A fluorescent carbapenem for structure function studies of penicillin-binding proteins, ?-lactamases, and ?-lactam sensors.

ABSTRACT: By reacting fluorescein isothiocyanate with meropenem, we have prepared a carbapenem-based fluorescent ?-lactam. Fluorescein-meropenem binds both penicillin-binding proteins and ?-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 ?-lactam and the position of its fluorescent modification render it a useful complement to other fluorescent ?-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.

SUBMITTER: June CM 

PROVIDER: S-EPMC4167909 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature