Abstract

AmpC, a class C β-lactamase, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. The current proposed mechanism of action involves an acyl-intermediate, where the enzyme becomes covalently attached to the drug at serine-64, before an activated water hydrolyzes the bond and regenerates the enzyme. Although this mechanism is generally accepted, the exact roles that the other active site residues play in recognition and breakdown of the substrate are not fully understood. Here, we investigate the role of the active site residue asparagine-152 (Asn152) in E. coli AmpC by mutating it to a glycine, serine, or threonine residue and examining the effect that these mutations have on kinetic and structural properties, when acting upon three different β-lactam drugs: cefotaxime, cefoxitin, and oxacillin. We found that the mutations cause 50 to 200 times higher k_cat values against cefotaxime and also allow the enzyme to break down oxacillin, which is not hydrolyzed at a detectable rate by wild type AmpC. We obtained the crystal structure of wild type AmpC and AmpC N152G bound to cefotaxime and found a rotation of glutamine-120 and lysine-67 to be the only significant differences in the active site residues as well as a slight conformational change in the drug itself. Uncovering the specific role of Asn152 in the function of AmpC in conjunction with work done to understand the roles of other active site residues will be useful in the development of inhibitors to these enzymes that may help combat antibiotic resistance.