzyme concentration.