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

Protein Sci. 2011 Jun;20(6):941-58. doi: 10.1002/pro.612. Epub 2011 May 3.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Anti-Bacterial Agents / metabolism*
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / antagonists & inhibitors
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism*
  • 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
  • beta-Lactam Resistance*
  • beta-Lactamase Inhibitors
  • beta-Lactamases / chemistry*
  • beta-Lactamases / metabolism*
  • beta-Lactams / metabolism
  • beta-Lactams / pharmacology


  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Boronic Acids
  • Cephalosporins
  • Enzyme Inhibitors
  • beta-Lactamase Inhibitors
  • beta-Lactams
  • Cephalosporinase
  • AmpC beta-lactamases
  • beta-Lactamases
  • Cephalothin