title: Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases.
abstract: Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES β-lactamases are important contributors to carbapenem resistance in clinical bacterial pathogens. A single amino acid difference at position 170 of the GES-1, GES-2, and GES-5 enzymes is responsible for the expansion of their substrate profile to include carbapenem antibiotics. This highlights the increasing need to understand the mechanisms by which the GES β-lactamases function to aid in development of novel therapeutics. We demonstrate that the catalytic efficiency of the enzymes with carbapenems meropenem, ertapenem, and doripenem progressively increases (100-fold) from GES-1 to -5, mainly due to an increase in the rate of acylation. The data reveal that while acylation is rate limiting for GES-1 and GES-2 for all three carbapenems, acylation and deacylation are indistinguishable for GES-5. The ertapenem-GES-2 crystal structure shows that only the core structure of the antibiotic interacts with the active site of the GES-2 β-lactamase. The identical core structures of ertapenem, doripenem, and meropenem are likely responsible for the observed similarities in the kinetics with these carbapenems. The lack of a methyl group in the core structure of imipenem may provide a structural rationale for the increase in turnover of this carbapenem by the GES β-lactamases. Our data also show that in GES-2 an extensive hydrogen-bonding network between the acyl-enzyme complex and the active site water attenuates activation of this water molecule, which results in poor deacylation by this enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Nichole K
authors.initials: NK
authors.lastname: Stewart
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Smith
authors.forename: Hilary
authors.initials: H
authors.lastname: Frase
authors.forename: D J
authors.initials: DJ
authors.lastname: Black
authors.forename: Sergei B
authors.initials: SB
authors.lastname: Vakulenko
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D015780:Carbapenems / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000077726:Doripenem; D000077727:Ertapenem; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D000077731:Meropenem; D008958:Models, Molecular; D013845:Thienamycins / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D013845:Thienamycins; D047090:beta-Lactams; D000077726:Doripenem; C435769:beta-lactamase GES-2; D001618:beta-Lactamases; C063912:carbapenemase; D000077731:Meropenem; D000077727:Ertapenem