title: Exploring the inhibition of CTX-M-9 by beta-lactamase inhibitors and carbapenems.
abstract: Currently, CTX-M β-lactamases are among the most prevalent and most heterogeneous extended-spectrum β-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by β-lactamase inhibitors. However, it is unknown if the pathway to inhibition by β-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV β-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 β-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. K(i) values ranged from 1.6 ± 0.3 μM (mean ± standard error) for tazobactam to 0.02 ± 0.01 μM for penem 1. Before and after tryptic digestion of the CTX-M-9 β-lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 ± 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 β-lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C(3) carboxylate of inhibitors and that the localization of this positive charge to a "region of the active site" rather than a specific residue represents an important evolutionary strategy used by β-lactamases.
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106, USA. robert.bonomo@va.gov
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.forename: Magdalena
authors.initials: M
authors.lastname: Taracila
authors.forename: Teresa
authors.initials: T
authors.lastname: Shyr
authors.forename: Jodi M
authors.initials: JM
authors.lastname: Thomson
authors.forename: Anne M
authors.initials: AM
authors.lastname: Distler
authors.forename: Kristine M
authors.initials: KM
authors.lastname: Hujer
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hujer
authors.forename: Andrea
authors.initials: A
authors.lastname: Endimiani
authors.forename: Krisztina
authors.initials: K
authors.lastname: Papp-Wallace
authors.forename: Richard
authors.initials: R
authors.lastname: Bonnet
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015780:Carbapenems / Q000494:pharmacology*; D019818:Clavulanic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D007700:Kinetics; D000077731:Meropenem; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013407:Sulbactam / Q000494:pharmacology; D000078142:Tazobactam; D013845:Thienamycins / Q000494:pharmacology; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D013845:Thienamycins; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D010397:Penicillanic Acid; C412613:CTX-M-9 protein, E coli; D001618:beta-Lactamases; D000077731:Meropenem; D013407:Sulbactam; D000078142:Tazobactam