[en] OBJECTIVES: The new metallo-beta-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 beta-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 bla(VIM-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 k(cat)/K(m) values (M(-1)s(-1)) for ceftazidime of 3.7 E(4), 1.9 E(4) and 10 E(4), respectively, and revealed k(cat)/K(m) values for cefepime of 3.5 E(5), 3 E(4) and 1.5 E(5), 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-beta-lactamases.
Research Center/Unit :
CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Merino, Maria
Perez-Llarena, Francisco J
Kerff, Frédéric ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Poza, Margarita
Mallo, Susana
Rumbo-Feal, Soraya
Beceiro, Alejandro
Juan, Carlos
Oliver, Antonio
Bou, German
Language :
English
Title :
Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo-beta-lactamases.
Ambler RP. The structure of β-lactamases. Philos Trans R Soc Lond B Biol Sci 1980; 289: 321-31.
Bush K, Jacoby GA. Updated functional classification of β-lactamases. Antimicrob Agents Chemother 2010; 54: 969-76.
Docquier JD, Lamotte-Brasseur J, Galleni M et al. On functional and structural heterogeneity of VIM-type metallo-β-lactamases. J Antimicrob Chemother 2003; 51: 257-66.
Crowder MW, Spencer J, Vila AJ. Metallo-β-lactamases: novel weaponry for antibiotic resistance in bacteria. Acc Chem Res 2006; 39: 721-8.
Walsh TR, Toleman MA, Poirel L et al. Metallo-β-lactamases: the quiet before the storm? Clin Microbiol Rev 2005; 18: 306-25.
Perez-Llarena FJ, Bou G. β-Lactamase inhibitors: the story so far. Curr Med Chem 2009; 16: 3740-65.
Poirel L, Pitout JD, Nordmann P. Carbapenemases: molecular diversity and clinical consequences. Future Microbiol 2007; 2: 501-12.
Lee K, Yum JH, Yong D et al. Novel acquired metallo-β-lactamase gene, blaSIM-1, in a class 1 integron from Acinetobacter baumannii clinical isolates from Korea. Antimicrob Agents Chemother 2005; 49: 4485-91.
Sekiguchi J, Morita K, Kitao T et al. KHM-1, a novel plasmid-mediated metallo-β-lactamase from a Citrobacter freundii clinical isolate. Antimicrob Agents Chemother 2008; 52: 4194-7.
Yong D, Walsh TR, Bell J et al. Novel ISCR element ISCR15 is responsible for the movement of the B3 subgroup mobile metallo-β-lactamase (MBL) gene blaAim-1. In: Abstracts of the Forty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008. Abstract C1-110, p. 88. American Society for Microbiology, Washington, DC, USA.
Yong D, Toleman MA, Giske CG et al. Characterization of a new metallo-β-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009; 53: 5046-54.
Poirel L, Rodriguez-Martinez JM, Al Naiemi N et al. Characterization of DIM-1, an integron-encoded metallo-β-lactamase from a Pseudomonas stutzeri clinical isolate in the Netherlands. Antimicrob Agents Chemother 2010; 54: 2420-4.
Rodriguez-Martinez JM, Nordmann P, Fortineau N et al. VIM-19, a metallo-β-lactamase with increased carbapenemase activity from Escherichia coli and Klebsiella pneumoniae. Antimicrob Agents Chemother 2009; 54: 471-6.
Samuelsen O, Castanheira M, Walsh TR et al. Kinetic characterization of VIM-7, a divergent member of the VIM metallo-β-lactamase family. Antimicrob Agents Chemother 2008; 52: 2905-8.
Juan C, Beceiro A, Gutierrez O et al. Characterization of the new metallo-β-lactamase VIM-13 and its integron-borne gene from a Pseudomonas aeruginosa clinical isolate in Spain. Antimicrob Agents Chemother 2008; 52: 3589-96.
Galleni M, Lamotte-Brasseur J, Rossolini GM et al. Standard numbering scheme for class B β-lactamases. Antimicrob Agents Chemother 2001; 45: 660-3.
Marchiaro P, Tomatis PE, Mussi MA et al. Biochemical characterization of metallo-β-lactamase VIM-11 from a Pseudomonas aeruginosa clinical strain. Antimicrob Agents Chemother 2008; 52: 2250-2.
Ho SN, Hunt HD, Horton RM et al. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77: 51-9.
Gacar GG, Midilli K, Kolayli F et al. Genetic and enzymatic properties of metallo-β-lactamase VIM-5 from a clinical isolate of Enterobacter cloacae. Antimicrob Agents Chemother 2005; 49: 4400-3.
Pournaras S, Ikonomidis A, Tzouvelekis LS et al. VIM-12, a novel plasmid-mediated metallo-β-lactamase from Klebsiella pneumoniae that resembles a VIM-1/VIM-2 hybrid. Antimicrob Agents Chemother 2005; 49: 5153-6.
Franceschini N, Caravelli B, Docquier JD et al. Purification and biochemical characterization of the VIM-1 metallo-β-lactamase. Antimicrob Agents Chemother 2000; 44: 3003-7.
Yamaguchi Y, Jin W, Matsunaga K et al. Crystallographic investigation of the inhibition mode of a VIM-2 metallo-β-lactamase from Pseudomonas aeruginosa by a mercaptocarboxylate inhibitor. J Med Chem 2007; 50: 6647-53.
Garcia-Saez I, Docquier JD, Rossolini GM et al. The three-dimensional structure of VIM-2, a Zn-β-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form. J Mol Biol 2008; 375: 604-11.
Concha NO, Janson CA, Rowling P et al. Crystal structure of the IMP-1 metallo β-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor. Biochemistry 2000; 39: 4288-98.