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Crystal Structure of the Pseudomonas aeruginosa BEL-1 Extended-Spectrum β-Lactamase and its Complex with Moxalactam and Imipenem

DOI: 10.1128/AAC.00936-16 DOI Help

Authors: Cecilia Pozzi (Università di Siena) , Filomena De Luca (Università di Siena) , Manuela Benvenuti (Università di Siena) , Laurent Poirel (University of Fribourg) , Patrice Nordmann (University of Fribourg) , Gian Maria Rossolini (Università di Siena; SOD Microbiologia e Virologia, Azienda Ospedaliera Universitaria Careggi; Università di Firenze) , Stefano Mangani (Università di Siena; Università di Firenze) , Jean-denis Docquier (Università di Siena)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Antimicrobial Agents And Chemotherapy

State: Published (Approved)
Published: September 2016
Diamond Proposal Number(s): 8574

Abstract: BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α-substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower Km values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties.

Journal Keywords: Pseudomonas aeruginosa BEL-1; Extended- Spectrum beta-Lactamase; Moxalactam; Imipenem

Subject Areas: Chemistry, Biology and Bio-materials, Medicine


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Other Facilities: ESRF