[en] The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Vanhove, Marc
Zakhem, M.
Devreese, B.
Franceschini, N.
Anne, Christine ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Bebrone, Carine ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Amicosante, G.
Rossolini, G. M.
Van Beeumen, J.
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Galleni, Moreno ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques
Language :
English
Title :
Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase.
Ambler R. P. (1980). The structure of β-lactamases. Phil. Trans. R. Soc. Lond. B 289: 321-331
Matagne A., Dubus A., Galleni M. and Frère J. M. (1999) The beta-lactamase cycle: a tale of selective pressure and bacterial ingenuity. Nat. Prod. Rep. 6: 1-19
Galleni M., Lamotte-Brasseur J., Rossolini G. M., Spencer J., Dideberg O., Frère J. M. et al. (2001) Standard numbering scheme for class B beta-lactamases. Antimicrob. Agents Chemother. 45: 660-663
Sabath L. D. and Abraham E. P. (1966) Zinc as a cofactor for cephalosporinase from Bacillus cereus 569. Biochem. J. 98: 11C-13C
Livermore D. M. and Woodford N. (2000) Carbapenemases: a problem in waiting? Curr. Opin. Microbiol. 3: 489-495
Carfi A., Pares S., Duée E., Galleni M., Duez C., Frère J. M. et al. (1995) The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold. EMBO J. 14: 4914-4921
Carfi A., Duée E., Paul-Soto R., Galleni M., Frère J. M. and Dideberg O. (1998) X-ray structure of the Zn(II) beta-lactamase from Bacteroides fragilis in an orthorhombic crystal form. Acta Crystallogr. Sect. D 54: 45-57
Carfi A., Duée E., Galleni M., Frère J. M. and Dideberg O. (1998) 1.85 Å resolution structure of the zinc (II) beta-lactamase from Bacillus cereus. Acta Crystallogr. Sect. D 54: 313-323
Fabiane S. M., Sohi M. K., Wan T., Payne D. J., Bateson J. H., Mitchell T. et al. (1998) Crystal structure of the zinc-dependent beta-lactamase from Bacillus cereus at 1.9 Å resolution: binuclear active site with features of a mononuclear enzyme. Biochemistry 37: 12404-12411
Concha N. O., Rasmussen B. A., Bush K. and Herzberg O. (1996) Crystal structure of the wide-spectrum binuclear zinc beta-lactamase from Bacteroides fragilis. Structure 4: 823-836
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 beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor. Biochemistry 39: 4288-4298
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-beta- lactamase from Stenotrophomonas maltophilia at 1.7 Å resolution. J. Mol. Biol. 284: 125-136
Hernandez Valladares 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-beta-lactamase activity and stability. Biochemistry 36: 11534-11541
Segatore B., Massidda O., Satta G., Setacci D. and Amicosante G. (1993) High specificity of cphA-encoded metallo-beta-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to beta-lactam resistance. Antimicrob. Agents Chemother. 37: 1324-1328
Felici A. and Amicosante G. (1995) Kinetic analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-beta-lactamases. Antimicrob. Agents Chemother. 39: 192-199.
Massidda O., Rossolini G. M. and Satta G. (1991) The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta- lactamases. J. Bacteriol. 173: 4611-4617
De Meester F., Joris B., Reckinger G., Bellefroid-Bourguignon C., Frère J. M. and Waley S. G. (1987) Automated analysis of enzyme inactivation phenomena: application to beta-lactamases and DD-peptidases. Biochem. Pharmacol. 36: 2393-2403
Zervosen A., Valladares M. H., Devreese B., Prosperi-Meys C., Adolph H. W., Mercuri P. S. et al. (2001) Inactivation of Aeromonas hydrophila metallo-beta-lactamase by cephamycins and moxalactam. Eur. J. Biochem. 268: 3840-3850
Jacob F., Joris B. and Frère J. M. (1991) Active-site serine mutants of the Streptomyces albus G beta-lactamase. Biochem. J. 277: 647-652
Xie P., Parsons S. H., Speckhard D. C., Bosron W. F. and Hurley T. D. (1997) X-ray structure of human class IV sigmasigma alcohol dehydrogenase: structural basis for substrate specificity. J. Biol. Chem. 272: 18558-18563
Hernandez Valladares M., Kiefer M., Heinz U., Soto R. P., Meyer-Klaucke W., Nolting H. F. et al. (2000) Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036. FEBS Lett. 467: 221-225
Paul Soto R., Bauer R., Frère J.-M., Galleni M., Meyer-Klaucke W., Nolting H. et al. (1999) Mono- and binuclear Zn2+-β-lactamase. J. Biol. Chem. 274: 13242-13249
Yang Y., Keeney D., Tang X., Canfield N. and Rasmussen B. A. (1999) Kinetic properties and metal content of the metallo-β-lactamase CcrA harboring selective amino acid substitutions. J. Biol. Chem. 274: 15706-15711
Paul-Soto R., Hernandez Valladares M., Galleni M., Bauer R., Zeppezauer M., Frère J. M. et al. (1998) Mono-and binuclear Zn-beta-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions. FEBS Lett. 438: 137-140
Wang Z. and Benkovic S. J. (1998) Purification, characterization, and kinetic studies of a soluble Bacteroides fragilis metallo-beta-lactamase that provides multiple antibiotic resistance. J. Biol. Chem. 273: 22402-22408
Hemmingsen L., Damblon C., Antony J., Jensen M., Adolph H. W, Wommer S. et al. (2001) Dynamics of mononuclear cadmium beta-lactamase revealed by the combination of NMR and PAC spectroscopy. J. Am. Chem. Soc. 123: 10329-10335
Heinz U., Bauer R., Wommer S., Meyer-Klaucke W., Papamichael C., Bateson J. et al. (2003) Coordination geometries of metal ions in d-or l-captopril-inhibited metallo-beta-lactamases. J. Biol. Chem. 278: 20659-20666