Two non-active site residues W165 and L166 prominently influence the beta-lactam hydrolytic ability of OXA-23 beta-lactamase

Dissemination of class D OXA-type carbapenemases is one of the significant causes of beta-lactam resistance in Gram-negative bacteria. The amino acid residues present near the active site are involved in hydrolytic mechanism of class D carbapenemases, though it is not identified in OXA-23. Here, with the help of site-directed mutagenesis, we aimed to explicate the importance of the residues W165, L166 and V167 of the possible omega loop and residue D222 in the short beta 5-beta 6 loop on the activity of OXA-23. All the residues were substituted with alanine. The resultant proteins were assayed for the changes in activity in E. coli cells and purified for in vitro activity, and stability assessment. E. coli cells harboring OXA-23_W165A and OXA-23_L166A, individually, exhibited a significant decrease in resistance towards beta-lactam antibiotics as compared to OXA-23. Further, purified OXA-23_W165A and OXA-23_L166A imparted about >4-fold decrease in catalytic efficiency and displayed reduced thermal stability as compared to OXA-23. Bocillin-FL binding assay revealed that W165A substitution results in improper N-carboxylation of K82, leading to deacylation deficient OXA-23. Therefore, we infer that the residue W165 maintains the integrity of N-carboxylated lysine (K82) of OXA-23 and the residue L166 might be responsible for properly orientating the antibiotic molecules.

Automated summary

The dissemination of class D OXA-type carbapenemases is a significant cause of beta-lactam resistance in Gram-negative bacteria. We conducted a study to determine the importance of specific amino acid residues in the activity of OXA-23 carbapenemase. Site-directed mutagenesis was used to substitute the residues W165, L166, V167, and D222 with alanine, and the resultant proteins were assessed for changes in activity and stability. Our findings suggest that W165 plays a role in maintaining the integrity of OXA-23, while L166 may be responsible for orientating the antibiotic molecules correctly.