Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo- -lactamases
Open Access
- 11 July 2010
- journal article
- research article
- Published by Oxford University Press (OUP) in Journal of Antimicrobial Chemotherapy
- Vol. 65 (9), 1950-1954
- https://doi.org/10.1093/jac/dkq259
Abstract
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. 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. 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−1s−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, 3 E4 and 1.5 E5, respectively. 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.Keywords
This publication has 22 references indexed in Scilit:
- Characterization of DIM-1, an Integron-Encoded Metallo-β-Lactamase from a Pseudomonas stutzeri Clinical Isolate in the NetherlandsAntimicrobial Agents and Chemotherapy, 2010
- Updated Functional Classification of β-LactamasesAntimicrobial Agents and Chemotherapy, 2010
- Characterization of a New Metallo-beta-Lactamase Gene, bla(NDM-1), and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella pneumoniae Sequence Type 14 from IndiaAntimicrobial Agents and Chemotherapy, 2009
- KHM-1, a Novel Plasmid-Mediated Metallo-β-Lactamase from a Citrobacter freundii Clinical IsolateAntimicrobial Agents and Chemotherapy, 2008
- Characterization of the New Metallo-β-Lactamase VIM-13 and Its Integron-Borne Gene from a Pseudomonas aeruginosa Clinical Isolate in SpainAntimicrobial Agents and Chemotherapy, 2008
- Kinetic Characterization of VIM-7, a Divergent Member of the VIM Metallo-β-Lactamase FamilyAntimicrobial Agents and Chemotherapy, 2008
- Biochemical Characterization of Metallo-β-Lactamase VIM-11 from a Pseudomonas aeruginosa Clinical StrainAntimicrobial Agents and Chemotherapy, 2008
- The Three-Dimensional Structure of VIM-2, a Zn-β-Lactamase from Pseudomonas aeruginosa in Its Reduced and Oxidised FormJournal of Molecular Biology, 2008
- Metallo-β-Lactamases: the Quiet before the Storm?Clinical Microbiology Reviews, 2005
- Site-directed mutagenesis by overlap extension using the polymerase chain reactionGene, 1989