[en] Bacteria can defend themselves against beta-lactam antibiotics through the expression of class B beta-lactamases, which cleave the beta-lactam amide bond and render the molecule harmless. There are three subclasses of class B beta-lactamases (B1, B2 and B3), all of which require Zn(2+) for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-beta-lactamases are most active as di-zinc enzymes, subclass B2 enzymes such as Aeromonas hydrophila CphA are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the di-zinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the "cysteine" site, as previously determined for the mono-zinc form of the enzyme. The second zinc ion occupies a slightly modified "histidine" site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed in enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically-important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bebrone, Carine ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Abriata, L. A., L. J. Gonzalez, L. I. Llarrull, P. E. Tomatis, W. K. Myers, A. L. Costello, D. L Tierney, and A. J. Vila. 2008. Engineered mononuclear variants in Bacillus cereus metallo-β-lactamase are inactive. Biochemistry 47:8590-8599.
Bebrone, C., C. Anne, K. De Vriendt, B. Devresse, J. Van Beeumen, J. M. Frère, and M. Galleni. 2005. Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis. J. Biol. Chem. 17:180-188.
Bebrone, C. 2007. Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem. Pharmacol. 74:1686-1701.
Bebrone, C., C. Anne, F. Kerff, G. Garau, K. De Vriendt, R. Lantin, B. Devreese, J. Van Beeumen, O. Dideberg, J. M. Frère, and M. Galleni. 2008. Mutational analysis of the zinc and substrate binding sites in the CphA metallo-β-lactamase from Aeromonas hydrophila. Biochem. J. 114:151-159.
Carfi, A., S. Parès, E. Duée, M. Galleni, C. Duez, J. M. Frère, and O. Dideberg. 1995. The 3-D structure of a zinc metallo-β-lactamase from Bacillus cereus reveals a new type of protein fold. EMBO J. 14:4914-4921.
Collaborative Computational Project, Number 4. 1994. CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50:760-763.
Costello, A. L., N. P. Sharma, K. W. Yang, M. W. Crowder, and D. L. Tierney. 2006. X-ray absorption spectroscopy of the zinc-binding sites in the class B2 metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria. Biochemistry 45:13650-13658.
Crawford, P. A., K. W. Yang, N. Sharma, B. Bennett, and M. W. Crowder. 2005. Spectroscopic studies on cobalt(II)-substituted metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria. Biochemistry 44:5168-5176.
Daiyasu, H., K. Osaka, Y. Ishino, and H. Toh. 2001. Expansion of the zinc metallo-hydrolase family of the β-lactamase fold. FEBS Lett. 503:1-6. (Pubitemid 32769945)
Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60:2126-2132. (Pubitemid 41742764)
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-lactamases. Biochem. J. 291:151-155. (Pubitemid 23107763)
Fonseca, F., A. Correia, and J. Spencer. 2008. Structural and kinetic characterisation of Sfh-1, the B2 metallo-β-lactamase of Serratia fonticola, p. 46. In Proceedings of the 10th β-Lactamase Meeting, Eretria, Greece.
Galleni, M., J. Lamotte-Brasseur, G. M. Rossolini, J. Spencer, O. Dideberg, J.-M. Frère, and the Metallo-β-Lactamases Working Group. 2001. Standard numbering scheme for class B β-lactamases. Antimicrob. Agents Chemother. 45:660-663.
Garau, G., I. Garcia-Saez, C. Bebrone, C. Anne, P. S. Mercuri, M. Galleni, J. M. Frère, and O. Dideberg. 2004. Update of the standard numbering scheme for class B β-lactamases. Antimicrob. Agents Chemother. 48:2347-2349.
Garau, G., C. Bebrone, C. Anne, M. Galleni, J. M. Frère, and O. Dideberg. 2005. A metallo-β-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem. J. Mol. Biol. 345:785-795.
Garcia-Saez, I., J. D. Docquier, G. M. Rossolini, and O. Dideberg. 2008. The three-dimensional structure of VIM-2, a Zn-β-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form. J. Mol. Biol. 375:604-611.
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 AE036 metallo-β- lactamase activity and stability. Biochemistry 36:11534-11541.
Hernandez-Valladares, M., M. Kiefer, U. Heinz, R. Paul Soto, W. Meyer- Klaucke, H. Friederich Nolting, M. Zeppezauer, M. Galleni, J. M. Frère, G. M. Rossolini, G. Amicosante, and H. W. Adolph. 2000. Kinetic and spectroscopic characterization of native and metal-substituted β-lactamase from Aeromonas hydrophila AE036. FEBS Lett. 467:221-225.
Hooft, R. W. W., G. Vriend, C. Sander, and E. E. Abola. 1996. Errors in protein structures. Nature 381:272.
Horsfall, L. E., G. Garau, B. M. Liénard, O. Dideberg, C. J. Schofield, J. M. Frère, and M. Galleni. 2007. Competitive inhibitors of the CphA metallo- β-lactamase from Aeromonas hydrophila. Antimicrob. Agents Chemother. 51:2136-2142.
Kabsch, W. 1993. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26:795-800.
Laskowski, R. A., M. W. MacArthur, D. S. Moss, and J. M. Thornton. 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26:283-291.
Liénard, B. M. R., G. Garau, L. Horsfall, A. I. Karsisiotis, C. Damblon, P. Lassaux, C. Papamicael, G. C. K. Roberts, M. Galleni, O. Dideberg, J. M. Frère, and C. J. Schofield. 2008. Structural basis for the broad-spectrum inhibition of metallo-β-lactamases by thiols. Org. Biomol. Chem. 6:2282-2294.
Llarull, L. I., M. F. Tioni, and A. J. Vila. 2008. Metal content and localization during turnover in Bacillus cereus metallo-β-lactamase. J. Am. Chem. Soc. 130:15842-15851.
Morán-Barrio, J., J. M. González, M. N. Lisa, A. L. Costello, M. Dal Peraro, P. Carloni, B. Bennett, D. L. Tierney, A. S. Limansky, A. M. Viale, and A. J. Vila. 2007. The metallo-β-lactamase GOB is a mono-Zn(II) enzyme with a novel active site. J. Biol. Chem. 282:18286-18293.
Murphy, T. A., L. E. Catto, S. E. Halford, A. T. Hadfield, W. Minor, T. R. Walsh, and J. Spencer. 2006. Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-β- lactamases. J. Mol. Biol. 357:890-903.
Murshudov, G. N., A. A. Vagin, and E. J. Dodson. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53:240-255.
Painter, J., and E. A. Merritt. 2006. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62:439-450.
Perrakis, A., R. Morris, and V. S. Lamzin. 1999. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6:458-463.
Segatore, B., O. Massida, G. Satta, D. Setacci, and G. Amicosante. 1993. High specificity of cphA-encoded metallo-β-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to β-lactam resistance. Antimicrob. Agents Chemother. 37:1324-1328.
Sharma, N. P., C. Hajdin, S. Chandrasekar, B. Bennett, K. W. Yang, and M. W. Crowder. 2006. Mechanistic studies on the mononuclear Zn(II)- containing metallo-β-lactamase ImiS from Aeromonas sobria. Biochemistry 45:10729-10738.
Tioni, M. F., L. I. Llarull, A. A. Poeylaut-Palena, M. A. Marti, M. Saggu, G. R. Periyannan, E. G. Mata, B. Bennett, D. H. Murgida, and A. J. Vila. 2008. Trapping and characterization of a reaction intermediate in carbapenem hydrolysis by Bacillus cereus metallo-β-lactamase. J. Am. Chem. Soc. 130:15852-15863.
Ullah, J. H., T. R. Walsh, I. A. Taylor, D. C. Emery, C. S. Verma, S. J. Gamblin, and J. Spencer. 1998. The crystal structure of the L1 metallo-β- lactamase from Stenotrophomonas maltophilia at 1.7 Å resolution. J. Mol. Biol. 284:125-136.
Vanhove, M., M. Zakhem, B. Devreese, N. Franceschini, C. Anne, C. Bebrone, G. Amicosante, G. M. Rossolini, J. Van Beeumen., J. M. Frère, and M. Galleni. 2003. Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-β-lactamase. Cell. Mol. Life Sci. 60:2501-2509.
Wommer, S., S. Rival, U. Heinz, M. Galleni, J. M. Frère, N. Franceschini, G. Amicosante, B. Rasmussen, R. Bauer, and H. W. Adolph. 2002. Substrate-activated zinc binding of metallo-β-lactamases: physiological importance of mononuclear enzymes. J. Biol. Chem. 277:24142-24147.
Xu, D., D. Xie, and H. Guo. 2006. Catalytic mechanism of class B2 metallo- β-lactamase. J. Biol. Chem. 281:8740-8747.
Yanchak, M. P., R. A. Taylor, and M. W. Crowder. 2000. Mutational analysis of metallo-β-lactamase CcrA from Bacteroides fragilis. Biochemistry 39: 11330-11339.
