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Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe 19F-NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM-1) from β-lactam-resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di-ZnII active site, were selectively 19F-labeled using 3-bromo-1,1,1-trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β-lactam products to SPM-1. These results have implications for the mechanisms and inhibition of MBLs by β-lactams and non-β-lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers. [ABSTRACT FROM AUTHOR]

The New Delhi metallo-β-lactamase (NDM-1) is involved in the emerging antibiotic resistance problem. Development of metallo-β-lactamases (MBLs) inhibitors has proven challenging, due to their conformational flexibility. Here we report site-selective labeling of NDM-1 with 1,1,1-trifluoro-3-bromo acetone (BFA), and its use to study binding events and conformational changes upon ligand-metal binding using 19F NMR spectroscopy. The results demonstrate different modes of binding of known NDM-1 inhibitors, including L- and D-captopril by monitoring the changing chemical environment of the active-site loop of NDM-1. The method described will be applicable to other MBLs and more generally to monitoring ligand-induced conformational changes. [ABSTRACT FROM AUTHOR]

Antibiotikaresistenz ist ein weltweites Gesundheitsproblem. Metallo ‐ β ‐ lactamasen katalysieren die Hydrolyse von nahezu allen β ‐ Lactam ‐ Antibiotika. T. D. W. Claridge, C. J. Schofield et al. zeigen in ihrer Zuschrift auf S. 3193 ff., wie 19F ‐ NMR ‐ Spektroskopie genutzt werden kann, um die Bindungsweise von Inhibitoren der New ‐ Delhi ‐ Metallo ‐ β ‐ lactamase I (NDM ‐ 1) zu untersuchen: Man markiert eine Schleife im aktiven Zentrum mit 19F und kann dann Änderungen der lokalen chemischen Umgebung, die durch die Bindung des Inhibitors verursacht werden, NMR ‐ spektroskopisch verfolgen. [ABSTRACT FROM AUTHOR]