Inhibition of the Broad Spectrum Nonmetallocarbapenamase of Class A (NMC-A) β-Lactamase from Enterobacter cloacae by Monocyclic β-Lactams
1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês
10.1074/jbc.274.36.25260
ISSN1083-351X
AutoresLionel Mourey, Lakshmi P. Kotra, John R. Bellettini, Alexey Bulychev, E O’Brien, Marvin J. Miller, Shahriar Mobashery, Jean‐Pierre Samama,
Tópico(s)Antibiotics Pharmacokinetics and Efficacy
Resumoβ-Lactamases hydrolyze β-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to β-lactam antibiotics. The class A β-lactamases form the largest group. A novel class A β-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) β-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A β-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 Å resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented. β-Lactamases hydrolyze β-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to β-lactam antibiotics. The class A β-lactamases form the largest group. A novel class A β-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) β-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A β-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 Å resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented. nonmetallocarbapenamase of class A; 4-morpholineethanesulfonate β-Lactamases are the primary cause of resistance to β-lactam antibiotics (1Bush K. Mobashery S. Rosen B.P. Mobashery S. Resolving the Antibiotic Paradox: Progress in Understanding Drug Resistance and Development of New Antibiotics. Plenum Press, New York1998: 71-98Google Scholar). These enzymes hydrolyze the β-lactam moiety of β-lactam antibiotics, and by so-doing, render them inactive. There are four classes of these enzymes, of which the class A is the largest group (1Bush K. Mobashery S. Rosen B.P. Mobashery S. Resolving the Antibiotic Paradox: Progress in Understanding Drug Resistance and Development of New Antibiotics. Plenum Press, New York1998: 71-98Google Scholar). The active-site serine in the class A β-lactamases undergoes acylation by the substrate and the acyl-enzyme intermediate is subsequently hydrolyzed to give substrate turnover (1Bush K. Mobashery S. Rosen B.P. Mobashery S. Resolving the Antibiotic Paradox: Progress in Understanding Drug Resistance and Development of New Antibiotics. Plenum Press, New York1998: 71-98Google Scholar,2Matagne A. Lamotte-Brasseur J. Frère J.-M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (318) Google Scholar). Class A enzymes perform this task with their preferred substrates, penicillins, at the diffusion limit (3Hardy L.W. Kirsch J.F. Arch. Biochem. Biophys. 1989; 268: 338-348Crossref PubMed Scopus (4) Google Scholar). Many of the “parental” β-lactamases of class A, such as the TEM-1 β-lactamase, have undergone mutations that impart to them an increase in the breadth of their substrate profile (1Bush K. Mobashery S. Rosen B.P. Mobashery S. Resolving the Antibiotic Paradox: Progress in Understanding Drug Resistance and Development of New Antibiotics. Plenum Press, New York1998: 71-98Google Scholar), as well as the ability to avoid being inhibited by the known clinical inhibitors. This is currently a serious clinical challenge.One new class A β-lactamase, designated NMC-A1 (4Nordmann P. Mariotte S. Naas T. Labia R. Nicolas M.H. Antimicrob. Agents Chemother. 1993; 37: 939-946Crossref PubMed Scopus (156) Google Scholar), and the highly homologous Sme-1 (5Naas T. Vandel L. Sougakoff W. Livermore D.M. Nordmann P. Antimicrob. Agents Chemother. 1994; 38: 1262-1270Crossref PubMed Scopus (180) Google Scholar) and IMI-1 (6Rasmussen B.A. Bush K. Keeney D. Yang Y. Hare R. O'Gara C. Medeiros A.A. Antimicrob. Agents Chemother. 1996; 40: 2080-2086Crossref PubMed Google Scholar) β-lactamases, enjoy an unusually broad substrate profile, which includes penicillins, cephalosporins, and carbapenems (4Nordmann P. Mariotte S. Naas T. Labia R. Nicolas M.H. Antimicrob. Agents Chemother. 1993; 37: 939-946Crossref PubMed Scopus (156) Google Scholar, 7Rasmussen B.A. Bush K. Antimicrob. Agents Chemother. 1997; 41: 223-232Crossref PubMed Google Scholar). Currently, carbapenem antibiotics such as imipenem are considered antibiotics of last resort, and the advent of enzymes that turn them over efficiently bodes poorly for the prospects of continued clinical utility of these versatile antibacterials.The x-ray structure of the NMC-A enzyme, and its comparison to that of the classical class A enzyme (8Swarén P. Maveyraud L. Raquet X. Cabantous S. Duez C. Pedelacq J.D. Mariotte-Boyer S. Mourey L. Labia R. Nicolas-Chanoine M.H. Nordmann P. Frère J.M. Samama J.P. J. Biol. Chem. 1998; 273: 26714-26721Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar), showed that the carbapenamase activity of the NMC-A β-lactamase could be attributed to the displacement of Asn-132. The subtle relocation of this residue in the active site by a mere 1 Å, enlarges the substrate-binding site to accommodate the 6α-hydroxyethyl substituent of carbapenems, allowing the turnover process to take place (9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar). These observations were the basis for the design of a novel inactivator for the NMC-A β-lactamase, namely 6α-hydroxypropylpenicillanate (9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar). The x-ray structure determination of the complex of the NMC-A β-lactamase and the inhibitor illustrated that inactivation of the enzyme arose from interactions between the protein and inhibitor that prevented the approach of the hydrolytic water to the ester of the acyl-enzyme intermediate (9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar).We disclose herein inhibition of the NMC-A β-lactamase by a set of monobactam inhibitors. These are effective inactivators for this enzyme, and their mechanism of action is distinct compared with that of 6α-hydroxypropylpenicillanate, which was reported earlier (9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar). The mechanistic implications of interactions of the NMC-A β-lactamase and one of the inhibitors of our design are addressed by the determination of the crystal structure of the complex, as well as by computational molecular dynamics simulations.EXPERIMENTAL PROCEDURESThe NMC-A β-lactamase was purified to homogeneity according to a literature procedure (8Swarén P. Maveyraud L. Raquet X. Cabantous S. Duez C. Pedelacq J.D. Mariotte-Boyer S. Mourey L. Labia R. Nicolas-Chanoine M.H. Nordmann P. Frère J.M. Samama J.P. J. Biol. Chem. 1998; 273: 26714-26721Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Cephaloridine was purchased from Sigma. Syntheses of compounds 1 and 6 were reported previously (10Bulychev A. O'Brien M.E. Massova I. Teng M. Gibson T.A. Miller M.J. Mobashery S. J. Am. Chem. Soc. 1995; 117: 5938-5943Crossref Scopus (37) Google Scholar), and those for the remainder of the inhibitors are given in the Supplementary Material available in the on-line version. Spectrophotometric studies were performed on a Hewlett-Packard 8453 diode array instrument. Calculations were performed by the MS Excel program.Kinetic ExperimentsA 1.0-ml assay mixture typically consisted of 0.5 mm cephaloridine in 100 mmsodium phosphate, pH 7.0. Hydrolysis of the β-lactam ring of cephaloridine was monitored at 290 nm (Δε290 = 2070m−1 cm−1) upon addition of the enzyme (final concentration was typically 5 nm).Inactivation experiments were performed as follows. An aliquot of the stock solution of the inactivator (100 μm in p-dioxane) was added to the NMC-A β-lactamase (0.4 μm final concentration) in 100 mm sodium phosphate, pH 7.0, at 4 °C (10% p-dioxane final). Portions (10 μl) were removed from the mixture at time intervals and were diluted 100-fold into the assay mixture containing 0.5 mm cephaloridine. The enzyme activity was monitored until cephaloridine was entirely consumed. The remaining enzyme activity was calculated from the initial linear portion of the hydrolysis curve.Rates of hydrolysis of the inactivated acyl-enzyme species (krec), and the attendant recovery of activity, were measured under conditions of excess substrate (0.5 mmcephaloridine) (11Koerber S.C. Fink A.L. Anal. Biochem. 1987; 165: 75-87Crossref PubMed Scopus (41) Google Scholar). A solution of a given inactivator in p-dioxane (typically 300 μm final concentration) was mixed with the NMC-A β-lactamase (0.7 μm final concentration). The mixture was incubated (30 min to 6 h, depending on the inhibitor) at room temperature until residual enzyme activity was less than 2%. A 10-μl portion of this mixture was added to the solution of cephaloridine (0.5 mm) in 100 mm sodium phosphate, pH 7.0. Hydrolysis of cephaloridine was monitored at 290 nm (Δε290 = 2070m−1 cm−1). The computation of the rate constant was performed according to the method of Glick et al. (12Glick B.R. Brubacher L.J. Leggett D.J. Can. J. Biochem. 1978; 56: 1055-1057Crossref PubMed Scopus (23) Google Scholar).Michaelis-Menten parameters for turnover (Km and kcat) for compounds 1-4 were evaluated by Lineweaver-Burk plots. The concentrations of the compounds were varied from 2.5 to 15 μm. A portion of the enzyme was added to a solution of the inhibitor to give a final concentration of 4 nm for the enzyme in a total volume of 1.0 ml; hydrolysis was monitored at 240 nm (1,Δε240 = 11,480 m−1cm−1; 2, Δε240 = 6140m−1 cm−1; 3,Δε240 = 12,700 m−1cm−1; 4, Δε240 = 15,100m−1 cm−1).Determination of the X-ray Structure of the Enzyme Inhibited by Compound 6Crystals of NMC-A (8Swarén P. Maveyraud L. Raquet X. Cabantous S. Duez C. Pedelacq J.D. Mariotte-Boyer S. Mourey L. Labia R. Nicolas-Chanoine M.H. Nordmann P. Frère J.M. Samama J.P. J. Biol. Chem. 1998; 273: 26714-26721Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) were soaked in 2 μl of a freshly prepared solution of the inhibitor containing 10 mmof 6 in 19% polyethylene glycol 1500, 190 mmMES, pH 5.25, and 10% (v/v) p-dioxane at 4 °C. After 15 min, crystals were mounted in cryoloops and were flash-frozen in a stream of nitrogen gas cooled to 120 K. A 1.95-Å data set was collected on the W32 wiggler beam line at LURE-DCI (Orsay, France), tuned at a wavelength of 0.97 Å, and equipped with a large MarResearch imaging plate. The crystal to detector distance was set to 270 mm. A total of 60 frames (1.5° oscillation per frame and 10-min exposure) were collected from a single NMC-A crystal. Data were processed with MOSFLM (13Leslie A.G.W. Joint Collaborative Computing Project 4 and European Science Foundation-European Association of the Crystallography of Biological Macromolecules Newsletter on Protein Crystallography. 26. SERC Daresbury Laboratory, Warrington, UK1992Google Scholar) and CCP4 (14Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar) packages (TableI). The space group was P 21212 with cell parameters a = 77.48 Å, b = 52.69 Å, c = 67.10 Å. There is one molecule per asymmetric unit. The structure was refined with the program X-PLOR, version 3.1 (15Brünger A.T. X-PLOR, A System for X-ray Crystallography and NMR , version 3.1. Yale University Press, New Haven, CT1992Google Scholar), applying a bulk solvent correction. A total of 5% of the reflections were randomly selected in order to provide a test set for the Rfree calculations (16Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3849) Google Scholar). These reflections were omitted during refinement, but were included in the electron density map calculations. Models and electron density maps were displayed with the program TURBO FRODO (17Roussel, A., Inisan, A.-G., and Cambillau, C., TURBO FRODO program, AFMB and Biographics, Marseille, FranceGoogle Scholar).Table IX-ray diffraction data processing statistics given for the entire resolution range and for the highest resolution shell36.5–1.95 Å2.06–1.95 ÅNumber of measurements68,8489216Number of unique reflections20,5662898Completeness (%)99.698.5Rsym (%)6.311.4〈I/sd(I)〉8.16.2 Open table in a new tab Molecular replacement calculations, carried out with the AMoRe program (18Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar) using the 1.64-Å refined NMC-A β-lactamase structure (8Swarén P. Maveyraud L. Raquet X. Cabantous S. Duez C. Pedelacq J.D. Mariotte-Boyer S. Mourey L. Labia R. Nicolas-Chanoine M.H. Nordmann P. Frère J.M. Samama J.P. J. Biol. Chem. 1998; 273: 26714-26721Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) as a model, were performed to account for the 1.2-Å variation of the cell parameters along the crystallographic a axis. Rigid body refinement, performed between 8.0 and 3.0 Å (Rfactor = 0.36), was followed by molecular dynamics refinement in the resolution range 31.2–1.95 Å, using a slow cooling protocol starting from 3000 K, energy minimization, and individual thermal factor refinement. Water molecules were added as neutral oxygen atoms when they appeared as positive peaks above 4.0 ς in the (Fobs −Fcalc) exp(iαcalc) map in acceptable hydrogen-bonding geometry. Hereafter, the simulated annealing was performed from 500 K. The electron density maps revealed two conformations for the inhibitor covalently bound in the active site to Ser-70. The starting geometry of the inhibitor was obtained by optimization using DISCOVER (MSI). The final model of the enzyme−inhibitor complex, composed of all protein atoms with the exception of the solvent-exposed side chains of three lysine residues, includes 266 crystallographic water molecules and three molecules of MES buffer molecules. Alternate conformations were assigned to two side chains. The average B factors were 11.7 Å2 for protein atoms (10.5 Å2 and 12.9 Å2 for main chains and side chains, respectively), 17.2 Å2 for Ser-70 acylated by 6, 23.1 Å2 for solvent atoms, and 58.8 Å2 for MES buffer atoms. The final crystallographic R and Rfree values were 0.179 and 0.226, respectively.Molecular Modeling and Dynamics SimulationsThe coordinates of the native NMC-A β-lactamase were obtained by removing the bound inhibitor from the active site of the enzyme in the x-ray structure. This native structure was utilized to construct an immediate acyl-enzyme species for structure 11. The immediate acyl-enzyme species refers to what would be generated after serine acylation, whereby the ester carbonyl is ensconced in the oxyanion hole with strong hydrogen bonds to the backbone amines of Ser-70 and Ser-237. There is ample structural evidence for the existence of such a species from our earlier work (19Maveyraud L. Mourey L. Kotra L.P. Pédelacq J.D. Guillet V. Mobashery S. Samama J.P. J. Am. Chem. Soc. 1998; 120: 9748-9752Crossref Scopus (125) Google Scholar, 20Maveyraud L. Massova I. Birck C. Miyashita K. Samama J.-P. Mobashery S. J. Am. Chem. Soc. 1996; 118: 7435-7440Crossref Scopus (113) Google Scholar). The model for the immediate acyl-enzyme intermediate included the crystallographic waters and an additional box of water molecules up to 8-Å thickness from the surface of the enzyme. The model was energy-minimized and was utilized for the molecular dynamics simulations as the starting conformation. Molecular dynamics were performed using the Amber 5.0 software package (Oxford Molecular Inc.) (21Case D.A. Pearlman D.A. Caldwell J.W. Cheatham III, T.E. Ross W.S. Simmerling C.L. Darden T.A. Merz K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Ferguson D.M. Radmer R.J. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER 5. University of California, San Francisco, CA1997Google Scholar, 22Pearlman D.A. Case D.A. Caldwell J.W. Ross W.S. Cheatham III, T.E. DeBolt S. Ferguson D. Seibel G. Kollman P.A. Comp. Phys. Commun. 1995; 91: 1-41Crossref Scopus (2628) Google Scholar), at constant pressure using periodic boundary conditions. Calculations were performed on a Silicon Graphics Octane workstation, and the graphical analysis was performed using the SYBYL molecular modeling software version 6.4 (23SYBYL, Molecular Modeling Software, version 6.4, Tripos Associates, St. Louis, MOGoogle Scholar). The complete system was warmed from 0 to 300 K in steps of 20 K per 5 ps and then equilibrated at 300 K for 25 ps (a total of 100 ps). Snapshots of the structures were collected from here on for every 0.2 ps for 128 ps, and the resulting structures were analyzed. β-Lactamases are the primary cause of resistance to β-lactam antibiotics (1Bush K. Mobashery S. Rosen B.P. Mobashery S. Resolving the Antibiotic Paradox: Progress in Understanding Drug Resistance and Development of New Antibiotics. Plenum Press, New York1998: 71-98Google Scholar). These enzymes hydrolyze the β-lactam moiety of β-lactam antibiotics, and by so-doing, render them inactive. There are four classes of these enzymes, of which the class A is the largest group (1Bush K. Mobashery S. Rosen B.P. Mobashery S. Resolving the Antibiotic Paradox: Progress in Understanding Drug Resistance and Development of New Antibiotics. Plenum Press, New York1998: 71-98Google Scholar). The active-site serine in the class A β-lactamases undergoes acylation by the substrate and the acyl-enzyme intermediate is subsequently hydrolyzed to give substrate turnover (1Bush K. Mobashery S. Rosen B.P. Mobashery S. Resolving the Antibiotic Paradox: Progress in Understanding Drug Resistance and Development of New Antibiotics. Plenum Press, New York1998: 71-98Google Scholar,2Matagne A. Lamotte-Brasseur J. Frère J.-M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (318) Google Scholar). Class A enzymes perform this task with their preferred substrates, penicillins, at the diffusion limit (3Hardy L.W. Kirsch J.F. Arch. Biochem. Biophys. 1989; 268: 338-348Crossref PubMed Scopus (4) Google Scholar). Many of the “parental” β-lactamases of class A, such as the TEM-1 β-lactamase, have undergone mutations that impart to them an increase in the breadth of their substrate profile (1Bush K. Mobashery S. Rosen B.P. Mobashery S. Resolving the Antibiotic Paradox: Progress in Understanding Drug Resistance and Development of New Antibiotics. Plenum Press, New York1998: 71-98Google Scholar), as well as the ability to avoid being inhibited by the known clinical inhibitors. This is currently a serious clinical challenge. One new class A β-lactamase, designated NMC-A1 (4Nordmann P. Mariotte S. Naas T. Labia R. Nicolas M.H. Antimicrob. Agents Chemother. 1993; 37: 939-946Crossref PubMed Scopus (156) Google Scholar), and the highly homologous Sme-1 (5Naas T. Vandel L. Sougakoff W. Livermore D.M. Nordmann P. Antimicrob. Agents Chemother. 1994; 38: 1262-1270Crossref PubMed Scopus (180) Google Scholar) and IMI-1 (6Rasmussen B.A. Bush K. Keeney D. Yang Y. Hare R. O'Gara C. Medeiros A.A. Antimicrob. Agents Chemother. 1996; 40: 2080-2086Crossref PubMed Google Scholar) β-lactamases, enjoy an unusually broad substrate profile, which includes penicillins, cephalosporins, and carbapenems (4Nordmann P. Mariotte S. Naas T. Labia R. Nicolas M.H. Antimicrob. Agents Chemother. 1993; 37: 939-946Crossref PubMed Scopus (156) Google Scholar, 7Rasmussen B.A. Bush K. Antimicrob. Agents Chemother. 1997; 41: 223-232Crossref PubMed Google Scholar). Currently, carbapenem antibiotics such as imipenem are considered antibiotics of last resort, and the advent of enzymes that turn them over efficiently bodes poorly for the prospects of continued clinical utility of these versatile antibacterials. The x-ray structure of the NMC-A enzyme, and its comparison to that of the classical class A enzyme (8Swarén P. Maveyraud L. Raquet X. Cabantous S. Duez C. Pedelacq J.D. Mariotte-Boyer S. Mourey L. Labia R. Nicolas-Chanoine M.H. Nordmann P. Frère J.M. Samama J.P. J. Biol. Chem. 1998; 273: 26714-26721Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar), showed that the carbapenamase activity of the NMC-A β-lactamase could be attributed to the displacement of Asn-132. The subtle relocation of this residue in the active site by a mere 1 Å, enlarges the substrate-binding site to accommodate the 6α-hydroxyethyl substituent of carbapenems, allowing the turnover process to take place (9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar). These observations were the basis for the design of a novel inactivator for the NMC-A β-lactamase, namely 6α-hydroxypropylpenicillanate (9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar). The x-ray structure determination of the complex of the NMC-A β-lactamase and the inhibitor illustrated that inactivation of the enzyme arose from interactions between the protein and inhibitor that prevented the approach of the hydrolytic water to the ester of the acyl-enzyme intermediate (9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar). We disclose herein inhibition of the NMC-A β-lactamase by a set of monobactam inhibitors. These are effective inactivators for this enzyme, and their mechanism of action is distinct compared with that of 6α-hydroxypropylpenicillanate, which was reported earlier (9Mourey L. Miyashita K. Swarén P. Bulychev A. Samama J.P. Mobashery S. J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (50) Google Scholar). The mechanistic implications of interactions of the NMC-A β-lactamase and one of the inhibitors of our design are addressed by the determination of the crystal structure of the complex, as well as by computational molecular dynamics simulations. EXPERIMENTAL PROCEDURESThe NMC-A β-lactamase was purified to homogeneity according to a literature procedure (8Swarén P. Maveyraud L. Raquet X. Cabantous S. Duez C. Pedelacq J.D. Mariotte-Boyer S. Mourey L. Labia R. Nicolas-Chanoine M.H. Nordmann P. Frère J.M. Samama J.P. J. Biol. Chem. 1998; 273: 26714-26721Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Cephaloridine was purchased from Sigma. Syntheses of compounds 1 and 6 were reported previously (10Bulychev A. O'Brien M.E. Massova I. Teng M. Gibson T.A. Miller M.J. Mobashery S. J. Am. Chem. Soc. 1995; 117: 5938-5943Crossref Scopus (37) Google Scholar), and those for the remainder of the inhibitors are given in the Supplementary Material available in the on-line version. Spectrophotometric studies were performed on a Hewlett-Packard 8453 diode array instrument. Calculations were performed by the MS Excel program.Kinetic ExperimentsA 1.0-ml assay mixture typically consisted of 0.5 mm cephaloridine in 100 mmsodium phosphate, pH 7.0. Hydrolysis of the β-lactam ring of cephaloridine was monitored at 290 nm (Δε290 = 2070m−1 cm−1) upon addition of the enzyme (final concentration was typically 5 nm).Inactivation experiments were performed as follows. An aliquot of the stock solution of the inactivator (100 μm in p-dioxane) was added to the NMC-A β-lactamase (0.4 μm final concentration) in 100 mm sodium phosphate, pH 7.0, at 4 °C (10% p-dioxane final). Portions (10 μl) were removed from the mixture at time intervals and were diluted 100-fold into the assay mixture containing 0.5 mm cephaloridine. The enzyme activity was monitored until cephaloridine was entirely consumed. The remaining enzyme activity was calculated from the initial linear portion of the hydrolysis curve.Rates of hydrolysis of the inactivated acyl-enzyme species (krec), and the attendant recovery of activity, were measured under conditions of excess substrate (0.5 mmcephaloridine) (11Koerber S.C. Fink A.L. Anal. Biochem. 1987; 165: 75-87Crossref PubMed Scopus (41) Google Scholar). A solution of a given inactivator in p-dioxane (typically 300 μm final concentration) was mixed with the NMC-A β-lactamase (0.7 μm final concentration). The mixture was incubated (30 min to 6 h, depending on the inhibitor) at room temperature until residual enzyme activity was less than 2%. A 10-μl portion of this mixture was added to the solution of cephaloridine (0.5 mm) in 100 mm sodium phosphate, pH 7.0. Hydrolysis of cephaloridine was monitored at 290 nm (Δε290 = 2070m−1 cm−1). The computation of the rate constant was performed according to the method of Glick et al. (12Glick B.R. Brubacher L.J. Leggett D.J. Can. J. Biochem. 1978; 56: 1055-1057Crossref PubMed Scopus (23) Google Scholar).Michaelis-Menten parameters for turnover (Km and kcat) for compounds 1-4 were evaluated by Lineweaver-Burk plots. The concentrations of the compounds were varied from 2.5 to 15 μm. A portion of the enzyme was added to a solution of the inhibitor to give a final concentration of 4 nm for the enzyme in a total volume of 1.0 ml; hydrolysis was monitored at 240 nm (1,Δε240 = 11,480 m−1cm−1; 2, Δε240 = 6140m−1 cm−1; 3,Δε240 = 12,700 m−1cm−1; 4, Δε240 = 15,100m−1 cm−1).Determination of the X-ray Structure of the Enzyme Inhibited by Compound 6Crystals of NMC-A (8Swarén P. Maveyraud L. Raquet X. Cabantous S. Duez C. Pedelacq J.D. Mariotte-Boyer S. Mourey L. Labia R. Nicolas-Chanoine M.H. Nordmann P. Frère J.M. Samama J.P. J. Biol. Chem. 1998; 273: 26714-26721Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) were soaked in 2 μl of a freshly prepared solution of the inhibitor containing 10 mmof 6 in 19% polyethylene glycol 1500, 190 mmMES, pH 5.25, and 10% (v/v) p-dioxane at 4 °C. After 15 min, crystals were mounted in cryoloops and were flash-frozen in a stream of nitrogen gas cooled to 120 K. A 1.95-Å data set was collected on the W32 wiggler beam line at LURE-DCI (Orsay, France), tuned at a wavelength of 0.97 Å, and equipped with a large MarResearch imaging plate. The crystal to detector distance was set to 270 mm. A total of 60 frames (1.5° oscillation per frame and 10-min exposure) were collected from a single NMC-A crystal. Data were processed with MOSFLM (13Leslie A.G.W. Joint Collaborative Computing Project 4 and European Science Foundation-European Association of the Crystallography of Biological Macromolecules Newsletter on Protein Crystallography. 26. SERC Daresbury Laboratory, Warrington, UK1992Google Scholar) and CCP4 (14Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar) packages (TableI). The space group was P 21212 with cell parameters a = 77.48 Å, b = 52.69 Å, c = 67.10 Å. There is one molecule per asymmetric unit. The structure was refined with the program X-PLOR, version 3.1 (15Brünger A.T. X-PLOR, A System for X-ray Crystallography and NMR , version 3.1. Yale University Press, New Haven, CT1992Google Scholar), applying a bulk solvent correction. A total of 5% of the reflections were randomly selected in order to provide a test set for the Rfree calculations (16Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3849) Google Scholar). These reflections were omitted during refinement, but were included in
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