Background. BEL-1 is a class A ESBL detected in P. aeruginosa clinical isolates from Belgium, which is quite divergent from other ESBLs (max. identity, 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem and moxalactam. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) and conferring a higher level of resistance to CAZ, CTX and FEP was also isolated, which showed much lower turnover rates and Km values as compared to those of BEL-1, especially with oxyiminocephalosporins. Methods. BEL-1 was produced in E. coli using a T7-based promoter expression plasmid and purified to near homogeneity by means of two cationic ion exchange chromatography steps. Crystals of BEL-1 were obtained at pH 5.6, and subsequently soaked with imipenem and moxalactam for 30-60 min. Diffraction data were collected and the structure of native BEL-1 determined by molecular replacement, using the coordinates of the Toho-1 beta-lactamase (PDB code, 1IYS) as the search model. Results. The structure of BEL-1, in the native form and in complex with imipenem and moxalactam, was obtained at 1.6-1.9-angstroms resolution. In the acyl-enzyme complexes with imipenem and moxalactam, the C6 α-substituent interacts with conserved residue Asn-132. More surprisingly, the omega-loop, which includes the catalytically-relevant residue Glu-166, was found in different conformations in the various subunits, resulting in a approx. 180° rotation outwards the active site of the Glu-166 side chain or displacements of its Cα atom up to approx. 10 angstroms. Leu-162, located at the beginning of the omega-loop, is surrounded by Phe-72, Leu-139, Leu-148 and Leu-169 (contact distances, 3.8-4 angstroms). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe-162 found in BEL-2 without altering neighbouring residues or the omega-loop itself, thus causing an important alteration of the enzyme kinetic properties. Conclusions. The structure of BEL-1 in complex with imipenem and moxalactam shows an important variation in the conformation adopted by the omega-loop. It also provides a structural rationale supporting the large impact of Leu162Phe substitution on beta-lactam hydrolysis.

Pozzi, C., DE LUCA, F., Benvenuti, M., Poirel, L., Rossolini, G.M., Mangani, S., et al. (2013). Crystal structure of the extended-spectrum β-lactamase BEL-1, in native form and in complex with imipenem and moxalactam.. In 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy Abstract Book. Washington : ASM Press.

Crystal structure of the extended-spectrum β-lactamase BEL-1, in native form and in complex with imipenem and moxalactam.

POZZI, CECILIA;DE LUCA, FILOMENA;BENVENUTI, MANUELA;ROSSOLINI, GIAN MARIA;MANGANI, STEFANO;DOCQUIER, JEAN DENIS
2013-01-01

Abstract

Background. BEL-1 is a class A ESBL detected in P. aeruginosa clinical isolates from Belgium, which is quite divergent from other ESBLs (max. identity, 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem and moxalactam. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) and conferring a higher level of resistance to CAZ, CTX and FEP was also isolated, which showed much lower turnover rates and Km values as compared to those of BEL-1, especially with oxyiminocephalosporins. Methods. BEL-1 was produced in E. coli using a T7-based promoter expression plasmid and purified to near homogeneity by means of two cationic ion exchange chromatography steps. Crystals of BEL-1 were obtained at pH 5.6, and subsequently soaked with imipenem and moxalactam for 30-60 min. Diffraction data were collected and the structure of native BEL-1 determined by molecular replacement, using the coordinates of the Toho-1 beta-lactamase (PDB code, 1IYS) as the search model. Results. The structure of BEL-1, in the native form and in complex with imipenem and moxalactam, was obtained at 1.6-1.9-angstroms resolution. In the acyl-enzyme complexes with imipenem and moxalactam, the C6 α-substituent interacts with conserved residue Asn-132. More surprisingly, the omega-loop, which includes the catalytically-relevant residue Glu-166, was found in different conformations in the various subunits, resulting in a approx. 180° rotation outwards the active site of the Glu-166 side chain or displacements of its Cα atom up to approx. 10 angstroms. Leu-162, located at the beginning of the omega-loop, is surrounded by Phe-72, Leu-139, Leu-148 and Leu-169 (contact distances, 3.8-4 angstroms). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe-162 found in BEL-2 without altering neighbouring residues or the omega-loop itself, thus causing an important alteration of the enzyme kinetic properties. Conclusions. The structure of BEL-1 in complex with imipenem and moxalactam shows an important variation in the conformation adopted by the omega-loop. It also provides a structural rationale supporting the large impact of Leu162Phe substitution on beta-lactam hydrolysis.
Pozzi, C., DE LUCA, F., Benvenuti, M., Poirel, L., Rossolini, G.M., Mangani, S., et al. (2013). Crystal structure of the extended-spectrum β-lactamase BEL-1, in native form and in complex with imipenem and moxalactam.. In 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy Abstract Book. Washington : ASM Press.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/975420