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

• Published in: Protein science 2011-06, Vol.20 (6), p.941-958
• Main Authors: Drawz, Sarah M, Taracila, Magdalena, Caselli, Emilia, Prati, Fabio, Bonomo, Robert A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anti-Bacterial Agents - metabolism; Anti-Bacterial Agents - pharmacology; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; beta-Lactam Resistance; beta-Lactamase Inhibitors; beta-Lactamases - chemistry; beta-Lactamases - metabolism; beta-Lactams - metabolism; beta-Lactams - pharmacology; Binding Sites; Boronic Acids - pharmacology; Cephalosporinase - chemistry; Cephalosporinase - metabolism; Cephalosporins - metabolism; Cephalosporins - pharmacology; Cephalothin - metabolism; Cephalothin - pharmacology; Enzyme Inhibitors - pharmacology; Models, Molecular; Molecular Sequence Data; Protein Binding; Pseudomonas aeruginosa - chemistry; Pseudomonas aeruginosa - enzymology; Sequence Alignment; Substrate Specificity
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.612

In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-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 β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C₃/C₄ carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C₃/C₄ carboxylate characteristic of β-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 C₃/C₄ β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C₃/C₄ β-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 C₃/C₄ carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.