Acyl-intermediate Structures of an Extended Spectrum Clinically-Derived Class D β-lactamase Variant, OXA-160, in Complex with Cefotaxime, Ceftazidime, and Aztreonam

OXA-24 is a carbapenem-hydrolyzing class D β-lactamase (CHDL) that poses a serious medical threat by destroying carbapenem class antibiotics. OXA-160 is a clinically-derived OXA-24 variant with a Pro→Ser substitution. Previously, it was shown that OXA-160 has higher catalytic activity against third-generation cephalosporins compared to OXA-24 and is able to maintain normal activity against penicillins and carbapenems. To slow deacylation, we introduced a second mutation (Val130Asp) to allow us to capture a drug-complex structure. We examined the OXA-160/Val130Asp variant in complex with the substrates cefotaxime, ceftazidime, and aztreonam using X-ray crystallography. Our analysis shows that all three of these bulky antibiotics require β5-β6 and/or omega loop deviations, and we propose that these conformational changes are made possible by replacing the restricted proline with the more flexible serine. These crystallographic structures reveal that a Pro227Ser mutation enlarges the active site, better accommodating advanced cephalosporin drugs

Structural Basis of Activity against Aztreonam and Extended Spectrum Cephalosporins for Two Carbapenem-Hydrolyzing Class D β‑Lactamases from <i>Acinetobacter baumannii</i>

The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for β-lactam antibiotic resistance in <i>Acinetobacter</i> 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 backgrounds, 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 <i>K</i>_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 β5-β6 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 β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases but may also provide a map for β-lactam improvement