[en] The blaIMP gene coding for the IMP-1 metallo-beta-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 metallo-beta-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 metallo-beta-lactamase was not pH dependent.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Laraki, N.
Franceschini, N.
Rossolini, G. M.
Santucci, P.
Meunier, C.
De Pauw, Edwin ; Université de Liège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Amicosante, G.
Frère, Jean-Marie ; Université de Liège > Département des sciences de la vie > Centre d'ingénierie des protéines
Ansorge, W., B. Sproat, J. Stegemann, C. Schwager, and M. Zenke. 1987. Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis. Nucleic Acids Res. 15:4593-1602.
Bounaga, S., A. P. Laws, M. Galleni, and M. I. Page. 1998. Unusual pH dependence of the class B β-lactamase catalysed hydrolysis of substrates and their inhibition by thiols. Biochem. J. 331:703-711.
Bush, K., and G. Jacoby. 1995. A functional classification for β-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-1233.
Carfi, A., S. Pares, E. Duée, M. Galleni, C. Duez, J. M. Frère, and O. Dideberg. 1995. The 3D structure of a zinc metallo-β-lactamase from Bacillus cereus reveals a new type of protein fold. EMBO J. 14:4914-4921.
Carfi, A., E. Duée, R. P. Soto, M. Galleni, C. Duez, J. M. Frère, and O. Dideberg. 1998. X-ray structure of the Zn II β-lactamase from Bacteroides fragilis in an orthorhombic crystal form. Acta Crystallogr. D54:47-57.
Concha, N. O., B. A. Rasmussen, K. Bush, and O. Herzberg. 1996. Crystal structure of the wide-spectrum hinuclear zinc β-lactamase from Bacteroides fragilis. Structure 4:823-836.
Crowder, M. W., T. R. Walsh, L. Banovic, M. Pettit, and J. Spencer. 1998. Overexpression, purification, and characterization of the cloned metallo-β-lactamase L1 from Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 42:921-926.
Crowder, M. W., Z. Wang, S. L. Franklin, E. P. Zovinka, and S. J. Benkovic. 1996. Characterisation of the metal binding sites of the β-lactamase from Bacteroides fragilis. Biochemistry 35:12126-12132.
Cuchural, G. J., Jr., M. H. Malamy, and F. P. Tally. 1986. β-Lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrob. Agents Chemother. 30:645-648.
De Meester, F., B. Joris, G. Reckinger, C. Bellefroid-Bourguignon, J. M. Frère, and S. G. Waley. 1987. Automated analysis of enzyme inactivator phenomena. Biochem. Pharmacol. 36:2393-2403.
Felici, A., and G. Amicosante. 1995. Kinetic analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-β-lactamases. Antimicrob. Agents Chemother. 39:192-199.
Felici, A., G. Amicosante, A. Oratore, R. Strom, P. Ledent, B. Joris, L. Fanuel, and J. M. Frère. 1993. An overview of the kinetic parameters of class B β-lactamase. Biochem. J. 291:151-155.
Felici, A., M. Perilli, B. Segatore, N. Franceschini, D. Setacci, A. Oratore, S. Stefani, M. Galleni, and G. Amicosante. 1995. Interactions of biapenem with active-site serine and metallo-β-lactamases. Antimicrob. Agents Chemother. 39:1300-1305.
Frère, J. M. 1995. Beta-lactamases and bacterial resistance to antibiotics. Mol. Microbiol. 16:385-395.
Galleni, M., N. Franceschini, B. Quinting, L. Fattorini, G. Orefici, A. Orators, J. M. Frère, and G. Amicosante. 1994. Use of the chromosomal class A β-lactamase of Mycobacterium fortuitum D316 to study potentially poor substrates and inhibitory β-lactam compounds. Antimicrob. Agents Chemother. 38:1608-1614.
Hernandez Valladares, M., A. Felici, G. Weber, H. W. Adolph, M. Zeppezauer, G. M. Rossolini, G. Amicosante, J. M. Frère, and M. Galleni. 1997. Zn (II) dependence of the Aeromonas hydrophila AEO36 metallo-β-lactamase activity and stability. Biochemistry 36:11534-11541.
Hussain, M., A. Carlino, M. J. Madonna, and O. Lampen. 1985. Cloning and sequencing of the metallothioprotein β-lactamase II gene of Bacillus cereus 569H in Escherichia coli. J. Bacteriol. 164:223-229.
Ito, H., Y. Arakawa, S. Ohsuka, R. Wachorotayankun, N. Kato, and M. Ohta. 1995. Plasmid-mediated dissemination of the metallo-β-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens. Antimicrob. Agents Chemother. 39:824-829.
Laraki, N., M. Galleni, I. Thamm, M. L. Riccio, G. Amicosante, J.-M. Frere, and G. M. Rossolini. 1999. Structure of In101, a blaIMP-containing Pseudomonas aeruginosa integron phyletically related to In5, which carries an unusual array of gene cassettes. Antimicrob. Agents Chemother. 43:818-829.
Massida, O., G. M. Rossolini, and G. Satta. 1991. The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-β-lactamases. J. Bacteriol. 173:4611-4617.
Muramo, K., A. Takeda, Y. Nakamura, and K. Nakaya. 1995. Purification and characterization of metallo-β-lactamase from Serratia marcescens. Microbiol. Immunol. 39:27-33.
Osano, E., Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato. 1994. Molecular characterization of an enterobacterial metallo β-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance. Antimicrob. Agents Chemother. 38:71-78.
Rasmussen, B. A., and K. Bush. 1997. Carbapenem-hydrolyzing beta-lactamases. Antimicrob. Agents Chemother. 41:223-232.
Rossolini, G. M., N. Franceschini, M. L. Riccio, P. S. Mercuri, M. G. Perilli, M. Galleni, J. M. Frère, and G. Amicosante. 1998. Characterisation and sequence of the Chryseobacterium (Flavobacterium) meningosepticum carbapenemase: a new molecular class B β-lactamase showing a broad substrate profile. Biochem. J. 332:145-152.
Sanschagrin, F., J. Dufresne, and R. C. Levesque. 1998. Molecular heterogeneity of the L-1 metallo-beta-lactamase family from Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 42:1245-1248.
Sendo, H., Y. Arakawa, K. Nakeshima, H. Ito, S. Ichiyama, K. Shimakotu, N. Kato, and M. Ohta. 1986. Multifocal outbreak of metallo-β-lactamase producing Pseudomonas aeruginosa resistant to broad spectrum β-lactam including carbapenems. Antimicrob. Agents Chemother. 40:349-353.
Waley, S. G. 1992. β-Lactamase: mechanism of action, p. 198-228. In M. I. Page (ed.). The chemistry of β-lactam. Blackie A. et P., London, United Kingdom.
Walsh, T. R., L. Hall, S. J. Assinda, W. W. Nichols, S. J. Cartwright, A. P. MacGowan, and P. M. Bennett. 1994. Sequence and analysis of the L1 metallo-β-lactamase from Xanthomonas maltophilia. Biochem. Biophys. Acta 1218:199-201.
Watanabe, M., S. Iyobe, M. Inoue, and S. Mitsuhashi. 1991. Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35:147-151.
Yamaguchi, H., M. Nukaya, and T. Sawai. 1994. Sequence of Klebsiella pneumoniae RDK4 metallo-β-lactamase. EMBO database accession no. D29636. EMBO, Heidelberg, Germany.
Yang, Y., B. A. Rasmussen, and K. Bush. 1992. Biochemical characterization of an imipenem-hydrolyzing metallo-β-lactamase from Bacteroides fragilis TAL3636. Antimicrob. Agents Chemother. 36:1155-1157.
