Crystal Structure of the Pseudomonas aeruginosa BEL-1 Extended-Spectrum β-Lactamase and Its Complexes with Moxalactam and Imipenem.
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 Kmvalues 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.
Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Siena, Italy.
Cecilia
C
Pozzi
Dipartimento di Biotecnologie Mediche, Università di Siena, Siena, Italy.
Filomena
F
De Luca
Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Siena, Italy.
Manuela
M
Benvenuti
INSERM European Unit (LEA), University of Fribourg, Fribourg, Switzerland.|Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland.
Laurent
L
Poirel
INSERM European Unit (LEA), University of Fribourg, Fribourg, Switzerland.|Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland.
Patrice
P
Nordmann
Dipartimento di Biotecnologie Mediche, Università di Siena, Siena, Italy.|SOD Microbiologia e Virologia, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.|Dipartimento di Medicina Sperimentale e Clinica, Università di Firenze, Florence, Italy.
Gian Maria
GM
Rossolini
Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Siena, Italy stefano.mangani@unisi.it jddocquier@unisi.it.|Magnetic Resonance Center CERM, Università di Firenze, Sesto Fiorentino, Italy.
Stefano
S
Mangani
Dipartimento di Biotecnologie Mediche, Università di Siena, Siena, Italy stefano.mangani@unisi.it jddocquier@unisi.it.
Jean-Denis
JD
Docquier
D000900:Anti-Bacterial Agents / Q000737:chemistry; D020134:Catalytic Domain; D019343:Citric Acid / Q000737:chemistry; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D015378:Imipenem / Q000737:chemistry*; D009070:Moxalactam / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004220:Disulfides; D019343:Citric Acid; D015378:Imipenem; D001618:beta-Lactamases; C505391:beta-lactamase BEL-1, Pseudomonas aeruginosa; D009070:Moxalactam
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