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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]

Increasing antibiotic resistance is a global health concern. Metallo‐β‐lactamases catalyze the hydrolysis of almost all β‐lactam antibiotics. T. D. W. Claridge, C. J. Schofield et al. show in their Communication on page 3129 ff. how 19F NMR spectroscopy can be used to investigate the binding mode of inhibitors to the New Delhi Metallo‐β‐lactamase I (NDM‐1). By labeling an active‐site loop with 19F, changes in the local chemical environment caused by inhibitor binding can be readily monitored by NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe 19 F-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-Zn II active site, were selectively 19 F-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., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Metallo-β-lactamases (MBLs) are of increasing clinical significance; the development of clinically useful MBL inhibitors is challenged by the rapid evolution of variant MBLs. The Verona integron-borne metallo-β-lactamase (VIM) enzymes are among the most widely distributed MBLs, with >40 VIM variants having been reported. We report on the crystallographic analysis of VIM-5 and comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38. Recombinant VIM variants were produced and purified, and their secondary structure and thermal stabilities were investigated by circular dichroism analyses. Steady-state kinetic analyses with a representative panel of β-lactam substrates were carried out to compare the catalytic efficiencies of the VIM variants. Furthermore, a set of metalloenzyme inhibitors were screened to compare their effects on the different VIM variants. The results reveal only small variations in the kinetic parameters of the VIM variants but substantial differences in their thermal stabilities and inhibition profiles. Overall, these results support the proposal that protein stability may be a factor in MBL evolution and highlight the importance of screening MBL variants during inhibitor development programs., (Copyright © 2016 Makena et al.)

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 (19) F 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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)