Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m309852200
ISSN1083-351X
AutoresJames D. Garrity, Anne L. Carenbauer, Lissa R. Herron, Michael W. Crowder,
Tópico(s)Arsenic contamination and mitigation
ResumoMetallo-β-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a β-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 ± 0.2, 1.8 ± 0.2, and 1.1 ± 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10- to 1000-fold drops in kcat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pKa values outside the range of pH 5–10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction. Metallo-β-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a β-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 ± 0.2, 1.8 ± 0.2, and 1.1 ± 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10- to 1000-fold drops in kcat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pKa values outside the range of pH 5–10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction. The ability of bacteria to acquire resistance to antibiotics is a serious problem that continues to challenge modern society (1.Neu H.C. Science. 1992; 257: 1064-1073Crossref PubMed Scopus (2303) Google Scholar). Excessive use and often misuse of antibiotics in the clinic and for agricultural purposes has resulted in tremendous selective pressure for antibiotic-resistant bacteria (2.Levy S.B. Sci. Am. 1998; 3: 47-53Google Scholar). These bacteria utilize a variety of methods to become resistant, including modification of cell wall components to prevent antibiotic binding, production of efflux pumps that transport the antibiotic out of the cell, and the production of enzymes that hydrolyze and render the antibiotic ineffective (1.Neu H.C. Science. 1992; 257: 1064-1073Crossref PubMed Scopus (2303) Google Scholar, 2.Levy S.B. Sci. Am. 1998; 3: 47-53Google Scholar). The most common and least expensive effective antibiotics currently used are the β-lactams, such as carbapenems, cephalosporins, and penicillins (3.Kotra L.P. Mobashery S. Arch. Immunol. Ther. Exp. 1999; 47: 211-216PubMed Google Scholar, 4.Bush K. Mobashery S. Mobashery R.A. Resolving the Antibiotic Paradox. Kluwer Academic/Plenum Publishers, New York1998: 71-98Google Scholar). These antibiotics are mechanism-based inhibitors of transpeptidase, a bacterial enzyme required for the production of a strong viable cell wall (5.Eberhardt C. Kuerschner L. Weiss D.S. J. Bacteriol. 2003; 185: 3726-3734Crossref PubMed Scopus (50) Google Scholar, 6.Therrien C. Levesque R.C. FEMS Microbiol. Rev. 2000; 24: 251-262Crossref PubMed Google Scholar, 7.Knox J.R. Moews P.C. Frere J.M. Chem. Biol. 1996; 3: 937-947Abstract Full Text PDF PubMed Scopus (143) Google Scholar). 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Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar, 37.Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spenser J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (292) Google Scholar). The enzyme exists as a homotetramer of ∼118 kDa in solution and in the crystalline state, tightly binding two Zn(II) ions per subunit. The Zn1 site has 3 histidine residues and 1 bridging hydroxide as ligands, and the Zn2 site has 2 histidines, 1 aspartic acid, 1 terminally bound water, and the bridging hydroxide as ligands (see Fig. 1). Efforts to solve the crystal structure of one of the metallo-β-lactamases with a bound substrate molecule have failed, most likely due to the high activity of the enzymes, even in the crystalline state, toward all β-lactam-containing antibiotics (37.Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spenser J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (292) Google Scholar, 38.Carfi A. Paul-Soto R. 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With L1, three key assumptions were made: 1) the bridging hydroxide functions as the nucleophile during catalysis, 2) Zn1 coordinates the β-lactam carbonyl, and 3) Zn2 coordinates the amide nitrogen of the β-lactam ring (37.Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spenser J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (292) Google Scholar). One of the residues identified through computational studies to be catalytically important in L1 is the aspartic acid at position 120 (the standard numbering scheme for class B β-lactamases is utilized herein (20.Galleni M. Lamotte-Brasseur J. Rossolini G.M. Spencer J. Dideberg O. Frere J.M. Antimicrob. Agents Chemother. 2001; 45: 660-663Crossref PubMed Scopus (323) Google Scholar)). From the crystal structure, Asp-120 clearly coordinates Zn2, with its unbound oxygen located directly under the bridging group in the active site (37.Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spenser J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (292) Google Scholar). This is a geometry shared by many dinuclear metal-containing hydrolytic enzymes, including other metallo-β-lactamases and dioxygenases (46.Daiyasu H. Osaka K. Ishino Y. Toh H. FEBS Lett. 2001; 503: 1-6Crossref PubMed Scopus (269) Google Scholar). Therefore, we believe that the findings of this work are applicable beyond L1 from S. maltophilia. In addition to its role as a metal-binding ligand, it has been hypothesized that Asp-120 electrostatically interacts with the bridging hydroxide, properly orienting it for nucleophilic attack on the substrate (37.Ullah J.H. Walsh T.R. Taylor I.A. Emery D.C. Verma C.S. Gamblin S.J. Spenser J. J. Mol. Biol. 1998; 284: 125-136Crossref PubMed Scopus (292) Google Scholar). This work describes our efforts to test this prediction and further our understanding the role of Asp-120 in both metal binding and substrate turnover. To probe the importance of this residue, three mutant enzymes were generated. Asp-120 was changed to a cysteine, an asparagine, and a serine to create D120C, D120N, and D120S, respectively (Fig. 1). Cysteine was substituted to allow for continued binding of Zn2 but eliminate any interaction of the residue with the bridging hydroxide/water. Asparagine was chosen as a chemically different but structurally similar surrogate for aspartic acid, allowing for continued binding of Zn2 and providing a moiety for interaction with the bridging hydroxide/water. Replacement of aspartic acid with serine was intended to remove both the metal-binding ability of the residue at this position and any ability to interact with the bridging hydroxide/water. Escherichia coli strains DH5α and BL21(DE3) were obtained from Invitrogen and Novagen, respectively. Plasmids pET26b and pUC19 were purchased from Novagen. Primers for sequencing and mutagenesis studies were purchased from Integrated DNA Technologies. Deoxynucleotide triphosphates (dNTPs), MgSO4, Thermopol buffer, Deep Vent DNA polymerase, and restrictions enzymes were purchased from Promega or New England Biolabs. Polymerase chain reaction was conducted using a Thermolyne Amplitron II unit. DNA was purified using the Qiagen QIAquick gel extraction kit or Plasmid Purification kit with Qiagen-tip 100 (Midi) columns. Wizard Plus Minipreps were acquired from Promega. Luria-Bertani media in powder form was purchased from Invitrogen. Isopropyl-β-thiogalactoside, Biotech grade, was procured from Anatrace. Phenylmethylsulfonyl fluoride was purchased from Sigma. Protein solutions were concentrated with an Amicon ultrafiltration cell equipped with YM-10 DIAFLO membranes from Amicon, Inc. Dialysis tubing was prepared using Spectra/Por-regenerated cellulose molecular porous membranes with a molecular weight cut-off of 6–8000 g/mol. Q-Sepharose Fast Flow was purchased from Amersham Biosciences. Nitrocefin was purchased from BD Biosciences, and solutions of nitrocefin were filtered through a Fisherbrand 0.45-μm syringe filter. Cefaclor, cefoxitin, and cephalothin were purchased from Sigma; penicillin G and ampicillin were purchased from Fisher. Imipenem, meropenem, and biapenem were generously supplied by Merck, Zeneca Pharmaceuticals, and Lederle (Japan), respectively. All buffers and media were prepared using Barnstead NANOpure ultrapure water. The overexpression plasmid for L1, pUB5832, was digested with NdeI and HindIII, and the resulting ∼900-bp piece was gel-purified and ligated using T4 ligase into pUC19, which was also digested with NdeI and HindIII, to yield the cloning plasmid pL1pUC19. Mutations were introduced into the L1 gene by using the overlap extension method of Ho et al. (47.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar), as described previously (48.Carenbauer, A. L., Garrity, J. A., Periyannan, G., Yates, R. B., and Crowder, M. W. (2002) BMC Biochemistry3:4http://www.biomedcentral.com/content/pdf1/1471-2091-3-4.pdfGoogle Scholar, 49.Crowder M.W. Yang K.W. Carenbauer A.L. Periyannan G. Seifert M.A. Rude N.E. Walsh T.R. J. Biol. Inorg. Chem. 2001; 6: 91-99Crossref PubMed Scopus (26) Google Scholar). The oligonucleotides used for the preparation of the mutants are as follows: D120C forward, CACgCACACgCCTgCCATgCCggACCggTg; D120C reverse, CACCggTCCggCATggCAggCgTgTgCgTg; D120N forward, CACgCACACgCCAACCATgCCggACCggTg; D120N reverse, CACCggTCCggCATggTTggCgTgTgCgTg; D120S forward, CACgCACACgCCAgCCATgCCggACCggTg; and D120S reverse, CACCggTCCggCATggCTggCgTgTgCgTg. The ∼900-bp PCR products were digested with NdeI and HindIII and ligated into pUC19. The DNA sequences were analyzed by the Biosynthesis and Sequencing Facility in the Department of Biological Chemistry at Johns Hopkins University. After confirmation of the sequence, the mutated pL1pUC19 plasmid was digested with NdeI and HindIII, and the 900-bp, mutated L1 gene was gel-purified and ligated into pET26b to create the mutant overexpression plasmids. To test for overexpression of the mutant enzymes, E. coli BL21(DE3)pLysS cells were transformed with the mutated overexpression plasmids, and small scale growth cultures were used (48.Carenbauer, A. L., Garrity, J. A., Periyannan, G., Yates, R. B., and Crowder, M. W. (2002) BMC Biochemistry3:4http://www.biomedcentral.com/content/pdf1/1471-2091-3-4.pdfGoogle Scholar). Large-scale (4 liters) preparations of the L1 mutants were performed as described previously (36.Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar). Protein purity was ascertained by SDS-PAGE. The concentrations of L1 and the mutants were determined by measuring the proteins' absorbances at 280 nm and using the published extinction coefficient of ϵ280 nm = 54,804 m–1·cm–1 (36.Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar) or by using the method of Pace (50.Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Prot. Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3373) Google Scholar). Before metal analyses, the "as isolated" protein samples were dialyzed versus 3× 1 liter of metal-free, 50 mm HEPES, pH 7.5, over 96 h at 4 °C. A Varian Inductively Coupled Plasma Spectrometer with atomic emission spectroscopy detection (ICP-AES) 1The abbreviations used are: ICP-AES, inductively coupled plasmaatomic emission spectroscopy; L1, metallo-β-lactamase from S. maltophilia; MTCN, MES-TRIS-CHES-NaCl buffer; CD, circular dichroism; MES, 4-morpholineethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid. was used to determine the metal content of multiple preparations of wild-type L1 and L1 mutants. Calibration curves were based on four standards and had correlation coefficient limits of at least 0.9950. The final dialysis buffer was used as a blank. The emission line of 213.856 nm is the most intense for zinc and was used to determine the zinc content in the samples. The errors in metal content data reflect the standard deviation (σn–1) of multiple enzyme preparations. A second analysis of metal content was preformed on enzyme samples that were incubated for 1 h, on ice, in buffer containing a final concentration of 100 μm ZnCl2. These "metal-saturated" samples were then dialyzed versus 2× 1-liter metal-free buffer for a total of 4 h, and metal content was analyzed by ICP-AES as described above. Circular dichroism samples were prepared by dialyzing the purified enzyme samples versus 3 × 2 liters of 5 mm phosphate buffer, pH 7.0, over 6 h. The samples were diluted with final dialysis buffer to ∼75 μg/ml. A JASCO J-810 CD spectropolarimeter operating at 25 °C was used to collect CD spectra. Assays were conducted at 25 °C in 50 mm cacodylate buffer, pH 7.0, containing 100 μm ZnCl2 on an HP 5480A diode array UV-visible spectrophotometer. The changes in molar absorptivities (Δϵ) used to quantitate products were (in m–1 cm–1): nitrocefin, Δϵ485 = 17,420; cephalothin, Δϵ265 = –8,790; cefoxitin, Δϵ265 = –7,000; cefaclor, Δϵ280 = –6,410; imipenem, Δϵ300 = –9,000; meropenem, Δϵ293 = –7,600; biapenem, Δϵ293 = –8,630; ampicillin, Δϵ235 = –809; and penicillin G, Δϵ235 = –936. When possible, substrate concentrations were varied between 0.1 and 10 times the Km value, and changes in absorbance (ΔA) versus time data were measured for a period of 60 s for each substrate concentration. In kinetic studies using substrates with low Km values (cefoxitin, nitrocefin, and cephalothin) or with small Δϵ values (penicillin and ampicillin), substrate concentrations were varied between ∼Km and 10 times Km, and as much of the linear portion of the ΔA versus time data as possible was used to determine the velocity. Steady-state kinetics constants, Km and kcat, were determined by fitting initial velocity versus substrate concentration data directly to the Michaelis equation using Igor Pro (36.Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar). The reported errors reflect fitting uncertainties. All steady-state kinetic studies were performed in triplicate with recombinant L1 from at least three different enzyme preparations. pH dependence studies were performed as described above but using a buffer system containing 25 mm MES, 50 mm Tris, 25 mm CHES, 10 mm NaCl, and 100 μm ZnCl2 (MTCN). Buffers for each pH tested were made from a common 10× stock of MTCN buffer. The pH of each was then adjusted to the desired value using either 6 m HCl or 10 m NaOH, and the appropriate volume of aqueous ZnCl2 was added to reach a concentration of 100 μm. Km and kcat were determined as described above, and log plots of those values versus pH were generated using Igor Pro. Steady-state kinetic assays were conducted at 25 °C in 50 mm cacodylate buffer, pH 7.0, containing 100 μm ZnCl2 and ranging in D2O concentrations from 0 to 100%, on a HP 5480A diode array UV-visible spectrophotometer. Steady-state kinetics constants, Km and kcat, were determined by fitting initial velocity versus substrate concentration data directly to the Michaelis equation using Igor Pro (36.Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar). Plots of kcat versus %D2O were generated using Igor Pro. The reported errors reflect fitting uncertainties. Rapid-scanning visible spectra of nitrocefin hydrolysis by L1, and the L1 mutants were collected on a Applied Photophysics SX.18MV stopped-flow spectrophotometer equipped with an Applied Photophysics PD.1 photodiode array detector and a 2-mm path length optical cell. The wild-type L1 experiment consisted of 25 μm enzyme and 5 μm nitrocefin in 50 mm cacodylate buffer, pH 7.0, containing 100 μm ZnCl2, the reaction temperature was thermostated at 25 °C, and the spectra were collected between 300 and 725 nm. Data from at least three experiments were collected and averaged. Absorbance data were converted to concentration data as described previously by McMannus and Crowder (51.McMannus-Munoz S. Crowder M.W. Biochemistry. 1999; 38: 1547-1553Crossref PubMed Scopus (96) Google Scholar). Due to weaker binding and slower turnover of substrate with the L1 mutants, enzyme concentrations of 50 μm were used with 5 μm nitrocefin utilizing the same buffer system and experimental conditions as in the wild-type L1 experiment. Stopped-flow fluorescence studies of nitrocefin hydrolysis by L1 were performed on an Applied Photophysics SX.18MV spectrophotometer, using an excitation wavelength of 295 nm and a WG320 nm cut-off filter on the photomultiplier. These experiments were conducted at 10 °C using the same buffer as in the rapid-scanning visible studies. Fluorescence data were fitted to kobs = {(kf [S])/KS + [S])} + kr as described previously (52.Spencer J. Clark A.R. Walsh T.R. J. Biol. Chem. 2001; 276: 33638-33644Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) or to kobs = kf[S] + kr by using CurveFit version 1.0. Wild-type L1, D120C, D120N, and D120S were overexpressed in E. coli and purified as previously described (36.Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar). This procedure produced an average of 50–60 mg of >95% pure, active protein per 4 liters of growth culture. Circular dichroism spectra were collected on samples of wild-type, and each of the mutants to ensure the proteins produced using the pET26b overexpression system had the correct secondary structure. The CD spectra (data not shown) of wild-type L1 and the mutants were identical. Metal analyses on multiple preparations of wild-type L1 demonstrated that the enzyme binds 1.9 ± 0.2 Zn(II) ions per monomer (Table I), in agreement with previous results (36.Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar). Metal analysis on multiple preparations of D120C, D120N, and D120S showed 1.6 ± 0.2, 1.8 ± 0.2, and 1.1 ± 0.2 Zn(II) ions per monomer, respectively.Table IMetal analysis and KS and kH/kD values with nitrocefin of wild-type L1 and L1 mutantsEnzyme Zn(II) contentKS with nitrocefinSolvent isotope kH/kDmol Zn(II)/mol monomerμmWt1.9 ± 0.238 ± 52.08 ± 0.03D120C1.6 ± 0.237 ± 61.47 ± 0.05D120N1.8 ± 0.297 ± 145.36 ± 0.22D120S1.1 ± 0.2Unable to determine1.87 ± 0.06 Open table in a new tab Steady-state kinetic constants Km and kcat were determined for wild-type L1 and each of the mutants with nine substrates. These values are presented in Tables II, III, IV. When using nitrocefin as substrate and 50 mm cacodylate, pH 7.0, as the buffer, wild-type L1 exhibited a kcat value of 38 ± 1s–1 and a Km value of 12 ± 1 μm. The inclusion of 100 μm ZnCl2 in the assay buffer resulted in slightly lower values of Km and higher values for kcat (36.Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar). The inclusion of higher concentrations of Zn(II) did not further affect the steady-state kinetic constants. Four cephalosporins (cefaclor, cefoxitin, cephalothin, and nitrocefin), three carbapenems (biapenem, imipenem, and meropenem), and two penicillins (penicillin G and ampicillin) were utilized as repres
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