Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo-β-lactamases

Abstract

Objectives: The new metallo-β-lactamase VIM-13 has been recently characterized. In comparison with the VIM-1 enzyme, VIM-13 showed 19 amino acid differences, 2 of which were located in the active site centre. The main objective of the present study was to assess whether differences between VIM-1 and VIM-13 β-lactamases in the active site, at His224Leu and Ser228Arg, are necessary and sufficient to explain the microbiological and biochemical differences between the two enzymes. Methods: Single mutants VIM-13 (Leu224His) and VIM-13 (Arg228Ser) and double mutant VIM-13 (Leu224His, Arg228Ser) were created by site-directed mutagenesis with the blaVIM-13 gene as template. VIM-1, VIM-13 and VIM-13 (Leu224His, Arg228Ser) were purified by affinity chromatography, and kinetic parameters for these enzymes were obtained with ceftazidime, cefepime and ampicillin. Results: Ceftazidime and cefepime MICs (mg/L) for Escherichia coli TG1 expressing VIM-1, VIM-13, VIM-13 (Leu224His), VIM-13 (Arg228Ser) and VIM-13 (Leu224His, Arg228Ser) were .256 and 64, 6 and 4, 8 and 1, .256 and 8, and .256 and 48, respectively. VIM-1, VIM-13 and VIM-13 (Leu224His, Arg228Ser) revealed kcat/Km values (M-1 s-1) for ceftazidime of 3.7 E4, 1.9 E4 and 10 E4, respectively, and revealed kcat/Km values for cefepime of 3.5 E5,3E4 and 1.5 E5, respectively. Conclusions: Overall, the results showed that the two residues located in the L3 loop are sufficient to confer the substrate specificity of each enzyme, thus highlighting the importance of the L3 loop of the active site in the evolution of VIM-type metallo-β-lactamases. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.