Structural milestones in the reaction pathway of an amide hydrolase: Substrate, acyl, and product complexes of cephalothin with AmpC β-lactamase

β-lactamases hydrolyze β-lactam antibiotics and are the leading cause of bacterial resistance to these drugs. Although β-lactamases have been extensively studied, structures of the substrate-enzyme and product-enzyme complexes have proven elusive. Here, the structure of a mutant AmpC in complex with the β-lactam cephalothin in its substrate and product forms was determined by X-ray crystallography to 1.53 Å resolution. The acyl-enzyme intermediate between AmpC and cephalothin was determined to 2.06 Å resolution. The ligand undergoes a dramatic conformational change as the reaction progresses, with the characteristic six-membered dihydrothiazine ring of cephalothin rotating by 109°. These structures correspond to all three intermediates along the reaction path and provide insight into substrate recognition, catalysis, and product expulsion.