Characterization and structural analysis of class D β-lactamases: variants and mutants of OXA-type carbapenemases

Abstract

The rise and spread of antibiotic resistant bacteria is posing a serious threat to the global health; antibiotic drugs are rendered ineffective and common infectious diseases become harder or impossible to treat. The beta-lactamase enzymes, with their ability to hydrolyze the beta-lactam antibiotics, are a major cause of resistant bacteria. The OXA-beta-lactamase enzymes are one of the most alarming group, as some members of this group have the ability to hydrolyze and inactivate the carbapenem type beta-lactams, which are considered a last resort drug. OXA-23, OXA-24 and OXA-48 are all OXA-enzymes with carbapenem activity.

This study focused on the three enzymes OXA-23, OXA-24 and OXA-48. It was attempted to clone and express the OXA-23 and OXA-24 proteins from clinical isolates, using a restriction free cloning method.

For the OXA-48 enzyme, the three mutations S118G, R206A and R250A were made in order to study the importance of these enzymes with respect to activity, avibactam binding, and protein dimerization. The S118G and R250A mutations are both in the active site, and enzyme kinetics studies on these mutations showed a significant decrease in the enzyme’s ability to hydrolyze carbapenem and penicillin type antibiotics, which gives evidence to the importance of these to residues with respect to the activity of the enzyme. The R206 residue is in a dimer interface interaction, but size exclusion chromatography on the mutant showed that the mutation did not have any effect on the protein dimerization.