title: Role of the Conserved Disulfide Bridge in Class A Carbapenemases.
abstract: Some members of the class A β-lactamase family are capable of conferring resistance to the last resort antibiotics, carbapenems. A unique structural feature of these clinically important enzymes, collectively referred to as class A carbapenemases, is a disulfide bridge between invariant Cys69and Cys238residues. It was proposed that this conserved disulfide bridge is responsible for their carbapenemase activity, but this has not yet been validated. Here we show that disruption of the disulfide bridge in the GES-5 carbapenemase by the C69G substitution results in only minor decreases in the conferred levels of resistance to the carbapenem imipenem and other β-lactams. Kinetic and circular dichroism experiments with C69G-GES-5 demonstrate that this small drop in antibiotic resistance is due to a decline in the enzyme activity caused by a marginal loss of its thermal stability. The atomic resolution crystal structure of C69G-GES-5 shows that two domains of this disulfide bridge-deficient enzyme are held together by an intensive hydrogen-bonding network. As a result, the protein architecture and imipenem binding mode remain unchanged. In contrast, the corresponding hydrogen-bonding networks in NMCA, SFC-1, and SME-1 carbapenemases are less intensive, and as a consequence, disruption of the disulfide bridge in these enzymes destabilizes them, which causes arrest of bacterial growth. Our results demonstrate that the disulfide bridge is essential for stability but does not play a direct role in the carbapenemase activity of the GES family of β-lactamases. This would likely apply to all other class A carbapenemases given the high degree of their structural similarity.
authors: From the Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, California 94025 and csmith@slac.stanford.edu.
authors: Clyde A
authors: CA
authors: Smith
authors: the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556.
authors: Zahra
authors: Z
authors: Nossoni
authors: the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556.
authors: Marta
authors: M
authors: Toth
authors: the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556.
authors: Nichole K
authors: NK
authors: Stewart
authors: the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556.
authors: Hilary
authors: H
authors: Frase
authors: the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 svakulen@nd.edu.
authors: Sergei B
authors: SB
authors: Vakulenko
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D020125:Mutation, Missense; D000072417:Protein Domains; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D001618:beta-Lactamases; C063912:carbapenemase; D003545:Cysteine
keywords: antibiotic resistance; carbapenemase; crystal structure; disulfide; enzyme kinetics; enzyme stability; hydrogen bond