[en] The VIM metallo-beta-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens. In this work a T7-based expression system for overproduction of the VIM-2 enzyme by Escherichia coli was developed, which yielded similar to80 mg of protein per litre of culture. The enzyme was mostly released into the medium, from which it was recovered at >99% purity by an initial ammonium sulphate precipitation followed by two chromatography steps, with almost 80% efficiency. Determination of kinetic parameters of VIM-2 under the same experimental conditions previously used for VIM-1 (the first VIM-type enzyme detected in clinical isolates, which is 93% identical to VIM-2) revealed significant differences in K-m values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Molecular modelling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behaviour.
Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39, 1211-33.
Bush, K. (1999). β-Lactamases of increasing clinical importance. Current Pharmaceutical Design 11, 839-45.
Livermore, D. M. & Woodford, N. (2000). Carbapenemases: a problem in waiting? Current Opinion in Microbiology 5, 489-95.
Carfi, A., Pares, S., Duée, E., Galleni, M., Duez, C., Frère, J. M. et al. (11995). The 3-D structure of a zinc metallo-β-lactamase from Bacillus cereus reveals a new type of protein fold. EMBO Journal 14, 4914-21.
Concha, N. O., Rasmussen, B. A., Bush, K. & Herzberg, O. (1996). Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis. Structure 4, 823-36.
Ullah, J. H., Walsh, T. R., Taylor, I. A., Emery, D. C., Verma, C. S., Gamblin, S. J. et al. (1998). The crystal structure of the L1 metallo-β-lactamase from Stenotrophomonas maltophilia at 1.7 Å resolution. Journal of Mololecular Biology 284, 125-36.
Concha, N. O., Janson, C. A., Rowling, P., Pearson, S., Cheever, C. A., Clarke, B. P. et al. (2000). 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 39, 4288-98.
Watanabe, M., Iyobe, S., Inoue, M. & Mitsuhashi, S. (1991). Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 35, 147-51.
Osano, E., Arakawa, Y., Wacharotayankun, R., Ohta, M., Horii, T., Ito, H. et al. (1994). Molecular characterization of an entero-bacterial metallo-β-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance. Antimicrobial Agents and Chemotherapy 38, 71-8.
Muramo, K., Takeda, A., Nakamura, Y. & Nakaya, K. (1995). Purification and characterization of metallo-β-lactamase from Serratia marcescens. Microbiology and Immunology 39, 27-33.
Laraki, N., Franceschini, N., Rossolini, G. M., Santucci, P., Meunier, C., de Pauw, E. et al. (1999). Biochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-β-lactamase IMP-1 produced by Escherichia coli. Antimicrobial Agents and Chemotherapy 43, 902-6.
Prosperi-Meys, C., Llabres, G., de Seny, D., Paul Soto, R., Hernandez Valladares, M., Laraki, N. et al. (1999). Interaction between class B β-lactamases and suicide substrates of active-site serine β-lactamases. FEBS Letters 443, 109-11.
Haruta, S., Yamaguchi, H., Yamamoto, E. T., Eriguchi, Y., Nukaga, M., O'Hara, K. et al. (2000). Functional analysis of the active site of a metallo-β-lactamase proliferating in Japan. Antimicrobial Agents and Chemotherapy 44, 2304-9.
Riccio, M. L., Franceschini, N., Boschi, L., Caravelli, B., Cornaglia, G., Fontana, R. et al. (2000). Characterization of the metallo-β-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of blaIMP allelic variants carried by gene cassettes of different phylogeny. Antimicrobial Agents and Chemotherapy 44, 1229-35.
Haruta, S., Yamamoto, E. T., Eriguchi, Y. & Sawai, T. (2001). Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo β-lactamase. FEMS Microbiology Letters 197, 85-9.
Lauretti, L., Riccio, M. L., Mazzariol, A., Cornaglia, G., Amicosante, G. Fontana, R. et al. (1999). Cloning and characterization of blaVIM, a new integron-borne metallo-β-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrobial Agents and Chemotherapy 43, 1584-90.
Poirel, L., Naas, T., Nicholas, D., Collet, L., Bellais, S., Cavallo, J. D. et al. (2000). Characterization of VIM-2, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrobial Agents and Chemotherapy 44, 891-7.
Franceschini, N., Caravelli, B., Docquier, J. D., Galleni, M., Frère, J. M., Amicosante, G. et al. (2000). Purification and biochemical characterization of the VIM-1 metallo-β-lactamase. Antimicrobial Agents and Chemotherapy 44, 3003-7.
Yan, J. J., Hsueh, P. R., Ko, W. C., Luh, K. T., Tsai, S. H., Wu, H. M. et al. (2001). Metallo-β-lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme. Antimicrobial Agents and Chemotherapy 45, 2224-8.
Mavroidi, A., Tsakris, A., Tzelepi, E., Pournaras, S., Loukova, V. & Tzouvelekis, L. S. (2000) Carbapenem-hydrolysing VIM-2 metallo-β-lactamase in Pseudomonas aeruginosa from Greece. Journal of Antimicrobial Chemotherapy 46, 1041-2.
Pallecchi, L., Riccio, M. L., Docquier, J. D., Fontana, R. & Rossolini, G. M. (2001). Molecular heterogeneity of blaVIM-2-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-β-lactamase. FEMS Microbiology Letters 195, 145-50.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (2001). Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
Pace, C. N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Science 4, 2411-23.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-5.
Hagel, J. (1989). Gel filtration. In Principles, High Resolution Methods and Applications (Janson, J. C. & Rydén, L., Eds), pp. 63-106. VCH Publishers Inc., New York, NY, USA.
Segel, I. H. (1975). Rapid equilibrium partial and mixed-type inhibition. In Enzyme Kinetics, Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, pp. 210-2. John Wiley & Sons, New York, NY, USA.
De Meester, F., Joris, B., Reckinger, G., Bellefroid-Bourguignon, C., Frère, J. M. & Waley, S. G. (1987). Automated analysis of enzyme inactivation phenomena. Application to β-lactamases and DD-peptidases. Biochemical Pharmacology 36, 2393-403.
Hernandez Valladeres, M., Felici, A., Weber, G., Adolph, H. W., Zeppezauer, M., Rossolini, G. M. et al. (1997). Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-β-lactamase activity and stability. Biochemistry 36, 11534-41.
Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J. M. & Dideberg, O. (1998). X-ray structure of the ZnII β-lactamase from Bacteroides fragilis in an orthorhombic crystal form. Acta Crystallographica Section D Biological Crystallography 54, 45-57.
Pearlman, D., Case, D. A., Caldwell, J. A., Ross, W. S., Cheathman, T. E., Ferguson, D. M. et al. (1995). AMBER 4.1. University of California, San Francisco, CA, USA.
Banci, L., Schröder, S. & Kollman, P. A. (1992). Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts. Proteins 13, 288-305.
Koradi, R., Billeter, M. & Wüthrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. Journal of Molecular Graphics 14, 51-5.
Galleni, M., Lamotte-Brasseur, J., Rossolini, G. M., Spencer, J., Dideberg, O. & Frère, J. M. (2001). Standard numbering scheme for class B β-lactamases. Antimicrobial Agents and Chemotherapy 45, 660-3.
Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185, 60-89.
Rossolini, G. M., Franceschini, N., Riccio, M. L., Mercuri, P. S., Perilli, M., Galleni, M. et al. (1998). Characterization and sequence of the Chryseobacterium (Flavobacterium) meningosepticum carbapenemase: a new molecular class B β-lactamase showing a broad substrate profile. Biochemical Journal 332, 145-52.
Wang, Z., Fast, W., Valentine, A. M. & Benkovic, S. J. (1999). Metallo-β-lactamase: structure and mechanism. Current Opinion in Chemical Biology 3, 614-22.
Prosperi-Meys, C., Wouters, J., Galleni, M. & Lamotte-Brasseur, J. (2001). Substrate binding and catalytic mechanism of class B β-lactamases: a molecular modelling study. Cellular and Molecular Life Sciences 58, 2136-43.