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The Metallo-β-lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site

2007; Elsevier BV; Volume: 282; Issue: 25 Linguagem: Inglês

10.1074/jbc.m700467200

ISSN

1083-351X

Autores

Jorgelina Morán-Barrio, Javier González, María‐Natalia Lisa, Alison L. Costello, Matteo Dal Peraro, Paolo Carloni, Brian Bennett, David L. Tierney, Adriana S. Limansky, Alejandro M. Viale, Alejandro J. Vila,

Tópico(s)

Antibiotics Pharmacokinetics and Efficacy

Resumo

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs. Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs. The expression of β-lactam degrading enzymes (β-lactamases) is the most common mechanism of antibiotic resistance among bacteria (1Fisher J.F. Meroueh S.O. Mobashery S. Chem. Rev. 2005; 105: 395-424Crossref PubMed Scopus (760) Google Scholar, 2Wilke M.S. Lovering A.L. Strynadka N.C. Curr. Opin. Microbiol. 2005; 8: 525-533Crossref PubMed Scopus (285) Google Scholar). These enzymes have been grouped into four classes (A–D) according to sequence homology (3Frere J.M. Galleni M. Bush K. Dideberg O. J. Antimicrob. Chemother. 2005; 55: 1051-1053Crossref PubMed Scopus (37) Google Scholar, 4Hall B.G. Salipante S.J. Barlow M. J. Mol. Evol. 2003; 57: 249-254Crossref PubMed Scopus (50) Google Scholar). Class A, C, and D enzymes use an active site serine residue as a nucleophile, whereas class B lactamases (generically termed metallo-β-lactamases, MβLs) 9The abbreviations used are: MβL, metallo-β-lactamase; EXAFS, extended x-ray absorption fine structure; MES, 4-morpholineethanesulfonic acid. employ one or two Zn(II) ions to cleave the β-lactam ring. MβLs have particular importance in the clinical setting since they can hydrolyze a broader spectrum of β-lactam substrates than the serine-type enzymes and are resistant to most clinically employed inhibitors (5Galleni M. Lamotte-Brasseur J. Rossolini G.M. Spencer J. Dideberg O. Frere J.M. Antimicrob. Agents Chemother. 2001; 45: 660-663Crossref PubMed Scopus (335) Google Scholar, 6Garau G. Di Guilmi A.M. Hall B.G. Antimicrob. Agents Chemother. 2005; 49: 2778-2784Crossref PubMed Scopus (64) Google Scholar, 7Walsh T.R. Toleman M.A. Poirel L. Nordmann P. Clin. Microbiol. Rev. 2005; 18: 306-325Crossref PubMed Scopus (1223) Google Scholar, 8Wang Z. Fast W. Valentine A.M. Benkovic S.J. Curr. Opin. Chem. Biol. 1999; 3: 614-622Crossref PubMed Scopus (269) Google Scholar, 9Cricco J.A. Rasia R.M. Orellano E.G. Ceccarelli E.A. Vila A.J. Coord. Chem. Rev. 1999; 190–192: 519-535Crossref Scopus (72) Google Scholar, 10Cricco J.A. Vila A.J. Curr. Pharm. Des. 1999; 5: 915-927PubMed Google Scholar, 11Crowder M.W. Spencer J. Vila A.J. Acc. Chem. Res. 2006; 39: 721-728Crossref PubMed Scopus (328) Google Scholar). The design of an efficient pan-MβL inhibitor has been mostly limited by a striking diversity in the active site structures, catalytic features, and metal ion requirements for activity among different enzymes. Based on this heterogeneity, MβLs have been classified into three subclasses: B1, B2, and B3 (3Frere J.M. Galleni M. Bush K. Dideberg O. J. Antimicrob. Chemother. 2005; 55: 1051-1053Crossref PubMed Scopus (37) Google Scholar, 6Garau G. Di Guilmi A.M. Hall B.G. Antimicrob. Agents Chemother. 2005; 49: 2778-2784Crossref PubMed Scopus (64) Google Scholar). Subclass B1 includes several chromosomally encoded enzymes such as BcII from Bacillus cereus (12Carfi A. Pares S. Duee E. Galleni M. Duez C. Frère J.M. Dideberg O. EMBO J. 1995; 14: 4914-4921Crossref PubMed Scopus (404) Google Scholar, 13Orellano E.G. Girardini J.E. 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Dideberg O. J. Biol. Chem. 2003; 278: 23868-23873Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), as well as the transferable VIM (20Docquier J.D. Lamotte-Brasseur J. Galleni M. Amicosante G. Frere J.M. Rossolini G.M. J. Antimicrob. Chemother. 2003; 51: 257-266Crossref PubMed Scopus (142) Google Scholar)-, IMP (21Materon I.C. Beharry Z. Huang W. Perez C. Palzkill T. J. Mol. Biol. 2004; 344: 653-663Crossref PubMed Scopus (56) Google Scholar, 22Toney J.H. Hammond G.G. Fitzgerald P.M. Sharma N. Balkovec J.M. Rouen G.P. Olson S.H. Hammond M.L. Greenlee M.L. Gao Y.D. J. Biol. Chem. 2001; 276: 31913-31918Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar)-, SPM (23Toleman M.A. Simm A.M. Murphy T.A. Gales A.C. Biedenbach D.J. Jones R.N. Walsh T.R. J. Antimicrob. Chemother. 2002; 50: 673-679Crossref PubMed Google Scholar, 24Murphy T.A. Catto L.E. Halford S.E. Hadfield A.T. Minor W. Walsh T.R. Spencer J. J. Mol. Biol. 2006; 357: 890-903Crossref PubMed Scopus (77) Google Scholar)-, and GIM-type enzymes. Subclass B2 includes the CphA (25Garau G. Bebrone C. Anne C. Galleni M. Frere J.M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar, 26Hernández Valladares M. Felici A. Weber G. Adolph H.W. Zeppezauer M. Rossolini G.M. Amicosante G. Frère J.M. Galleni M. Biochemistry. 1997; 36: 11534-11541Crossref PubMed Scopus (176) Google Scholar) and ImiS (27Crawford P.A. Yang K.W. Sharma N. Bennett B. Crowder M.W. Biochemistry. 2005; 44: 5168-5176Crossref PubMed Scopus (56) Google Scholar) lactamases from Aeromonas species. Subclass B3, originally represented only by L1 from Stenotrophomonas maltophilia (28Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spencer J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (297) Google Scholar, 29Spencer J. Clarke A.R. Walsh T.R. J. Biol. Chem. 2001; 276: 33638-33644Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 30Garrity J.D. Carenbauer A.L. Herron L.R. Crowder M.W. J. Biol. Chem. 2004; 279: 920-927Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), now includes enzymes from other opportunistic pathogens like FEZ-1 from Legionella gormanii (31Garcia-Saez I. Mercuri P.S. Papamicael C. Kahn R. Frere J.M. Galleni M. Rossolini G.M. Dideberg O. J. Mol. Biol. 2003; 325: 651-660Crossref PubMed Scopus (116) Google Scholar) and GOB from E. meningoseptica (32Bellais S. Aubert D. Naas T. Nordmann P. Antimicrob. Agents Chemother. 2000; 44: 1878-1886Crossref PubMed Scopus (121) Google Scholar), as well as from environmental bacteria such as CAU-1 from Caulobacter crescentus (33Docquier J.D. Pantanella F. Giuliani F. Thaller M.C. Amicosante G. Galleni M. Frere J.M. Bush K. Rossolini G.M. Antimicrob. Agents Chemother. 2002; 46: 1823-1830Crossref PubMed Scopus (53) Google Scholar) and THIN-B from Janthinobacterium lividum (34Docquier J.D. Lopizzo T. Liberatori S. Prenna M. Thaller M.C. Frere J.M. Rossolini G.M. Antimicrob. Agents Chemother. 2004; 48: 4778-4783Crossref PubMed Scopus (21) Google Scholar). Molecular structures of MβLs from the three subclasses have been solved by x-ray crystallography (12Carfi A. Pares S. Duee E. Galleni M. Duez C. Frère J.M. Dideberg O. EMBO J. 1995; 14: 4914-4921Crossref PubMed Scopus (404) Google Scholar, 14Fabiane S.M. Sohi M.K. Wan T. Payne D.J. Bateson J.H. Mitchell T. Sutton B.J. Biochemistry. 1998; 37: 12404-12411Crossref PubMed Scopus (216) Google Scholar, 15Concha N. Rasmussen B.A. Bush K. Herzberg O. Structure (Camb.). 1996; 4: 823-836Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 25Garau G. Bebrone C. Anne C. Galleni M. Frere J.M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar, 31Garcia-Saez I. Mercuri P.S. Papamicael C. Kahn R. Frere J.M. Galleni M. Rossolini G.M. Dideberg O. J. Mol. Biol. 2003; 325: 651-660Crossref PubMed Scopus (116) Google Scholar). Comparison of the tertiary structure of enzymes belonging to the different subclasses reveals a common αβ/βα sandwich fold, in which different insertions and deletions have resulted in different loop topologies and, ultimately, in different zinc coordination environments and metal site occupancies among B1, B2, and B3 enzymes (Fig. 1). MβLs bind up to two metal ions in their active sites. In B1 and B3 enzymes, one Zn(II) ion (Zn1) is tetrahedrally coordinated to three histidine ligands (His116, His118, and His196 in Fig. 1) and a water/OH– molecule (3-H site) (14Fabiane S.M. Sohi M.K. Wan T. Payne D.J. Bateson J.H. Mitchell T. Sutton B.J. Biochemistry. 1998; 37: 12404-12411Crossref PubMed Scopus (216) Google Scholar, 15Concha N. Rasmussen B.A. Bush K. Herzberg O. Structure (Camb.). 1996; 4: 823-836Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 28Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spencer J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (297) Google Scholar). The coordination polyhedron of Zn2 in B1 enzymes is provided by Asp120, Cys221, His263, and one or two-water molecules (DCH site). Notably, this site represents the active species in mono-Zn(II) B2 enzymes (25Garau G. Bebrone C. Anne C. Galleni M. Frere J.M. Dideberg O. J. Mol. Biol. 2005; 345: 785-795Crossref PubMed Scopus (187) Google Scholar). Instead, two mutations (Cys221 → Ser and Arg121 → His) affect the Zn2 coordination geometry in B3 MβLs, and the metal ion is bound to Asp120, His121, His263, and one- or two-water molecules, while Ser221 is no longer a metal ligand (DHH site) (28Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spencer J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (297) Google Scholar). A remarkable exception is provided by the deepest branching member of the MβL B3 subclass, GOB from E. meningoseptica (4Hall B.G. Salipante S.J. Barlow M. J. Mol. Evol. 2003; 57: 249-254Crossref PubMed Scopus (50) Google Scholar). In all reported GOB sequences, His116 and Ser221 are substituted by Gln and Met, respectively, suggesting the presence of an unusually perturbed metal binding site. Here we show that E. meningoseptica GOB-18 represents a novel type of broad spectrum MβL unique in being maximally active in the mono-Zn(II) form and in which the metal ion occupies only the Zn2, DHH site. This contrasts with two generally accepted ideas: broad spectrum MβLs are maximally active in the dinuclear form (B1 and B3 enzymes), and mono-Zn(II) enzymes are carbapenemases (B2 enzymes). Finally, the findings presented here confirm that Zn2 is central for MβL-mediated catalysis and that the attacking nucleophile could be provided either by a non-metal center or by the Zn2 site. This claims for a revisited strategy for the design of broad spectrum MβL inhibitors. Source of GOB-18 Coding Sequence—An E. meningoseptica clinical strain from the Hospital Clemente Alvarez (Rosario, Argentina), identified by API 20NE (bioMerieux, Marcy l'Etoile, France), was used as the source of the genomic DNA for cloning the GOB-18 coding gene. DNA Cloning and Construction of the Expression Vector for GOB-18—Genomic DNA from the E. meningoseptica clinical strain used here was isolated essentially as described in ref (35Ausubel K.M. Brent R. Kingston R.E. Moore R.E. Seidman I.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1987Google Scholar). The complete coding sequence of the gob-18 gene was amplified by employing primers 1 and 2 described in Ref. 32Bellais S. Aubert D. Naas T. Nordmann P. Antimicrob. Agents Chemother. 2000; 44: 1878-1886Crossref PubMed Scopus (121) Google Scholar. The DNA fragment was sequenced and two new primers were designed (see supplemental material) to clone the mature GOB-18 coding sequence cloned into BamHI-HindIII sites of pETGEXTerm vector (13Orellano E.G. Girardini J.E. Cricco J.A. Ceccarelli E.A. Vila A.J. Biochemistry. 1998; 37: 10173-10180Crossref PubMed Scopus (115) Google Scholar) in frame to the 3′-end of Schistosoma japonicus glutathione S-transferase gene, for expression purposes. The resulting recombinant pET-GOB-18 plasmid produces GOB-18 as a C-terminal fusion to glutathione S-transferase. The nucleotide sequence reported in this paper was assigned accession number DQ004496 in the combined EMBL/GenBank™/DDBJ sequence data base. Site-directed Mutagenesis—Site-directed mutagenesis was achieved using a rapid PCR-based method with modifications (36Costa G.L. Bauer J.C. McGowan B. Angert M. Weiner M.P. Trower M.K. In Vitro Mutagenesis Protocols. Humana Press, Totowa, NJ1996Google Scholar and see supplemental material). Protein Expression and Purification—GOB-18 wild-type and mutants were overproduced in Escherichia coli BL21(DE3) pLysS and E. coli BL21(DE3) Codon Plus RIL cells, respectively, transformed with plasmid pET-GOB-18, pET9a-Gln116 → His GOB-18, pET9a-Asp120 → Ser GOB-18, or pET9a-Cys201 → Ser GOB-18. We followed protein expression and purification procedures described before (13Orellano E.G. Girardini J.E. Cricco J.A. Ceccarelli E.A. Vila A.J. Biochemistry. 1998; 37: 10173-10180Crossref PubMed Scopus (115) Google Scholar) with modifications. The purification average yields were of ∼12 mg of GOB-18 or Cys201 → Ser GOB-18, 6 mg of Gln116 → His GOB-18, and 3 mg of Asp120 → Ser GOB-18, per liter of culture, and rendered polypeptides of 31 kDa at a purity higher than 95%, as estimated by SDS-PAGE (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Pure enzymes were dialyzed twice against 100 volumes of 15 mm Hepes, pH 7.5, NaCl 0.2 m, pooled, and then stored at 4 °C for immediate use. The concentration of purified enzymes was determined by measuring the absorbance at 280 nm in a Jasco V-550 spectrophotometer, using the theoretically calculated molar extinction coefficient (32,200 m–1 cm–1 for GOB-18, Gln116 → His GOB-18 or Asp120 → Ser GOB-18, and 32,075 m–1 cm–1 for Cys201 → Ser GOB-18) (38Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar). Biochemical Characterization of Wild-type GOB-18 and Mutants—Size exclusion chromatography was done on a Superdex 200 HR 10/30 column (Amersham Biosciences). The molecular mass of purified GOB-18 was measured by mass spectrometry/electrospray using an LCQ Duo Ion Trap mass spectrometer at the mass spectrometry facility LANAIS-PRO, University of Buenos Aires. Circular Dichroism spectra of protein samples in 10 mm Tris-HCl, pH 7.5, and 50 mm NaCl were measured at 25 °C, using a Jasco J-715 spectropolarimeter flushed with N2. The amount of solvent exposed cysteine thiols in GOB-18 was determined employing 5,5′-dithiobis-(2-nitrobenzoic acid) in native and SDS-unfolded samples (39Riener C.K. Kada G. Gruber H.J. Anal. Bioanal. Chem. 2002; 373: 266-276Crossref PubMed Scopus (447) Google Scholar). The GOB-18 and Gln116 → His GOB-18 apoproteins were prepared by treating samples ∼0.1 mm in 10 mm Tris-HCl, pH 7.0, with chelating agents in mild denaturing conditions (see supplemental material). The Zn(II) derivatives were prepared by dialyzing the apo-GOB-18 or apo-Gln116 → His GOB-18 against 100 volumes of 10 mm Tris-HCl, pH 7.0, 50 mm NaCl, with an equimolar concentration of ZnSO4. The Fe(II)-GOB-18 derivative was prepared in the same way using instead (NH4)Fe(SO4), under anaerobic conditions, with O2-free N2 efflux and addition of 1 mm sodium dithionite. The reduced state of the metal ion was verified colorimetrically using o-phenanthroline. The Fe(III)-GOB-18 derivative was prepared taking advantage of Zn(II) dissociation at acidic pH values retaining the iron content. Holo-GOB-18 was dialyzed against 100 volumes of polybuffer TAMS (50 mm Tris, 50 mm sodium acetate, 50 mm MES, and 500 mm NaCl) adjusted to pH 4.5 (twice). Then pH was raised by dialysis against 100 volumes of 10 mm Tris-HCl, pH 7, and 50 mm NaCl (twice). Metal Content Determination—The metal content of the purified enzymes was measured by inductively coupled plasmaatomic emission spectrometer or by atomic absorption spectroscopy in a Metrolab 250 instrument operating in the flame mode. Determination of IC50 Value—The concentration required to effect 50% inhibition of enzyme activity (IC50) was determined by preincubating GOB-18 (2 nm) in 15 mm Hepes, pH 7.5, 200 mm NaCl, 30 °C, with increasing EDTA concentrations for 1 min, prior to the initiation of the assay by the addition of 1 mm cefotaxime. A plot of steady-state hydrolysis rate versus inhibitor concentration provided the basis for the assessment of the IC50 value. Determination of the Kinetic Parameters—The hydrolysis of the antibiotics was monitored by following the absorbance variation resulting from the hydrolysis of the β-lactam ring. The reaction medium employed was 15 mm Hepes, pH 7.5, 0.2 m NaCl. All measurements were performed at 30 °C. The kinetic parameters Km and kcat were derived from initial rate measurements, recorded on a Jasco V-550 spectrophotometer, and were estimated by nonlinear data fitting to the integrated form of the Michaelis-Menten equation. pH Dependence—The pH dependence of GOB-18-mediated cefotaxime hydrolysis was determined by performing measurements in the polybuffer TAMS adjusted from pH 4.0 to pH 8.0 with increments of 0.5 pH units, at 1 mm substrate concentration (see supplemental material). X-ray Absorption Spectroscopy—Samples of GOB-18 (∼1 mm) were prepared with 20% (v/v) glycerol, and loaded in Lucite cuvettes with 6-μm polypropylene windows, before rapid freezing in liquid nitrogen. X-ray absorption spectra were measured at the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, NY), beamline X9B, with a Si(111) double crystal monochromator; harmonic rejection was accomplished using a nickel focusing mirror. Data collection and reduction were accomplished according to published procedures (40Thomas P.W. Stone E.M. Costello A.L. Tierney D.L. Fast W. Biochemistry. 2005; 44: 7559-7569Crossref PubMed Scopus (110) Google Scholar). XAS data were collected on two independently isolated samples. As each data set gave similar results, the spectra were averaged; the data in Fig. 3 represent the average of the two data sets (12 scans total). Fourier filtered EXAFS data (Δk = 1–13 Å–1; Δr = 0.5–2.1 Å, first shell or 0.1–4.5 Å for multiple scattering fits) were fit utilizing theoretical amplitude and phase functions calculated with FEFF v.8.00 (41Ankudinov A.L. Ravel B. Rehr J.J. Conradson S.D. Phys. Rev. B. 1998; 58: 7565-7576Crossref Scopus (4107) Google Scholar). The zinc-nitrogen scale factor and the threshold energy, ΔE0, were calibrated to the experimental spectrum for tetrakis-1-methylimidazole zinc(II) perchlorate, Zn(MeIm)4 (42McClure C.P. Rusche K.M. Peariso K. Jackman J.E. Fierke C.A. Penner-Hahn J.E. J. Inorg. Biochem. 2003; 94: 78-85Crossref PubMed Scopus (34) Google Scholar), and held fixed (at 0.78 and –21 eV, respectively), as were similarly determined values for iron (0.78 and –21 eV), in all subsequent fits to the data for GOB-18. First shell fits were then obtained for all reasonable coordination numbers, including mixed nitrogen/oxygen ligation, while allowing the absorber-scatterer distance, Ras, and the Debye-Waller factor, σ 2as, to vary; the best fits are presented in the supporting information. Multiple scattering contributions from histidine ligands were fit according to published procedures. Metal-metal (zinc-iron and iron-zinc) scattering was modeled with reference to the experimental EXAFS of Zn2(salpn)2 and Fe2(salpn)2. NMR—NMR spectra were recorded on a Bruker Avance II 600 spectrometer operating at 600.13 MHz at different temperatures, as indicated. 1H NMR spectra were recorded under conditions to optimize detection of the fast relaxing paramagnetic resonances, either using the superWEFT pulse sequence or water presaturation. Spectra were acquired over large spectral widths with acquisition times ranging from 16 to 80 ms and intermediate delays from 2 to 35 ms. One-dimensional experiments with solvent presaturation were used to record isotropically shifted signals closer to the diamagnetic envelope. EPR—EPR was recorded at 9.63 GHz with 2-milliwatt microwave power and 5-G (0.5 mT) magnetic field modulation at 100 kHz, using a Bruker EleXsys E500 spectrometer equipped with an ER4116DM cavity operating in perpendicular mode and an Oxford Instruments ESR900 helium flow cryostat and ITC503 temperature controller. Biochemical Characterization of GOB-18—The gene coding for a GOB-type MβL was cloned from a carpabenem-resistant E. meningoseptica clinical strain. Sequence analysis indicated a predicted molecular mass of 31.4 kDa for the encoded protein. At the time of sequencing there were 17 known variants of GOB enzymes. This protein differed from those reported previously and was named GOB-18. This enzyme differed from the firstly reported GOB enzyme, GOB-1 (32Bellais S. Aubert D. Naas T. Nordmann P. Antimicrob. Agents Chemother. 2000; 44: 1878-1886Crossref PubMed Scopus (121) Google Scholar), by three conserved mutations: Leu94 → Phe, Ala137 → Val, and Asp282 → Asn (standard consensus numbering) (5Galleni M. Lamotte-Brasseur J. Rossolini G.M. Spencer J. Dideberg O. Frere J.M. Antimicrob. Agents Chemother. 2001; 45: 660-663Crossref PubMed Scopus (335) Google Scholar). Recombinant GOB-18 was overproduced as a fusion to GST in the cytoplasm of E. coli BL21(DE3) pLysS cells, cleaved, and purified to homogeneity. GOB-18 is a monomeric enzyme according to size exclusion chromatography. Mass spectrometry confirmed the molecular mass expected from the gene sequence. The β-lactamase activity (measured as cefotaxime hydrolysis) was inhibited by EDTA with an IC50 of 2.3 ± 0.3 mm, indicating that GOB-18 holds a tightly bound divalent cation essential for activity. Inductively coupled plasma and atomic absorption analyses showed that recombinant GOB-18 contained significant amounts of zinc and, notably, iron. Although the relative amounts of these two metals varied among different enzyme preparations (0.45–0.75 iron/GOB-18 and 0.01–0.20 zinc/GOB-18), the total metal content never exceeded one metal ion per protein molecule. Addition of excess Zn(II) did not alter the CD spectrum of GOB-18 as isolated, neither in the near nor in the far UV (data not shown). Overall, these data suggest that GOB-18 is a mono-metallic enzyme, in sharp contrast to the other B3 MβLs. The apoprotein was devoid of lactamase activity that could be recovered by addition of Zn(II). Remarkably, apo-GOB-18 remetallated with Zn(II) bound only one equivalent of Zn(II) and its activity largely exceeded that of the enzyme as isolated (Fig. 2). On the other hand, Fe(II)-GOB-18 and Fe(III)-GOB-18 displayed negligible activities compared with that of Zn(II)-GOB-18. In fact, Fe(III)-GOB-18 was poorly active even upon addition of Zn(II), suggesting that both metal ions compete for the same binding site. The kinetic parameters of Zn(II)-GOB-18, reported in Table 1, show that mono-Zn(II)-GOB-18 presents a broad substrate spectrum for β-lactams, similarly to that shown by dinuclear B3 enzymes. Addition of 20 μm Zn(II) to the reaction medium did not modify the enzyme kinetic parameters.TABLE 1Kinetic parameters for the hydrolysis of different β-lactam substrates by fully loaded wild type Zn(II)-GOB-18 and Zn(II)-Gln116 → His GOB-18 mutantSubstrateGOB-18Gln116 → His GOB-18kcatKmkcat/KmkcatKmkcat/Kms-1μmμm-1 s-1s-1μmμm-1 s-1Penicillin G680 ± 80330 ± 302.1 ± 0.4664 ± 9380 ± 201.7 ± 0.1Cefaloridine30 ± 231.5 ± 0.50.95 ± 0.08NDNDCefotaxime83 ± 288 ± 60.94 ± 0.0913.1 ± 0.158 ± 20.23 ± 0.01Imipenem42 ± 926 ± 21.6 ± 0.519.3 ± 0.458 ± 30.33 ± 0.02Meropenem72.5 ± 0.540 ± 101.8 ± 0.5NDND Open table in a new tab The pH dependence of GOB-18-mediated cefotaxime hydrolysis showed a plateau between pH values of 6 and 8, and an acidic limb, resulting in a complete inactivation at pH ≤ 4.5. This loss of activity could be reverted by raising the pH to 6.5, indicating that the inactivation is reversible. Metal content analysis at different pH values showed that the decrease in activity at low pH paralleled the dissociation of Zn(II) from the enzyme, despite the fact that most of the Fe(III) remained bound up to pH 4 (see supplemental material). Overall, these data show that Zn(II)-GOB-18 is the active β-lactamase species. GOB-18 preparations with larger zinc/iron ratios were the most active ones, in line with this observation (see supplemental material). Expression of the complete GOB-18 gene (including a transit peptide sequence) in E. coli results in an enzyme secreted to the periplasm confering β-lactam resistance to the bacterial host. Isoelectrofocusing analysis of the periplasmic fraction after osmotic shock revealed that the enzyme is present exclusively in the Zn(II) form, suggesting that iron uptake could be due to an artifact of protein overexpression in E. coli cytoplasm. X-ray Absorption Spectroscopy—EXAFS data recorded at the zinc and iron edges, k3χ((k), and the corresponding Fourier transforms for GOB-18 are shown in Fig. 3. EXAFS curve-fitting results are presented as supporting information. Fits to Fourier-filtered first shell scattering (0.5–2.1 Å) in the case of Zn(II)-GOB-18 suggest coordination to 4 nitrogen/oxygen donors at a distance of 2.01 Å (Fit Zn-1). Fit residuals did not improve significantly upon inclusion of a sulfur, or a mixed shell of nitrogen/oxygen. Multiple scattering analysis indicates ligation to 2 histidine residues (Fit Zn-2). The low Debye-Waller factor for the second multiple scattering path (C1-N1) most likely reflects some rotation of the imidazole plane about the axis normal to the zinc-nitrogen bond. Inclusion of a single zinc-carbon scattering interaction at a distance of 2.71 Å, representing carboxylate carbon scattering, modestly improved the fit (∼15%) (Fit Zn-3). In the case of Fe(III)-GOB-18, single shell fits give an average of 5 nitrogen/oxygen scatterers at 2.11 Å (Fit Fe-1). Inclusion of a sulfur atom increased the fit residual. The larger metal-ligand distance is consistent with a higher coordination number for iron relative to zinc. Separate shells of 3 oxygen atoms at 2.04 Å and 2 nitrogen atoms at 2.20 Å could be resolved (Fit Fe-2), and multiple scattering fits indicate the presence of two His ligands (Fit Fe-3). Inclusion of an iron scatterer in fits for Zn(II)-GOB-18 (Fit Zn-4) and a zinc scatterer in fits for Fe(III)-GOB-18 (Fit Fe-3) slightly improved the fits but led to different metal-metal distances (3.61 and 3.55 Å, respectively). This allows us to discard the existence of a heterodimetallic site in GOB-18. Thus, EXAFS data, together with the biochemical studies, suggest that Zn(II) and Fe(III) compete for the only metal binding site in GOB-18. Based on this, we decided to exploit Fe(III) as a spectroscopic probe of the metal site. Spectroscopic Characterization of Fe(III)-GOB-18—The UV-visible spectrum of Fe(III)-GOB-18 revealed an absorption band centered at 375 nm, which was not present in the apoenzyme nor in the Zn(II)-reconstituted form (Fig. 4A). This feature can be attributed to a typical His-Fe(III) charge transfer band, similar to those observed in lipoxygenase, iron superoxide dismutase, and other non-heme, non-Fe-S iron proteins (43Averill B.A. Vincent J.B. Methods Enzymol. 1993; 226: 33-51Crossref PubMed Scopus (10) Google Scholar). EPR spectra of GOB-18 recorded at 6 K and 25 K (Fig. 4B) indicated an isolated, "rhombic" (i.e. mean E/D ∼ 1/3) Fe(III) ion. The almost complete loss of the MS = |±1/2 〉 (or MS = |±5/2 〉) signals at 25 K suggests a zero field splitting, Δ, of less than about 30 cm–1. The asymmetry of the lines at geff ∼ 4.3 and at geff ∼ 9 indicates significant strain in E/D, although the shoulder on the crossover at geff ∼ 4.3 (Fig. 4B, inset) indicates a lower degree of strain than in the often-seen "adventitious iron" signal and provides strong evidence for the Fe(III) being tightly protein-bound. In contrast to the iron-loaded glyoxylase II (44Maras

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