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

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 clas...

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Bibliographic Details
Published in:Analytical biochemistry 2014-10, Vol.463, p.70-74
Main Authors: June, Cynthia M., Vaughan, Robert M., Ulberg, Lucas S., Bonomo, Robert A., Witucki, Laurie A., Leonard, David A.
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
beta-Lactamases - chemistry
beta-Lactamases - metabolism
Biocatalysis
Boron Compounds - chemistry
Carbapenem
Carbapenems - analysis
Carbapenems - metabolism
Electrophoresis, Polyacrylamide Gel
Fluorescein-5-isothiocyanate - chemistry
Fluorescent modification
Hydrolysis
Kinetics
Penicillin-binding protein
Penicillin-Binding Proteins - chemistry
Penicillin-Binding Proteins - metabolism
Penicillins - chemistry
Thienamycins - chemistry
Thienamycins - metabolism
β-Lactamase
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2014.07.012