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

Sarah M. Drawz, Magdalena Taracila, Emilia Caselli, Fabio Prati, Robert A. Bonomo

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31 Scopus citations

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 C 3/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 C 3/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.

Original languageEnglish (US)
Pages (from-to)941-958
Number of pages18
JournalProtein Science
Volume20
Issue number6
DOIs
StatePublished - Jun 2011

Keywords

  • AmpC cephalosporinases
  • Pseudomonas aeruginosa
  • β-lactamase inhibitor resistance

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