Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases

Biochemistry. 2015 Jan 20;54(2):588-97. doi: 10.1021/bi501052t. Epub 2014 Dec 22.

Abstract

Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES β-lactamases are important contributors to carbapenem resistance in clinical bacterial pathogens. A single amino acid difference at position 170 of the GES-1, GES-2, and GES-5 enzymes is responsible for the expansion of their substrate profile to include carbapenem antibiotics. This highlights the increasing need to understand the mechanisms by which the GES β-lactamases function to aid in development of novel therapeutics. We demonstrate that the catalytic efficiency of the enzymes with carbapenems meropenem, ertapenem, and doripenem progressively increases (100-fold) from GES-1 to -5, mainly due to an increase in the rate of acylation. The data reveal that while acylation is rate limiting for GES-1 and GES-2 for all three carbapenems, acylation and deacylation are indistinguishable for GES-5. The ertapenem-GES-2 crystal structure shows that only the core structure of the antibiotic interacts with the active site of the GES-2 β-lactamase. The identical core structures of ertapenem, doripenem, and meropenem are likely responsible for the observed similarities in the kinetics with these carbapenems. The lack of a methyl group in the core structure of imipenem may provide a structural rationale for the increase in turnover of this carbapenem by the GES β-lactamases. Our data also show that in GES-2 an extensive hydrogen-bonding network between the acyl-enzyme complex and the active site water attenuates activation of this water molecule, which results in poor deacylation by this enzyme.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Anti-Bacterial Agents / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Carbapenems / metabolism*
  • Catalytic Domain
  • Crystallography, X-Ray
  • Doripenem
  • Ertapenem
  • Escherichia coli / chemistry
  • Escherichia coli / enzymology*
  • Escherichia coli / metabolism
  • Kinetics
  • Meropenem
  • Models, Molecular
  • Thienamycins / metabolism*
  • beta-Lactamases / chemistry
  • beta-Lactamases / metabolism*
  • beta-Lactams / metabolism*

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Carbapenems
  • Thienamycins
  • beta-Lactams
  • Doripenem
  • beta-lactamase GES-2
  • beta-Lactamases
  • carbapenemase
  • Meropenem
  • Ertapenem