Producción CyT
Libro de resumenes - The crystal structure of the carbapenemase OXA-24 in complex with tazobactam reveals evidences about the interaction with β-lactamase inhibitors and lysine carboxylation
Congreso
Fecha:
2014Editorial y Lugar de Edición:
ASM PressResumen *
Background. Class D (OXA) β-lactamases are classified in different sub-groups depending on their preferred substrates or kinetic behavior, and genetic origin. A particularly interesting group includes enzymes having increased hydrolytic activity towards carbapenems, and therefore called ?carbapenem hydrolyzing class D β-lactamases? (CHDL). Within this group, mainly found in Acinetobacter spp., the OXA-24/OXA-40 group is one of the most prevalent. In this study, we determined the structure of OXA-24 in complex with the mechanism-based inhibitor tazobactam, previously reported as a modest inhibitor by preliminary kinetic studies, and evaluated the role of some class D specific residues in the interaction with this inhibitor, in order to understand the weaker inhibition observed compared to other serine β-lactamases. Methods. Crystals from pure OXA-24 were obtained by hanging drop vapor diffusion at 20°C. The tazobactam adduct was obtained by diffusing the inhibitor into the crystals at room temperature. X-Ray diffraction was carried out at Soleil synchrotron (Paris, France) under cryogenic conditions (100oK). Indexing and integration were performed with XDS, the scaling of the intensity data with XSCALE, structure was obtained by molecular replacement and refinement by REFMAC5, TLS, and Coot, and models were with PyMol. Results. The structure of OXA-24 in complex with tazobactam was refined at 1.95 Å (Rfree: 0.209). The hydrolyzed inhibitor molecule is linked to active-site Ser81 as the trans-enamine form, and the thiazolidine ring is oriented in the opposite direction compared to what is observed for class A β-lactamases. This configuration seems to be partially due to the presence of Met223. The presence of a valine instead of an asparagine in the second conserved motif of class D enzymes modifies the hydrogen network stabilizing the acyl-enzyme complex. As observed in other class D β-lactamases, Lys84 is partially carboxylated in OXA-24/tazobactam. Conclusions. The presence of bulky hydrophobic residues (Tyr112, Trp115, Trp221 and Met225), as well as modifications in the hydrogen network within the active site of OXA-24 could reduce the affinity towards inhibitors like tazobactam and/or prevent the reorganization of the molecule required for an efficient inactivation. Información suministrada por el agente en SIGEVAPalabras Clave
X-ray crystallographycarbapenemasesclass D