Chromosomal class C beta-lactamases contain a hydrogen-bonded network that interacts with the cephalosporin C4-carboxylate. In plasmid-based class C enzymes, this network is frequently broken through mutations at position 346; these mutations correlate with resistance to cephamycins and third-generation cephalosporins. To better understand the mechanism of these mutations, AmpC from Enterobacter cloacae P99 was mutated and kinetic constants were determined for N346I and S289P. Compared to wild-type, the Km for nitrocefin increased three-fold in both S289P and N346I. The kcat values followed a different trend, where S289P decreased by 35 fold and N346I decreased by almost two fold. Overall, the kcat/Km values decreased in both mutations, by 116-fold in S289P and six-fold in N346I. Random mutagenesis of the N346I variant produced clones that grew on four times the amount of cefoxitin as the parent plasmids. Single mutations obtained from this experiment were constructed and tested by MIC to determine the source of increased activity.

Role of N346 in class C beta-lactamases

255th National Meeting (2018)

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