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• W2084421339 abstract "A structural feature shared by the metallo-β-lactamases is a flexible loop of amino acids that extends over their active sites and that has been proposed to move during the catalytic cycle of the enzymes, clamping down on substrate. To probe the movement of this loop (residues 152–164), a site-directed mutant of metallo-β-lactamase L1 was engineered that contained a Trp residue on the loop to serve as a fluorescent probe. It was necessary first, however, to evaluate the contribution of each native Trp residue to the fluorescence changes observed during the catalytic cycle of wild-type L1. Five site-directed mutants of L1 (W39F, W53F, W204F, W206F, and W269F) were prepared and characterized using metal analyses, CD spectroscopy, steady-state kinetics, stopped-flow fluorescence, and fluorescence titrations. All mutants retained the wild-type tertiary structure and bound Zn(II) at levels comparable with wild type and exhibited only slight (<10-fold) decreases in kcat values as compared with wild-type L1 for all substrates tested. Fluorescence studies revealed a single mutant, W39F, to be void of the fluorescence changes observed with wild-type L1 during substrate binding and catalysis. Using W39F as a template, a Trp residue was added to the flexile loop over the active site of L1, to generate the double mutant, W39F/D160W. This double mutant retained all the structural and kinetic characteristics of wild-type L1. Stopped-flow fluorescence and rapid-scanning UV-visible studies revealed the motion of the loop (kobs = 27 ± 2 s–1) to be similar to the formation rate of a reaction intermediate (kobs = 25 ± 2 s–1). A structural feature shared by the metallo-β-lactamases is a flexible loop of amino acids that extends over their active sites and that has been proposed to move during the catalytic cycle of the enzymes, clamping down on substrate. To probe the movement of this loop (residues 152–164), a site-directed mutant of metallo-β-lactamase L1 was engineered that contained a Trp residue on the loop to serve as a fluorescent probe. It was necessary first, however, to evaluate the contribution of each native Trp residue to the fluorescence changes observed during the catalytic cycle of wild-type L1. Five site-directed mutants of L1 (W39F, W53F, W204F, W206F, and W269F) were prepared and characterized using metal analyses, CD spectroscopy, steady-state kinetics, stopped-flow fluorescence, and fluorescence titrations. All mutants retained the wild-type tertiary structure and bound Zn(II) at levels comparable with wild type and exhibited only slight (<10-fold) decreases in kcat values as compared with wild-type L1 for all substrates tested. Fluorescence studies revealed a single mutant, W39F, to be void of the fluorescence changes observed with wild-type L1 during substrate binding and catalysis. Using W39F as a template, a Trp residue was added to the flexile loop over the active site of L1, to generate the double mutant, W39F/D160W. This double mutant retained all the structural and kinetic characteristics of wild-type L1. Stopped-flow fluorescence and rapid-scanning UV-visible studies revealed the motion of the loop (kobs = 27 ± 2 s–1) to be similar to the formation rate of a reaction intermediate (kobs = 25 ± 2 s–1). The ability of bacteria to acquire resistance to antibiotics is a serious problem that continues to challenge modern society (1Neu H.C. Science. 1992; 257: 1064-1073Crossref PubMed Scopus (2373) Google Scholar). Excessive and often misuse of antibiotics in the clinic and for agricultural purposes has resulted in tremendous selective pressure for antibiotic-resistant bacteria (2Levy 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, expression of efflux pumps that transport the antibiotic out of the cell, and the production of enzymes that hydrolyze and render antibiotics ineffective (1Neu H.C. Science. 1992; 257: 1064-1073Crossref PubMed Scopus (2373) Google Scholar, 2Levy S.B. Sci. Am. 1998; 3: 47-53Google Scholar). The most common, least expensive, and effective antibiotics currently used are the β-lactams, such as carbapenems, cephalosporins, and penicillins (3Kotra L.P. Mobashery S. Arch. Immunol. Ther. Exp. 1999; 47: 211-216PubMed Google Scholar, 4Bush 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 (5Bush K. Antimicrob. Agents Chemother. 1989; 33: 264-270Crossref PubMed Scopus (169) Google Scholar, 6Bush K. Antimicrob. Agents Chemother. 1989; 33: 271-276Crossref PubMed Scopus (130) Google Scholar). In response to their widespread use, an increasing number of bacterial strains have acquired the ability to produce β-lactamases, enzymes that hydrolyze and render β-lactam antibiotics ineffective. There are more than 300 distinct β-lactamases known, and Bush has classified these into four distinct groups based on their molecular properties (5Bush K. Antimicrob. Agents Chemother. 1989; 33: 264-270Crossref PubMed Scopus (169) Google Scholar, 6Bush K. Antimicrob. Agents Chemother. 1989; 33: 271-276Crossref PubMed Scopus (130) Google Scholar). One of the more troubling of these is group 3, the metallo-β-lactamases, which are Zn(II)-dependent enzymes that hydrolyze nearly all known β-lactams and for which there are no clinically useful inhibitors (7Siemann S. Evanoff D.P. Marrone L. Clarke A.J. Viswanatha T. Dmitrienko G.I. Antimicrob. Agents Chemother. 2002; 46: 2450-2457Crossref PubMed Scopus (99) Google Scholar, 8Toney J.H. Hammond G.G. Fitzgerald P.M.D. 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, 9Lee N.L.S. Yuen K.Y. Kumana C.R. J. Am. Med. Assoc. 2001; 285: 386-388Crossref PubMed Scopus (21) Google Scholar, 10Bounaga S. Galleni M. Laws A.P. Page M.I. Bioorg. Med. Chem. 2001; 9: 503-510Crossref PubMed Scopus (56) Google Scholar, 11Hammond G.G. Huber J.L. Greenlee M.L. Laub J.B. Young K. Silver L.L. Balkovec J.M. Pryor K.D. Wu J.K. Leiting B. Pompliano D.L. Toney J.H. FEMS Microbiol. Lett. 1999; 459: 289-296Google Scholar, 12Williams J.D. Int. J. Antimicrob. Agents. 1999; 12: 3-7Crossref PubMed Scopus (51) Google Scholar, 13Walter M.W. Valladares M.H. Adlington R.M. Amicosante G. Baldwin J.E. Frere J.M. Galleni M. Rossolini G.M. Schofield C.J. Bioorg. Chem. 1999; 27: 35-40Crossref Scopus (47) Google Scholar, 14Crowder M.W. Walsh T.R. Research Signpost. 1999; 3: 105-132Google Scholar). To date, there are no reports of a metallo-β-lactamase being isolated from a major pathogen (15Bush K. Clin. Infect. Dis. 2001; 32: 1085-1090Crossref PubMed Scopus (356) Google Scholar, 16Rice L.B. Bonomo R.A. Drug Resist. Updat. 2000; 3: 178-189Crossref PubMed Scopus (55) Google Scholar); however, these enzymes are produced by a variety of minor clinical pathogens such as Bacteroides fragilis, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, and the continued extensive use of β-lactam containing antibiotics will inevitably result in the production of a metallo-β-lactamase by a major pathogen (2Levy S.B. Sci. Am. 1998; 3: 47-53Google Scholar). There is significant diversity within the metallo-β-lactamases, and Bush, based on their amino acid sequence identities and substrate affinities, has further divided them into three subgroups (17Bush K. Clin. Infect. Dis. 1998; 27: 48-53Crossref PubMed Scopus (204) Google Scholar). A similar grouping scheme based on structural properties has also been offered (18Galleni 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). The diversity of these subgroups is best exemplified by their vastly differing efficacies toward non-clinical inhibitors; these differences lead to the prediction that finding a single inhibitor for all metallo-β-lactamases may not be possible (14Crowder M.W. Walsh T.R. Research Signpost. 1999; 3: 105-132Google Scholar, 19Payne D.J. J. Med. Microbiol. 1993; 39: 93-99Crossref PubMed Scopus (115) Google Scholar, 20Payne D.J. Bateson J.H. Gasson B.C. Proctor D. Khushi T. Farmer T.H. Tolson D.A. Bell D. Skett P.W. Marshall A.C. Reid R. Ghosez L. Combret Y. Marchand-Brynaert J. Antimicrob. Agents Chemother. 1997; 41: 135-140Crossref PubMed Google Scholar, 21Payne D.J. Bateson J.H. Gasson B.C. Khushi T. Proctor D. Pearson S.C. Reid R. FEMS Microbiol. Lett. 1997; 157: 171-175Crossref PubMed Scopus (61) Google Scholar, 22Yang K.W. Arch. Biochem. Biophys. 1999; 368: 1-6Crossref PubMed Scopus (27) Google Scholar, 23Felici A. Perilli M. Segatore B. Franceschini N. Setacci D. Oratore A. Stefani S. Galleni M. Amicosante G. Antimicrob. Agents Chemother. 1995; 39: 1300-1305Crossref PubMed Scopus (21) Google Scholar, 24Prosperi-Meys C. Llabres G. de Seny D. Soto R.P. Valladares M.H. Laraki N. Frere J.M. Galleni M. FEBS Lett. 1999; 443: 109-111Crossref PubMed Scopus (37) Google Scholar, 25Nagano R. Adachi Y. Imamura H. Yamada K. Hashizume T. Morishima H. Antimicrob. Agents Chemother. 1999; 43: 2497-2503Crossref PubMed Google Scholar, 26Toney J.H. Cleary K.A. Hammond G.G. Yuan X. May W.J. Hutchins S.M. Ashton W.T. Vanderwall D.E. Bioorg. Med. Chem. Lett. 1999; 9: 2741-2746Crossref PubMed Scopus (50) Google Scholar, 27Arakawa Y. Shibata N. Shibayama K. Kurokawa H. Yagi T. Fujiwara H. Goto M. J. Clin. Microbiol. 2000; 38: 40-43PubMed Google Scholar). To address this problem, we are currently characterizing a representative enzyme from each of the metallo-β-lactamase subgroups with the goal of identifying common structural and mechanistic similarities that can be targeted for the generation of clinically useful inhibitors. This work describes our efforts on characterizing metallo-β-lactamase L1 1The abbreviations used are: L1, metallo-β-lactamase from S. maltophilia; apo, metal-free; WT, wild type; PM, photomultiplier. from S. maltophilia. S. maltophilia is an important pathogen in nosocomial infections of immunocompromised patients suffering from cancer, cystic fibrosis, drug addition, and AIDS, and in patients with organ transplants and on dialysis (28Khardori N. Elting L. Wong E. Schable B. Bodey G.P. Rev. Infect. Dis. 1990; 12: 997-1003Crossref PubMed Scopus (177) Google Scholar, 29Muder R.R. Yu V.L. Dummer J.S. Vinson C. Lumish R.M. Arch. Intern. Med. 1987; 147: 1672-1674Crossref PubMed Scopus (96) Google Scholar, 30Villarino M.E. Stevens L.E. Schable B. Mayers G. Miller J.M. Burke J.P. Jarvis W.R. Infect. Control. Hosp. Epidemiol. 1992; 13: 201-206Crossref PubMed Scopus (90) Google Scholar). This organism is inherently resistant to most antibiotics due to its low outer membrane permeability (31Mett H. Rosta S. Schacher B. Frei R. Rev. Infect. Dis. 1988; 10: 765-819Crossref PubMed Scopus (66) Google Scholar) and to β-lactams, due to the production of a chromosomally expressed group 2e β-lactamase (L2) and a group 3c β-lactamase (L1) (32Walsh T.R. Payne D.J. Neville T. Tolson D. MacGowan A.P. Bennett P.M. Antimicrob. Agents Chemother. 1997; 41: 1460-1462Crossref PubMed Google Scholar, 33Walsh T.R. Hall L. Assinder S.J. Nichols W.W. Cartwright S.J. MacGowan A.P. Bennett P.M. Biochim. Biophys. Acta. 1994; 1218: 199-201Crossref PubMed Scopus (148) Google Scholar). L1 has been cloned, overexpressed, and partially characterized by kinetic and crystallographic studies (34Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar, 35Ullah 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 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 three histidine residues and one bridging hydroxide as ligands, and the Zn2 site has two histidines, one aspartic acid, one terminally bound water, and the bridging hydroxide as ligands (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 (35Ullah 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, 36Carfi A. Paul-Soto R. Martin L. Petillot Y. Frere J.M. Dideberg O. Acta Crystallogr. Sect. D. 1997; 53: 485-487Crossref PubMed Scopus (13) Google Scholar). Therefore, computational studies have been used extensively to study substrate binding, the role of the Zn(II) ions in catalysis, the protonation state of the active site, protein dynamics, and inhibitor binding (35Ullah 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, 37Bernstein N.J. Pratt R.F. Biochemistry. 1999; 38: 10499-10510Crossref PubMed Scopus (35) Google Scholar, 38Diaz N. Suarez D. Merz K.M. J. Am. Chem. Soc. 2000; 122: 4197-4208Crossref Scopus (87) Google Scholar, 39Caselli E. Powers R.A. Blasczcak L.C. Wu C.Y.E. Prati F. Shoichet B.K. Chem. Biol. 2000; 49: 1-16Google Scholar, 40Suarez D. Merz K.M. J. Am. Chem. Soc. 2001; 123: 3759-3770Crossref PubMed Scopus (76) Google Scholar, 41Concha N.O. Rasmussen B.A. Bush K. Herzberg O. Structure. 1996; 4: 823-836Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 42Salsbury F.R. Crowley M.F. Brooks C.L. Proteins Struct. Funct. Genet. 2001; 44: 448-459Crossref PubMed Scopus (47) Google Scholar). The crystal structure of L1 reveals a series of amino acids that form a flexible loop, a structural feature shared with other metallo-β-lactamases, which extends over its active site (residues 152–164). It has been suggested that this loop plays an important role during the catalytic cycle of the enzyme, clamping down on substrate, perhaps inducing strain to assist in hydrolysis or helping to stabilize a reaction intermediate (35Ullah 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). To monitor the motion of this loop in L1, we proposed to introduce a Trp residue on the loop to act as a fluorescent probe. However, this simple approach was complicated by the presence of five Trp residues in wild-type L1: Trp-39, Trp-53, Trp-204, Trp-206, and Trp-269 at 6.2, 25.2, 18.8, 39.8, and 19.6 Å from the active site, respectively (Fig. 1). Spencer and co-workers (43Spencer J. Clark A.R. Walsh T.R. J. Biol. Chem. 2001; 276: 33638-33644Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) had demonstrated previously that the quenching of fluorescence and subsequent return to original values in wild-type L1 could be used to monitor substrate binding and catalysis (43Spencer J. Clark A.R. Walsh T.R. J. Biol. Chem. 2001; 276: 33638-33644Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). It was, therefore, necessary to determine whether these fluorescence changes could be pinpointed to a single Trp residue. To this end, five mutant enzymes were generated in which each Trp residue, in turn, was changed to a Phe residue, resulting in five mutant enzymes: W39F, W53F, W204F, W206F, and W269F. Because of its proximity to the active site and predicted edge/face interaction with His-263, a metal binding ligand (35Ullah 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), we hypothesized that if indeed the observed fluorescence changes were due to a single Trp residue, it was likely Trp-39. If this strategy was successful a second mutation could be introduced into W39F, replacing Asp-160, a residue located on the flexible loop, with a Trp to act as a fluorescent probe, resulting in the double mutant W39F/D160W. This work describes our efforts to determine whether we could generate an enzyme free of fluorescence changes during substrate binding and catalysis and subsequently create a double mutant that could be used to monitor the dynamics of the flexible loop in L1. Escherichia coli strains DH5α and BL21(DE3) were obtained from Invitrogen and Novagen, respectively. The plasmid pET26b was purchased from Novagen. Primers for sequencing and mutagenesis studies were purchased from Integrated DNA Technologies. QuikChange™ mutagensis kit was purchased from Stratagene. 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. 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 (44Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY1989: E39Google Scholar). Q-Sepharose Fast Flow was purchased from Amersham Biosciences. Nitrocefin was purchased from BD Biosciences, and solutions of nitrocefin were filtered through a Fisher-brand 0.45-μm syringe filter (34Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar). Cephalothin and penicillin G were purchased from Sigma and Fisher, respectively. Meropenem was generously supplied by Zeneca Pharmaceuticals. All buffers and media were prepared using Barnstead NANOpure ultrapure water. The overexpression plasmid for L1, pUB5832, was mutated using the Stratagene QuikChange mutagenesis kit. The following oligonucleotides were used for the preparation of the mutants: W39Ffor, CACCgTggACgCCTCgTTCCTgCAgCCgATggCACCgC; W39Frev, gCggTgCCATCggCTgCAggAACgAggCgTCCACggTg; W53Ffor, CTgCAgATCgCCgACCACACCTTCCAgATCggCACCgAggACCTg; W53Frev, CAggTCCTCggTgCCgATCTggAAggTgTggTCggCgATCTgCAg; W204Ffor, CCCCgggCAgCACCgCgTTCACCTggACCgATACCCg; W204Frev, CgggTATCggTCCAggTgAACgCggTgCTgCCCgggg; W206Ffor, ggCAgCACCgCgTggACCTTCACCgATACCCgCAATggC; W206Frev, gCCATTgCgggTATCggTgAAggTCCACgCggTgCTgCC; W269Ffor, CATCCgggTgCCAgCAACTTCgACTACgCCgCgggTgCC; W269Frev, ggCACCCgCggCgTAgTCgAAgTTgCTggCACCCggATg, W39F/D160Wfor, CTgCACTTCggCTggggCATCACCTAC; W39F/D160Wrev, gTAggTgATgCCCCAgCCgAAgTgCAg. The DNA sequences were analyzed using a PerkinElmer Life Sciences ABI 5300 Genetic Analyzer. 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 (45Carenbauer A.L. Garrity J.A. Periyannan G. Yates R.B. Crowder M.W. BMC Biochem. 2002; 3: 4-10Crossref PubMed Scopus (40) Google Scholar). Large scale (4-liter) preparations of the L1 mutants were performed as described previously (34Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar), with the exception of W53F and W206F. With these two mutants we observed lower levels of isolatable protein using the established protocol and, therefore, reduced the temperature from 37 to 25 °C during cell growth and induction and subsequently were able to obtain amounts of isolatable protein comparable with those of wild-type L1. Protein purity was ascertained by SDS-PAGE. Extinction coefficients for the mutants were determined utilizing the BCA protein assay kit purchased from Fisher, with wild-type L1 used to create the calibration curve. The concentrations of L1 and the mutants were determined by measuring the protein absorbances at 280 nm and using the published extinction coefficient of ϵ280 nm = 54,606 m–1·cm–1 for wild-type L1 (34Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar) and the experimentally determined extinction coefficients for the mutants. Analysis of metal content was performed on enzyme samples that were incubated for 1 h on ice in 50 mm HEPES, pH 7.5, containing a final concentration of 100 μm ZnCl2. Weakly bound metal was removed by dialysis versus 2 × 1 liter of metal-free (chelexed) 50 mm HEPES, pH 7.5. A Varian Liberty 2 inductively coupled plasma spectrometer with atomic emission spectroscopy detection 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 S.D. (σn–1) of multiple enzyme preparations. 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 (34Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar, 45Carenbauer A.L. Garrity J.A. Periyannan G. Yates R.B. Crowder M.W. BMC Biochem. 2002; 3: 4-10Crossref PubMed Scopus (40) Google Scholar). Steady-state kinetic assays were conducted at 25 °C in 50 mm cacodylate buffer, pH 7.0, containing 100 μm ZnCl2 on a HP 5480A diode array UV-visible spectrophotometer. The changes in molar absorptivities (Δϵ) used to follow the reactions were (in m–1cm–1): nitrocefin, Δϵ485 = 17,420; cephalothin, Δϵ265 = –8,790; meropenem, Δϵ293 = –7,600; penicillin G, Δϵ235 = –936. When possible, substrate concentrations were varied between 0.1 to 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. Steady-state kinetic constants, Km and kcat, were determined by fitting initial velocity versus substrate concentration data directly to the Michaelis equation using Igor Pro (34Crowder 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 and L1 mutants from at least three different enzyme preparations. Fluorescence spectra of 2 μm wild-type L1 and L1 mutant samples were obtained at 25 °C using an excitation wavelength of 295 nm on a PerkinElmer Life Sciences LS 55 luminescence spectrometer. Apo (metal-free) enzymes for fluorescence titrations were prepared by dialysis of samples versus 5× 1 liter of 50 mm HEPES, pH 7.0, containing 10 mm phenanthroline followed by dialysis versus 5× 1 liters of metal-free (chelexed) 50 mm HEPES, pH 7.0, and metal analyses were used to verify that the enzymes were metal-free. Data from fluorescence titrations were fitted to f(x)=Finitial+(ΔF/[E])(0.5)(KD+[E]+x)−((KD+[E]+x)exp2−4xFinitial)(Eq. 1) where Finitial is the initial fluorescence reading for each sample, [E] is the apoenzyme concentration, KD is the dissociation constant, f(x) is fluorescence reading, and x is concentration (m) (46Stewart J.D. Roberts V.A. Thomas N.R. Getzoff E.D. Benkovic S.J. Biochemistry. 1994; 33: 1994-2003Crossref PubMed Scopus (65) Google Scholar). Stopped-flow fluorescence studies of nitrocefin hydrolysis by wild-type L1 and L1 mutants 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 2 and 25 °C depending on the enzyme studied using the same buffer as in the rapid-scanning visible studies. Rapid-scanning UV-visible studies were performed under identical conditions using the above-mentioned instrument and utilizing an Applied Photophysics diode array detector. Wild-type L1 and the L1 mutants were overexpressed in E. coli and purified as previously described (34Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar), with the changes for W53F and W206F as noted under “Experimental Procedures.” 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 L1 and each of the mutants to ensure the proteins produced using the pET26b overexpression system had the correct secondary structure. The CD spectra of wild-type L1 and the mutants were similar and showed an intense, broad feature at 190 nm and a smaller feature at 215 nm (data not shown). These features are consistent with a sample with significant α/β content (35Ullah 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). Analyses of the CD spectra were performed on-line using DICHROWEB utilizing an algorithm called CDSSTR (www.cryst.bbk.ac.uk/cdweb/html/home.html) (47Lobley A. Whitmore L. Wallace B.A. Bioinformatics. 2002; 18: 211-212Crossref PubMed Scopus (645) Google Scholar, 48Lobley A. Wallace B.A. Biophys. J. 2001; 80: 1570Google Scholar). Results are shown in Table I. The CD spectra demonstrate no significant changes in the amount of unordered content and only minor differences in α/β content that can be attributed to fitting errors using the CDSSTR program. Metal analyses on multiple preparations of wild-type L1 and the mutants demonstrated that the wild-type L1 and W39F, W53F, W204F, W206F, W269F, and W39F/D160W mutants bind 1.9 ± 0.2, 1.9 ± 0.2, 1.6 ± 0.3, 1.8 ± 0.2, 1.5 ± 0.3, 2.0 ± 0.1, and 1.9 ± 0.2 Zn(II) eq per monomer (Table I). Within the limits of error, the metal analyses demonstrate that the single point mutations did not result in significant changes in metal binding ability of the enzymes.Table IAnalysis of CD spectra using CDSSTR using a 43-protein reference set optimized for 190–240 nmα-Helixβ-Sheetβ-TurnUnorderedMetal analysisϵ%%%%mol of Zn(II)/mol of monomerm-1·cm-1WT L1311821301.9 ± 0.254,606W39F292020311.9 ± 0.241,256W53F182426321.6 ± 0.346,387W204F202424321.8 ± 0.241,910W206F182524331.5 ± 0.336,969W269F302020302.0 ± 0.154,588W39F/D160W301921301.9 ± 0.254,600 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 four substrates. These values are presented in Table II. Cephalothin and nitrocefin, meropenem, and penicillin G were utilized as representatives of the three major classes of β-lactam-containing antibiotics, cephalosporins, carbapenems, and penicillins, respectively. The L1 preference for penicillins and carbapenems over cephalosporins, as exemplified by the kcat values, is in agreement with previous studies (34Crowder M.W. Walsh T.R. Banovic L. Pettit M. Spencer J. Antimicrob. Agents Chemother. 1998; 42: 921-926Crossref PubMed Google Scholar) and supports the L1 placement in the β-lactamase Bc family (17Bush K. Clin. Infect. Dis. 1998; 27: 48-53Crossref PubMed Scopus (204) Google Scholar). For all substrates tested, the mutant enzymes exhibited slightly reduced kcat values (<10-fold) as compared with wild-type L1. Because Km values are often used as a first approximation of substrate binding (49Segel I.H. Enzyme Kinetics. John Wiley and Sons, Inc., New York1993: 44-53Google Scholar), the Km values exhibited by the mutants were compared with those of wild-type L1. All five mutants, with the exception of W39F and W39F/D160W, exhibited similar Km values to wild-type L1. Km values for W39F were typically 4-fold greater than those of wild-type L1, whereas those of the double mutant approached and in one case (cephalothin) exceeded 10-fold that of wild-type L1. Because changes of 10-fold or greater are the standard to indicate significant differences (50Fersht A. Enzyme Structure and Mechanism. 2nd Ed. W. H. Freeman and Co., New York1985: 420-455Google Scholar), steady-state kinetics indicate that only W39F and W39F/D160W approach this standard. However, both enzymes retained enough activity to be suitable for the purpose of this study.Table IISteady-state kinetics constants for wild-type L1 and L1 mutantsNitrocefinCephalothinMeropenemPenicillin GkcatKmkcat/KmkcatKmkcat/KmkcatKmkcat/KmkcatKmkcat/Kms-1μms-1μms-1μms-1μmWT L141 ± 14.1 ± 1.01082 ± 58.9 ± 1.59.2157 ± 915 ± 410.5600 ± 10038 ± 215.8W39F38 ± 418 ± 52.133 ± 233 ± 71.033 ± 29 ± 23.796 ± 4125 ± 180.77W53F36 ± 13.9 ± 0.59.253 ± 14.9 ± 0.510.876 ± 312 ± 26.3402 ± 724 ± 316.8W204F30 ± 13.3 ± 0.29.146 ± 15.4 ± 0.78.566 ± 210 ± 26.6397 ± 735 ± 411.3W206F36 ± 14.0 ± 0.59.049 ± 214 ± 23.564 ± 211 ± 15.8407 ± 1231 ± 513.1W269F21 ± 12.1 ± 0.31055 ± 16.4 ± 0.78.634 ± 18 ± 14.3376 ± 1234 ± 611.1W39F/D160W39 ± 323 ± 41.723 ± 195 ± 110.2417 ± 16 ± 12.8161 ± 5356 ± 310.45 Open table in a new tab Initial fluorescence scans of wild-type L1 and the L1 mutants (Fig. 2) revealed that W39F and W206F exhibited decrease" @default.
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• W2084421339 title "Probing the Dynamics of a Mobile Loop above the Active Site of L1, a Metallo-β-lactamase from Stenotrophomonas maltophilia, via Site-directed Mutagenesis and Stopped-flow Fluorescence Spectroscopy" @default.
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• W2084421339 doi "https://doi.org/10.1074/jbc.m406826200" @default.
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