title: Structural basis of extended spectrum TEM beta-lactamases. Crystallographic, kinetic, and mass spectrometric investigations of enzyme mutants.
abstract: The E166Y and the E166Y/R164S TEM-1 beta-lactamase mutant enzymes display extended spectrum substrate specificities. Electrospray mass spectrometry demonstrates that, with penicillin G as substrate, the rate-limiting step in catalysis is the hydrolysis of the E166Y acyl-enzyme complex. Comparison of the 1.8-A resolution x-ray structures of the wild-type and of the E166Y mutant enzymes shows that the binding of cephalosporin substrates is improved, in the mutant enzyme, by the enlargement of the substrate binding site. This enlargement is due to the rigid body displacement of 60 residues driven by the movement of the omega-loop. These structural observations strongly suggest that the link between the position of the omega-loop and that of helix H5, plays a central role in the structural events leading to extended spectrum TEM-related enzymes. The increased omega-loop flexibility caused by the R164S mutation, which is found in several natural mutant TEM enzymes, may lead to similar structural effects. Comparisons of the kinetic data of the E166Y, E166Y/R164S, and R164S mutant enzymes supports this hypothesis.
authors.affiliation: Groupe de Cristallographie Biologique, Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Toulouse, France.
authors.forename: L
authors.initials: L
authors.lastname: Maveyraud
authors.forename: I
authors.initials: I
authors.lastname: Saves
authors.forename: O
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.forename: P
authors.initials: P
authors.lastname: Swarén
authors.forename: J M
authors.initials: JM
authors.lastname: Masson
authors.forename: M
authors.initials: M
authors.lastname: Delaire
authors.forename: L
authors.initials: L
authors.lastname: Mourey
authors.forename: J C
authors.initials: JC
authors.lastname: Promé
authors.forename: J P
authors.initials: JP
authors.lastname: Samama
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018360:Crystallography, X-Ray; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D009838:Oligodeoxyribonucleotides; D001618:beta-Lactamases