Exploring sequence requirements for C3/C4 carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC β-lactamase

In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC beta-lactamase) is often responsible for high-level resistance to beta-lactam antibiotics. Despite years of study of these important beta-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both beta-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C-3/C-4 carboxylate of beta-lactams, we first examined a molecular model of a P. aeruginosa AmpC beta-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C-3/C-4 carboxylate characteristic of beta-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C-3/C-4 beta-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 beta-lactamase maintains a high level of activity despite the substitution of C-3/C-4 beta-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C-3/C-4 carboxylate of the beta-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.