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 are available since the 1970s, there are currently no structures in complex with class-A β -lactamases available. In order to support the rational development of new β -lactamase inhibitors, we have analysed the structure of the clinically relevant β -lactamase CTX-M-14 from Klebsiella pneumoniae near physiolog- ical temperatures. By utilizing 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 dichloxacillin. While the three derivatives differ only by one and two Cl atoms, respectively, they show marked differences in their binding mode.