title: Structural insights into substrate recognition and product expulsion in CTX-M enzymes.
abstract: beta-Lactamase-mediated resistance to beta-lactam antibiotics poses a major threat to our antibiotic armamentarium. Among beta-lactamases, a significant threat comes from enzymes that hydrolyze extended-spectrum cephalosporins such as cefotaxime. Among the enzymes that exhibit this phenotype, the CTX-M family is found worldwide. These enzymes have a small active site, which makes it difficult to explain how they hydrolyze the bulky extended-spectrum cephalosporins into the binding site. We investigated noncovalent substrate recognition and product release in CTX-M enzymes using steered molecular dynamics simulation and X-ray diffraction. An arginine residue located far from the binding site favors the capture and tracking of substrates during entrance into the catalytic pocket. We show that the accommodation of extended-spectrum cephalosporins by CTX-M enzymes induced subtle changes in the active site and established a high density of electrostatic interactions. Interestingly, the product of the catalytic reaction initiates its own release because of steric hindrances and electrostatic repulsions. This suggests that there exists a general mechanism for product release for all members of the beta-lactamase family and probably for most carboxypeptidases.
authors.affiliation: CHU Clermont-Ferrand, Laboratoire de Bactériologie, Clermont-Ferrand F-63003, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Delmas
authors.forename: David
authors.initials: D
authors.lastname: Leyssene
authors.forename: Damien
authors.initials: D
authors.lastname: Dubois
authors.forename: Catherine
authors.initials: C
authors.lastname: Birck
authors.forename: Emilie
authors.initials: E
authors.lastname: Vazeille
authors.forename: Frédéric
authors.initials: F
authors.lastname: Robin
authors.forename: Richard
authors.initials: R
authors.lastname: Bonnet
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002439:Cefotaxime / Q000737:chemistry / Q000378:metabolism; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017434:Protein Structure, Tertiary; D014961:X-Ray Diffraction; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D001618:beta-Lactamases; D002439:Cefotaxime