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

作者：June Cynthia M; Vaughan Robert M; Ulberg Lucas S; Bonomo Robert A; Witucki Laurie A; Leonard David A^*

来源：Analytical Biochemistry, 2014, 463: 70-74.

DOI：10.1016/j.ab.2014.07.012

• beta-Lactamase
• Penicillin-binding protein
• Fluorescent modification
• Carbapenem

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

• 出版日期2014-10-15