title: A fluorescent carbapenem for structure function studies of penicillin-binding proteins, β-lactamases, and β-lactam sensors.
abstract: By reacting fluorescein isothiocyanate with meropenem, we have prepared a carbapenem-based fluorescent β-lactam. Fluorescein-meropenem binds both penicillin-binding proteins and β-lactam sensors and undergoes a typical acylation reaction in the active site of these proteins. The probe binds the class D carbapenemase OXA-24/40 with close to the same affinity as meropenem and undergoes a complete catalytic hydrolysis reaction. The visible light excitation and strong emission of fluorescein render this molecule a useful structure-function probe through its application in sodium dodecyl sulfate-polyacrylamide gel electrophoresis assays as well as solution-based kinetic anisotropy assays. Its classification as a carbapenem β-lactam and the position of its fluorescent modification render it a useful complement to other fluorescent β-lactams, most notably Bocillin FL. In this study, we show the utility of fluorescein-meropenem by using it to detect mutants of OXA-24/40 that arrest at the acyl-intermediate state with carbapenem substrates but maintain catalytic competency with penicillin substrates.
authors: Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA.
authors: Cynthia M
authors: CM
authors: June
authors: Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA.
authors: Robert M
authors: RM
authors: Vaughan
authors: Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA.
authors: Lucas S
authors: LS
authors: Ulberg
authors: Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and Department of Pharmacology, Molecular Biology, and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors: Robert A
authors: RA
authors: Bonomo
authors: Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA.
authors: Laurie A
authors: LA
authors: Witucki
authors: Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA. Electronic address: leonardd@gvsu.edu.
authors: David A
authors: DA
authors: Leonard
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D001896:Boron Compounds / Q000737:chemistry; D015780:Carbapenems / Q000032:analysis* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D000077731:Meropenem; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D010406:Penicillins / Q000737:chemistry; D013845:Thienamycins / Q000737:chemistry / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: C118961:BOCILLIN FL; D001426:Bacterial Proteins; D001896:Boron Compounds; D015780:Carbapenems; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D013845:Thienamycins; D001618:beta-Lactamases; C063912:carbapenemase; D000077731:Meropenem; D016650:Fluorescein-5-isothiocyanate
keywords: Carbapenem; Fluorescent modification; Penicillin-binding protein; β-Lactamase