Mechanism of Imipenem Hydrolysis by NDM1

PALACIOS, A.R.; LLARRULL, L. I.; SPENCER, J.; BONOMO, R.A.; VILA, A.J.

San Diego

Congreso; ICAAC/ICC 2015; 2015

American Society for Microbiology

Background: The emergence and dissemination of Gram-negative bacterial pathogens resistant to beta-lactam antibiotics is a global public health issue. Metallo-beta-Lactamases (MBLs) confer broad spectrum resistance by using a Zn(II) center to catalyze hydrolytic cleavage of the beta-lactam ring. NDM-1 is one of the most potent and widespread MBLs. Methods: NDM-138-294 was overexpressed in Escherichia coli BL21(DE3) cells and purified as the di-Zn(II) enzyme. Apo-NDM-1 (non-metallated) was titrated with CoSO4 to obtain di-Co(II) NDM-1. Pre-steady state kinetic experiments were conducted and single-wavelength traces were fit to different kinetic models by means of nonlinear regression analysis.Results: Imipenem hydrolysis by Zn(II)-NDM-1 and Co(II)-NDM-1 was fitted to two indistinguishable branched mechanisms, both characterized by accumulation of two reaction intermediates, but differing according to whether one, or both, were productive (Fig.1). The absorption features of the Co(II) ions (550 nm) showed changes in metal coordination geometry during enzyme turnover. The absorption features of the reaction intermediates at 385 nm (Zn(II)-NDM-1) and 407 nm (Co(II)-NDM-1), compared to those already reported for deprotonated hydrolyzed imipenem, suggest that they correspond to anionic species that give rise to the product upon a final protonation step.Conclusions: NDM-1 catalyzes imipenem hydrolysis by a branched kinetic mechanism with two intermediates, similar to those observed for other MBLs, which demonstrates the existence of common reaction intermediates for MBL-catalyzed carbapenem hydrolysis. This information serves as a potential foundation for rational MBL inhibitor design.