Structural and mechanistic insights into the inhibition of class C β-lactamases through the adenylylation of the nucleophilic serine

J Antimicrob Chemother. 2017 Mar 1;72(3):735-743. doi: 10.1093/jac/dkw491.


Objectives: : Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C β-lactamases.

Methods: : The formation and the stability of the adenylate adduct were examined by X-ray crystallography and MS. Inhibition assays for kinetic parameters were performed by monitoring the hydrolytic activity of AmpC BER and CMY-10 using nitrocefin as a reporter substrate. The effect of adenosine 5'-(P-acetyl)monophosphate (acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates.

Results: : The crystal structures and MS analyses confirmed the acAMP-mediated adenylylation of the nucleophilic serine in AmpC BER and CMY-10. acAMP inhibited AmpC BER and CMY-10 through the adenylylation of the nucleophilic serine, which could be modelled as a two-step mechanism. The initial non-covalent binding of acAMP to the active site is followed by the covalent attachment of its AMP moiety to the nucleophilic serine. The inhibition efficiencies ( k inact / K I ) of acAMP against AmpC BER and CMY-10 were determined to be 320 and 140 M -1 s -1 , respectively. The combination of ceftazidime and acAMP reduced the MIC of ceftazidime against the tested bacteria.

Conclusions: : Our structural and kinetic studies revealed the detailed mechanism of adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C β-lactamase inhibitors on the basis of the nucleotide scaffold.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology*
  • Bacterial Proteins / metabolism
  • Ceftazidime / pharmacology
  • Crystallography, X-Ray
  • Kinetics
  • Microbial Sensitivity Tests
  • Serine / chemistry*
  • beta-Lactamase Inhibitors / pharmacology*
  • beta-Lactamases / chemistry*
  • beta-Lactamases / metabolism*


  • Anti-Bacterial Agents
  • Bacterial Proteins
  • beta-Lactamase Inhibitors
  • Serine
  • Ceftazidime
  • beta-Lactamases