Crystal Structure of the Extended-Spectrum B-Lactamase PER-2 and Simulated Modeling of its Interaction with B-Lactams and B-Lactamase Inhibitors

M. RUGGIERO; E. SAUVAGE; R. HERMAN; F.SAPUNARIC; M. GALLENI; G. GUTKIND; P. CHARLIER; P. POWER

Denver. Colorado

Congreso; 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy; 2013

American Society for Microbiology

Background: As we recently observed, mutations at R220 (equivalent to R244 in IRT-TEMs) have an impact on the inhibition susceptibility of PER-2, as well as towards the substrate profile. From the crystallographic structure of PER-2 and simulated models, we evaluated the possible role of several key residues in the structure and activity towards b-lactams and inhibitors. Methods: Crystals of pure PER-2 were obtained by hanging drop vapor diffusion at 20°C. X-Ray diffraction was carried out at Soleil synchrotron (Paris, France) under cryogenic conditions (100K). Indexing and integration were performed with XDS, the scaling of the intensity data with XSCALE, and enzymes structure was obtained by molecular replacement and refinement by REFMAC5, TLS, and Coot. Simulated models of PER-2 and derived mutants in combination with b-lactams were obtained with Coot and PyMol. Results: The structure of PER-2 was refined to 2.10 Å (PDB entry: 3znw (HPUB); Rfree: 24.2%; Ramachandran s favored residues: 96.7%). In PER-2, B3 strand is shifted up to ca. 5 Å towards the catalytic cleft in comparison with other class A b-lactamases, creating an apparently more favorable environment for stabilization of molecules like the oxyimino-cephalosporins through hydrogen bonds. This strand seems to be also stabilized by hydrogen bonds between T237 and R220, and mutations in the latter could disrupt the network leading to the already observed differences in the hydrolytic/inhibition behavior towards several drugs, especially cefotaxime, ceftazidime, benzyl-penicillin and clavulanic acid. Conclusions: The structural evidences suggest that the hydrogen-bond network including R220-T237-Ser238-b-lactam is important for the proper activity and inhibition of the enzyme. Mutations in these positions seem to also affect the whole hydrolytic behavior.