Binding mode of Isoxazolyl Penicillins to a Class-A β-lactamase at ambient conditions.

The predominant resistance mechanism observed in Gram-negative bacteria involves the production of β-lactamases, which catalyse the hydrolysis of β-lactam antibiotics, thereby rendering them ineffective. Although Isoxazolyl Penicillins have been available since the 1970s, there are currently no structures in complex with class-A β-lactamases available. Here we have analysed the structure of the clinically relevant β-lactamase CTX-M-14 from Klebsiella pneumoniae near physiological temperatures, via serial synchrotron crystallography. We demonstrate the acyl-enzyme intermediates of the catalytically impaired CTX-M-14 mutant E166A in complex with three Isoxazolyl-Penicillins: Oxacillin, Cloxacillin and Dicloxacillin. Structural comparisons of CTX-M-Penicillin complexes show that, while conserved active-site interactions are maintained, each Isoxazolyl-Penicillin adopts a distinct conformation. While the three derivatives differ only by one and two chlorine atoms, respectively, their conformational heterogeneity appears to be increased by chlorination of the phenyl ring.