Structural and Mechanistic Insights into NDM-1 Catalyzed Hydrolysis of Cephalosporins

Cephalosporins constitute a large class of beta-lactam antibiotics clinically used as antimicrobial drugs. New Dehli metallo-beta-lactamase (NDM-1) poses a global threat to human health as it confers on bacterial pathogen resistance to almost all beta-lactams, including penicillins, cephalosporins, and carbapenems. Here we report the first crystal structures of NDM-1 in complex with cefuroxime and cephalexin, as well as NMR spectra monitoring cefuroxime and cefixime hydrolysis catalyzed by NDM-1. Surprisingly, cephalosporoate intermediates were captured in both crystal structures determined at 1.3 and 2.0 A. These results provide detailed information concerning the mechanism and pathways of cephalosporin hydrolysis. We also present the crystal structure and enzyme assays of a D124N mutant, which reveals that D124 most likely plays a more structural than catalytic role.