title: Ligand-dependent disorder of the Omega loop observed in extended-spectrum SHV-type beta-lactamase.
abstract: Among Gram-negative bacteria, resistance to β-lactams is mediated primarily by β-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate β-lactam antibiotics. Substitutions at critical amino acid positions in the class A β-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an "extended-spectrum" β-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the Ω loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel β-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique β-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced Ki values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Ω loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Ω loop flexibility as a mechanism for accommodating and hydrolyzing β-lactam substrates.
authors.affiliation: Department of Biochemistry, Case Western University School of Medicine, Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH 44106, USA.
authors.forename: Jared M
authors.initials: JM
authors.lastname: Sampson
authors.forename: Wei
authors.initials: W
authors.lastname: Ke
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.forename: S R R
authors.initials: SR
authors.lastname: Pagadala
authors.forename: Michael D
authors.initials: MD
authors.lastname: Nottingham
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.forename: John D
authors.initials: JD
authors.lastname: Buynak
authors.forename: Focco
authors.initials: F
authors.lastname: van den Akker
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000078142:Tazobactam; D014961:X-Ray Diffraction; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D010397:Penicillanic Acid; D001618:beta-Lactamases; D000078142:Tazobactam