title: Exploring sequence requirements for C₃/C₄ carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC β-lactamase.
abstract: 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.
authors.affiliation: Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Drawz
authors.forename: Magdalena
authors.initials: M
authors.lastname: Taracila
authors.forename: Emilia
authors.initials: E
authors.lastname: Caselli
authors.forename: Fabio
authors.initials: F
authors.lastname: Prati
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001897:Boronic Acids / Q000494:pharmacology; D002510:Cephalosporinase / Q000737:chemistry / Q000378:metabolism*; D002511:Cephalosporins / Q000378:metabolism* / Q000494:pharmacology; D002512:Cephalothin / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001897:Boronic Acids; D002511:Cephalosporins; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D002510:Cephalosporinase; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D002512:Cephalothin