Structural Basis of Activity against Aztreonam and Extended Spectrum Cephalosporins for Two Carbapenem-Hydrolyzing Class D beta-Lactamases from Acinetobacter baumannii

The carbapenem-hydrolyzing class D beta-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for beta-lactam antibiotic resistance in Acinetobacter species. Many Variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P -> S substitution at homologous positions in the OXA-24/40 and OXA-23 background's, respectively. We purified OXA-160 and OXA-225 and used steady-State kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime, and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower K-m values, suggesting that the P -> S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the beta 5-beta 6 loop and the omega loop of the enzymes. The P -> S substitution found in OXA-160 and OXA-225 results in a deviation of the beta 5-beta 6 loop, relieving the steric dash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class beta-lactamases but may also provide a map for Chi-lactam improvement.