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Probing the role of N152 in the class C beta-lactamase AmpC


Chemistry Department


College of Liberal Arts and Sciences

Date Range



Physical Sciences and Mathematics


AmpC, a class C ²-lactamase, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. Although a mechanism involving acylation by S64 followed by hydrolytic cleavage has been generally accepted, the exact roles that some 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 (N152) in E. coli AmpC by mutating it to a G, S, or T residue and examining the effect that these mutations have on kinetic and structural properties with four different ²-lactam drugs: cefotaxime, cefoxitin, oxacillin, and a derivative of cephalothin (CENTA). We discovered that although the mutations cause higher Km values with all substrates, they result in 50 to 150 times higher kcat values against cefotaxime. In addition, the N152 mutations provided the enzyme the ability to break down oxacillin, which is not a viable substrate for the wild type AmpC. To probe the mechanism behind the observed kinetics changes, crystal structures were obtained of AmpC WT or N125G in acyl-enzyme complexes with cefotaxime, cefoxitin, and oxacillin. The small structural differences in the active site have been associated with the changes in the Km and kcat kinetic values as a way to uncover the specific role of N152 in the function of AmpC. This work was supported by the Office of Undergraduate Research and Scholarship at GVSU.

Conference Name

Experimental Biology

Conference Location

Boston, MA

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