Metallo-β-lactamases (MβLs) are bacterial Zn(II)-dependent hydrolases that confer broad-spectrum resistance to β-lactam antibiotics. These enzymes can be subdivided into three subclasses (B1, B2 and B3) that differ in their metal binding sites and their characteristic tertiary structure. To date there are no clinically useful pan-MβL inhibitors available, mainly due to the unawareness of key catalytic features common to all MβL brands. Here we have designed, expressed and characterized two double mutants of BcII, a di-Zn(II) B1-MβL from Bacillus cereus, namely BcII-R121H/C221D (BcII-HD) and BcII-R121H/C221S (BcII-HS). These mutants display modified environments at the so-called Zn2 site or DCH site, reproducing the metal coordination environments of structurally related metallohydrolases. Through a combination of structural and functional studies, we found that BcII-HD is an impaired β-lactamase even as a di-Zn(II) enzyme, whereas BcII-HS exhibits the ability to exist as mono or di-Zn(II) species in solution, with different catalytic performances. We show that these effects result from an altered position of Zn2, which is incapable of providing a productive interaction with the substrate β-lactam ring. These results indicate that the position of Zn2 is essential for a productive substrate binding and hydrolysis. © 2007 Elsevier Ltd. All rights reserved.373511411156Fisher, J.F., Meroueh, S.O., Mobashery, S., Bacterial resistance to beta-lactam antibiotics: compelling opportunism, compelling opportunity (2005) Chem. Rev., 105, pp. 395-424Frere, J.M., Beta lactamases and bacterial resstance to antibiotics (1995) Mol. Microbiol., 16, pp. 385-395Wilke, M.S., Lovering, A.L., Strynadka, N.C., beta-Lactam antibiotic resistance: a current structural perspective (2005) Curr. Opin. Microbiol., 8, pp. 525-533Page, M.I., Laws, A.P., The mechanism of catalysis and the inhibition of beta-lactamases (1998) J. Chem. Soc. Chem. Commun., 1998, pp. 1609-1617Strynadka, N.C., Adachi, H., Jensen, S.E., Johns, K., Sielecki, A., Betzel, C., Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 Å resolution (1992) Nature, 359, pp. 700-705Sulton, D., Pagan-Roderiguez, D., Zhou, X., Liu, Y., Hujer, A.M., Bethel, C.R., Clavulanic acid inactivation of SHV-1 and the inhibitor resistant SER130GLY SHV-1 beta-lactamase: insights into the mechanism of inhibition (2005) J. Biol. Chem., 280, pp. 35528-35536Crowder, M.W., Spencer, J., Vila, A.J., Metallo-beta-lactamases: novel weaponry for antibiotic resistance in bacteria (2006) Acc. Chem. Res., 39, pp. 721-728Wang, Z., Fast, W., Valentine, A.M., Benkovic, S.J., Metallo-β-lactamase: structure and mechanism (1999) Curr. Opin. Chem. Biol., 3, pp. 614-622Walsh, T.R., Toleman, M.A., Poirel, L., Nordmann, P., Metallo-beta-lactamases: the quiet before the storm? (2005) Clin. Microbiol. Rev., 18, pp. 306-325Heinz, U., Adolph, H.W., Metallo-beta-lactamases: two binding sites for one catalytic metal ion? (2004) Cell Mol. Life Sci., 61, pp. 2827-2839Toney, J.H., Moloughney, J.G., Metallo-beta-lactamase inhibitors: promise for the future? (2004) Curr. Opin. Investig. Drugs, 5, pp. 823-826Daiyasu, H., Osaka, K., Ishino, Y., Toh, H., Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold (2001) FEBS Letters, 503, pp. 1-6Carfi, A., Pares, S., Duee, E., Galleni, M., Duez, C., Frère, J.M., Dideberg, O., The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold (1995) EMBO J., 14, pp. 4914-4921Galleni, M., Lamotte-Brasseur, J., Rossolini, G.M., Spencer, J., Dideberg, O., Frere, J.M., Standard numbering scheme for class B beta-lactamases (2001) Antimicrob. Agents Chemother., 45, pp. 660-663Garau, G., Di Guilmi, A.M., Hall, B.G., Structure-based phylogeny of the metallo-beta-lactamases (2005) Antimicrob. Agents Chemother., 49, pp. 2778-2784Fabiane, S.M., Sohi, M.K., Wan, T., Payne, D.J., Bateson, J.H., Mitchell, T., Sutton, B.J., Crystal structure of the zinc-dependent beta lactamase from Bacillus cereus at 1.9 Å resolution: binuclear active site with features of a mononuclear enzime (1998) Biochemistry, 37, pp. 12404-12411Toney, J.H., Hammond, G.G., Fitzgerald, P.M., Sharma, N., Balkovec, J.M., Rouen, G.P., Succinic acids as potent inhibitors of plasmid-borne IMP-1 metallo-beta-lactamase (2001) J. Biol. Chem., 276, pp. 31913-31918Ullah, J.H., Walsh, T.R., Taylor, I.A., Emery, D.C., Verma, C.S., Gamblin, S.J., Spencer, J., The crystal strucuture of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 Å resolution (1998) J. Mol. Biol., 284, pp. 125-136Garcia-Saez, I., Mercuri, P.S., Papamicael, C., Kahn, R., Frere, J.M., Galleni, M., Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril (2003) J. Mol. Biol., 325, pp. 651-660Murphy, T.A., Catto, L.E., Halford, S.E., Hadfield, A. T., Minor, W., Walsh, T.R., Spencer, J., Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-beta-lactamases (2006) J. Mol. Biol., 357, pp. 890-903Garau, G., Bebrone, C., Anne, C., Galleni, M., Frere, J.M., Dideberg, O., A metallo-beta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem (2005) J. Mol. Biol., 345, pp. 785-795Sharma, N.P., Hajdin, C., Chandrasekar, S., Bennett, B., Yang, K.W., Crowder, M.W., Mechanistic studies on the mononuclear Zn(II)-containing metallo-beta-lactamase ImiS from Aeromonas sobria (2006) Biochemistry, 45, pp. 10729-10738Crawford, P.A., Yang, K.W., Sharma, N., Bennett, B., Crowder, M.W., Spectroscopic studies on cobalt(II)-substituted metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria (2005) Biochemistry, 44, pp. 5168-5176Bebrone, C., Anne, C., De Vriendt, K., Devreese, B., Rossolini, G.M., van Beeumen, J., Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-beta-lactamase by site-directed mutagenesis (2005) J. Biol. Chem., 280, pp. 28195-28202Bounaga, S., Laws, A.P., Galleni, M., Page, M.I., The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent beta-lactamase (1998) Biochem. J., 31, pp. 703-711Wang, Z., Fast, W., Benkovic, S.J., On the mechanism of the metallo-β-lactamase from Bacteroides fragilis (1999) Biochemistry, 38, pp. 10013-10023Spencer, J., Read, J., Sessions, R.B., Howell, S., Blackburn, G.M., Gamblin, S.J., Antibiotic recognition by binuclear metallo-beta-lactamases revealed by X-ray crystallography (2005) J. Am. Chem. Soc., 127, pp. 14439-14444Xu, D., Xie, D., Guo, H., Catalytic mechanism of class B2 metallo-beta-lactamase (2006) J. Biol. Chem., 281, pp. 8740-8747Rasia, R.M., Vila, A.J., Exploring the role and the binding affinity of a second zinc equivalent in B. cereus metallo-beta-lactamase (2002) Biochemistry, 41, pp. 1853-1860Morán-Barrio, J., González, J.M., Lisa, M.N., Costello, A.L., Peraro, M.D., Carloni, P., The metallo-beta-lactamase GOB is a mono-Zn(II) enzyme with a novel active site (2007) J. Biol. Chem., 282 (25), pp. 18286-18293Cameron, A.D., Ridderstrom, M., Olin, B., Mannervik, B., Crystal structure of human glyoxalase II and its complex with a glutathione thiolester substrate analogue (1999) Structure Fold. Des., 7, pp. 1067-1078Thomas, P.W., Stone, E.M., Costello, A.L., Tierney, D.L., Fast, W., The quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein (2005) Biochemistry, 44, pp. 7559-7569Hagelueken, G., Adams, T.M., Wiehlmann, L., Widow, U., Kolmar, H., Tummler, B., The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases (2006) Proc. Natl. Acad. Sci. USA, 103, pp. 7631-7636Park, H.S., Nam, S.H., Lee, J.K., Yoon, C.N., Mannervik, B., Benkovic, S.J., Kim, H.S., Design and evolution of new catalytic activity with an existing protein scaffold (2006) Science, 311, pp. 535-538Seny, D., Prosperi-Meys, C., Bebrone, C., Rossolini, G.M., Page, M.I., Noel, P., Mutational analysis of the two zinc-binding sites of the Bacillus cereus 569/H/9 metallo-beta-lactamase (2002) Biochem. J., 363, pp. 687-696Paul-Soto, R., Bauer, R., Frere, J.M., Galleni, M., Meyer-Klaucke, W., Nolting, H., Mono- and binuclear Zn2+-beta-lactamase. Role of the conserved cysteine in the catalytic mechanism (1999) J. Biol. Chem., 274, pp. 13242-13249Garrity, J.D., Carenbauer, A.L., Herron, L.R., Crowder, M.W., Metal binding Asp-120 in metallo-beta-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis (2004) J. Biol. Chem., 279, pp. 920-927Yanchak, M.P., Taylor, R.A., Crowder, M.W., Mutational analysis of metallo-beta-lactamase CcrA from Bacteroides fragilis (2000) Biochemistry, 39, pp. 11330-11339Vanhove, M., Zakhem, M., Devreese, B., Franceschini, N., Anne, C., Bebrone, C., Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase (2003) Cell Mol. Life Sci., 60, pp. 2501-2509Rasia, R.M., Ceolin, M., Vila, A.J., Grafting a new metal ligand in the cocatalytic site of B. cereus metallo-beta-lactamase: structural flexibility without loss of activity (2003) Protein Sci., 12, pp. 1538-1546de Seny, D., Heinz, U., Wommer, S., Kiefer, M., Meyer-Klaucke, W., Galleni, M., Metal ion binding and coordination geometry for wild type and mutants of metallo-beta-lactamase from Bacillus cereus 569/H/9 (BcII): a combined thermodynamic, kinetic, and spectroscopic approach (2001) J. Biol. Chem., 276, pp. 45065-45078Davies, A.M., Rasia, R.M., Vila, A.J., Sutton, B.J., Fabiane, S.M., Effect of pH on the active site of an Arg121Cys mutant of the metallo-beta-lactamase from Bacillus cereus: implications for the enzyme mechanism (2005) Biochemistry, 44, pp. 4841-4849Costello, A., Periyannan, G., Yang, K.W., Crowder, M.W., Tierney, D.L., Site-selective binding of Zn(II) to metallo-beta-lactamase L1 from Stenotrophomonas maltophilia (2006) J. Biol. Inorg. Chem., 11, pp. 351-358Yang, Y., Keeney, D., Tang, X., Canfield, N., Rasmussen, B.A., Kinetic properties and metal content of the metallo-β-lacatamase CcrA harboring selective amino acid substitutions (1999) J. Biol. Chem., 274, pp. 15706-15711Llarrull, L.I., Fabiane, S.M., Kowalski, J.M., Bennett, B., Sutton, B.J., Vila, A.J., Asp-120 locates Zn2 for optimal metallo-beta-lactamase activity (2007) J. Biol. Chem., 282 (25), pp. 18276-18285Liu, D., Lepore, B.W., Petsko, G.A., Thomas, P.W., Stone, E.M., Fast, W., Ringe, D., Three-dimensional structure of the quorum-quenching N-acyl homoserine lactone hydrolase from Bacillus thuringiensis (2005) Proc. Natl. Acad. Sci. USA, 102, pp. 11882-11887Dal Peraro, M., Vila, A.J., Carloni, P., Klein, M.L., Role of zinc content on the catalytic efficiency of B1 metallo-beta-lactamases (2007) J. Am. Chem Soc., 129, pp. 2808-2812Dal Peraro, M., Vila, A.J., Carloni, P., Substrate binding to mononuclear metallo-beta-lactamase from Bacillus cereus (2004) Proteins: Struct. Funct. Genet., 54, pp. 412-423Dal Peraro, M., Llarrull, L.I., Rothlisberger, U., Vila, A.J., Carloni, P., Water-assisted reaction mechanism of monozinc beta-lactamases (2004) J. Am. Chem. Soc., 126, pp. 12661-12668Chantalat, L., Duee, E., Galleni, M., Frere, J.M., Dideberg, O., Structural effects of the active site mutation cysteine to serine in Bacillus cereus zinc-beta-lactamase (2000) Protein Sci., 9, pp. 1402-1406Hall, B.G., Salipante, S.J., Barlow, M., Independent origins of subgroup Bl+ B2 and subgroup B3 metallo-beta-lactamases (2004) J. Mol. Evol., 59, pp. 133-141Orellano, E.G., Girardini, J.E., Cricco, J.A., Ceccarelli, E.A., Vila, A.J., Spectroscopic characterization of a binuclear metal site in Bacillus cereus beta-lactamase II (1998) Biochemistry, 37, pp. 10173-10180Hunt, J.B., Neece, S.H., Ginsburg, A., The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase (1985) Anal. Biochem., 146, pp. 150-157Kuzmic, P., Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase (1996) Anal. Biochem., 237, pp. 260-273Shulz, A.R., A closer look to the basic assumptions (1994) Enzyme Kinetics, from Diastase to Multi-enzyme Systems, pp. 22-48. , Cambridge University Press, CambridgeOtwinowski, Z., Minor, W., Processing of X-ray diffraction data collected in oscillation mode (1997) Methods Enzymol., 276, pp. 307-325Collaborative Computational Project Number 4, The CCP4 suite: programs for protein crystallography (1994) Acta Crystallog. sect. D, Biol. Crystallogr., 50, pp. 760-763Navaza, J., AMoRe: an automated package for molecular replacement (1994) Acta Crystallog. sect. D, Bi55, pp. 247-255Murshudov, G.N., Vagin, A.A., Dodson, E.J., Refinement of macromolecular structures by the maximum-likelihood method (1997) Acta Crystallog. sect. D, 53, pp. 240-255Laskowski, R.A., Mac Arthur, M.W., Moss, D.S., Thornton, J.M., PROCHECK: a program to check the stereochemical quality of protein structures (1993) J. Appl. Crystallog., 26, pp. 283-291Ankudinov, A.L., Ravel, B., Rehr, J.J., Conradson, S.D., Real-space multiple-scattering calculation and interpretation of x-ray-absorption near-edge structure (1998) Phys. Rev. ser. B, 58, pp. 7565-7576McClure, C.P., Rusche, K.M., Peariso, K., Jackman, J.E., Fierke, C.A., Penner-Hahn, J.E., EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) (2003) J. Inorg. Biochem., 94, pp. 78-85