ChemMedChemVolume 2, Issue 10 p. 1414-1417 Communication Naphthyl Tetronic Acids as Multi-Target Inhibitors of Bacterial Peptidoglycan Biosynthesis Tarek S. Mansour Dr., Tarek S. Mansour Dr. mansout@wyeth.com Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorCraig E. Caufield Dr., Craig E. Caufield Dr. Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorBeth Rasmussen Dr., Beth Rasmussen Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorRajiv Chopra Dr., Rajiv Chopra Dr. Wyeth Research, Cambridge, MA 02140, USASearch for more papers by this authorGirija Krishnamurthy Dr., Girija Krishnamurthy Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorKoi M. Morris, Koi M. Morris Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorKristine Svenson, Kristine Svenson Wyeth Research, Cambridge, MA 02140, USASearch for more papers by this authorJoel Bard Dr., Joel Bard Dr. Wyeth Research, Cambridge, MA 02140, USASearch for more papers by this authorClaudia Smeltzer, Claudia Smeltzer Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorShaughnessy Naughton, Shaughnessy Naughton Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorSchuyler Antane, Schuyler Antane Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorYoujun Yang Dr., Youjun Yang Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorAnatoly Severin Dr., Anatoly Severin Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorDominick Quagliato Dr., Dominick Quagliato Dr. Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorPeter J. Petersen, Peter J. Petersen Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorGuy Singh Dr., Guy Singh Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this author Tarek S. Mansour Dr., Tarek S. Mansour Dr. mansout@wyeth.com Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorCraig E. Caufield Dr., Craig E. Caufield Dr. Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorBeth Rasmussen Dr., Beth Rasmussen Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorRajiv Chopra Dr., Rajiv Chopra Dr. Wyeth Research, Cambridge, MA 02140, USASearch for more papers by this authorGirija Krishnamurthy Dr., Girija Krishnamurthy Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorKoi M. Morris, Koi M. Morris Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorKristine Svenson, Kristine Svenson Wyeth Research, Cambridge, MA 02140, USASearch for more papers by this authorJoel Bard Dr., Joel Bard Dr. Wyeth Research, Cambridge, MA 02140, USASearch for more papers by this authorClaudia Smeltzer, Claudia Smeltzer Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorShaughnessy Naughton, Shaughnessy Naughton Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorSchuyler Antane, Schuyler Antane Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorYoujun Yang Dr., Youjun Yang Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorAnatoly Severin Dr., Anatoly Severin Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorDominick Quagliato Dr., Dominick Quagliato Dr. Wyeth Research, CN 8000, Princeton, NJ 08543, USASearch for more papers by this authorPeter J. Petersen, Peter J. Petersen Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this authorGuy Singh Dr., Guy Singh Dr. Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA, Fax: (+1) 845-602-5580Search for more papers by this author First published: 26 September 2007 https://doi.org/10.1002/cmdc.200700094Citations: 37Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract A pathway screen targeting multiple muramyl peptide synthesis inhibitors identified the naphthyl tetronic acids series. Optimization of this series based on IC50, Kd and MIC values led to potent inhibitors. Compound 5 h was co-crystallized in the active site of E. coli Mur B. Citing Literature Volume2, Issue10October 8, 2007Pages 1414-1417 RelatedInformation

10.1128/AAC.50.2.556-564.2006. 2006, 50(2):556. DOI: Antimicrob. Agents Chemother. Tarek S. Mansour S. Shumsky, Kristina M. K. Kutterer, Adam Gilbert and David M. Shlaes, Beth A. Rasmussen, Amedeo A. Failli, Jay Kristine Svenson, Peter J. Petersen, Pornpen Labthavikul, Krishnamurthy, Guy Singh, William Hu, David Keeney, Youjun Yang, Anatoly Severin, Rajiv Chopra, Girija Gram-Positive Bacteria Reductase (MurB) with Activity against Acetylenolpyruvylglucosamine N UDP3,5-Dioxopyrazolidines, Novel Inhibitors of

BACKGROUND:Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library.RESULTS:Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport.CONCLUSION:Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes.

The reactions of class A β-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A β-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the β-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58–82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125–141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A β-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1Bonomo R.A Rudin S.E.A. Shlaes D.M. FEMS Microbiol. Lett. 1997; 148: 59-62Crossref PubMed Scopus (34) Google Scholar) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2Payne D.J. Cramp R. Winstanley D.J. Knowles D.J.C. Antimicrob. Agents Chemother. 1994; 38: 767-772Crossref PubMed Scopus (147) Google Scholar) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3Kuck N.A. Jacobus N.V. Petersen P.J. Weiss W.J. Testa R.T. Antimicrob. Agents Chemother. 1989; 33: 1964-1969Crossref PubMed Scopus (86) Google Scholar) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde. The reactions of class A β-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A β-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the β-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58–82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125–141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A β-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1Bonomo R.A Rudin S.E.A. Shlaes D.M. FEMS Microbiol. Lett. 1997; 148: 59-62Crossref PubMed Scopus (34) Google Scholar) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2Payne D.J. Cramp R. Winstanley D.J. Knowles D.J.C. Antimicrob. Agents Chemother. 1994; 38: 767-772Crossref PubMed Scopus (147) Google Scholar) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3Kuck N.A. Jacobus N.V. Petersen P.J. Weiss W.J. Testa R.T. Antimicrob. Agents Chemother. 1989; 33: 1964-1969Crossref PubMed Scopus (86) Google Scholar) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde. clavulanic acid tazobactam electrospray ionization mass spectrometry high performance liquid chromatography molecular weight fast atom bombardment tazobactam and PC1 covalent adduct tazobactam and TEM-1 covalent adduct acetonitrile Two structurally distinct classes of β-lactamase inhibitors, clavams represented by clavulanic acid (CA,1 see Structure Fs1) and penicillanic sulfones represented by sulbactam and tazobactam (TZB, see Structure Fs1), have been widely used clinically. In combination with a β-lactam antibiotic, these inhibitors have successfully overcome bacterial β-lactam resistance caused by β-lactamase-mediated β-lactam hydrolysis. In particular, tazobactam, a triazoly-substituted penicillanic sulfone, has potent inhibitory activity against class A β-lactamases, including some β-lactamases that are resistant to inactivation by CA and sulbactam (1Bonomo R.A Rudin S.E.A. Shlaes D.M. FEMS Microbiol. Lett. 1997; 148: 59-62Crossref PubMed Scopus (34) Google Scholar, 2Payne D.J. Cramp R. Winstanley D.J. Knowles D.J.C. Antimicrob. Agents Chemother. 1994; 38: 767-772Crossref PubMed Scopus (147) Google Scholar). Extensive studies have demonstrated that the combination of tazobactam-piperacillin is an effective antimicrobiological agent against class A β-lactamase producing isolates (3Kuck N.A. Jacobus N.V. Petersen P.J. Weiss W.J. Testa R.T. Antimicrob. Agents Chemother. 1989; 33: 1964-1969Crossref PubMed Scopus (86) Google Scholar, 4Murray P.R. Cantrell H.F. Lankford R.B. Diagn. Microbiol. Infect. Dis. 1994; 19 (the In Vitro Susceptibility Surveillance Group): 111-120Crossref PubMed Scopus (55) Google Scholar). However, direct evidence of the mechanism of inactivation of the class A β-lactamases by the penicillanic sulfone inhibitors has not been fully addressed. The mechanism of β-lactamase inactivation by CA has been studied extensively. Crystallographic studies performed on the clavulanic acid and PC1 covalent adduct revealed the structure of the acyl-enzyme complex to consist of a CA fragment covalently bound to the active site Ser-70, as either the cis or trans decarboxylated enamines (5Chen C.C. Herzberg O. J. Mol. Bacteriol. 1992; 224: 1103-1113Google Scholar). ESI/MS studies by Brown et al. (6Brown R.P. A Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar) provided direct evidence for the complexation of CA with the TEM-2 β-lactamase. CA fragments with masses of 70 and 88 Da were attached to the peptide fragment containing Ser-70, and a second 70-Da mass fragment, assigned as a β-linked acrylate, was localized to a peptide containing Ser-130. The mechanism of inactivation of TZB for all major classes of β-lactamases has been studied using UV spectrometric assays (7Bush K. Macalintal C. Rasmussen B.A. Lee V. Yang Y. Antimicrob. Agents Chemother. 1993; 37: 851-858Crossref PubMed Scopus (235) Google Scholar). The formation of a transient complex and generation of subsequent degradation products were observed. For the class A β-lactamases PC1 and TEM-2, a new chromophore absorbing at 288 nm indicated the formation of reaction intermediates. It was proposed that these intermediates were the products formed following deacylation. An analysis of the mechanism of inactivation of class A β-lactamases by CA and sulbactam (8Imtiaz U. Billings E.M. Knox J.R. Mobashery S. Biochemistry. 1994; 33: 5728-5738Crossref PubMed Scopus (76) Google Scholar) indicated that the main differences in β-lactamase inactivation by CA and sulbactam are in the events leading to β-elimination and opening of the five-membered ring. For CA, efficient β-elimination occurs following protonation. This proton is donated by an active site water molecule (9Imtiaz U. Billings E.M. Knox J.R. Manavathu E., K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar). The penicillanic sulfones do not require protonation to catalyze the β-elimination event and opening of the five-membered ring (8Imtiaz U. Billings E.M. Knox J.R. Mobashery S. Biochemistry. 1994; 33: 5728-5738Crossref PubMed Scopus (76) Google Scholar). This might account for the activity of tazobactam against the CA-resistant class A β-lactamases. Our studies were designed to provide detailed evidence regarding the mechanism of inhibition of class A β-lactamases by tazobactam, a penicillanic sulfone inhibitor. ESI/MS, HPLC/ESI/MS, and HPLC/ESI/MS/MS techniques were employed to identify the amino acid residues of PC1 directly involved in inactivation by tazobactam. In addition, the reaction products of tazobactam, following the initial acylation of the enzyme, were examined. From these findings, a mechanistic scheme for the inactivation of class A β-lactamases by TZB was proposed. The similarities and differences in the inactivation of β-lactamases by clavulanic acid and the penicillanic acid sulfones are discussed. PC1 β-lactamase (256 residues, MW 28,794.3 (10Ambler R.P. Biochem. J. 1975; 151: 197-218Crossref PubMed Scopus (58) Google Scholar,11McLaughlin J.R. Murray C.J. Rabinowitz J.C. J. Biol. Chem. 1981; 256: 11272-11282PubMed Google Scholar)) was produced by Proton Products (Berkshire, UK). TEM-1 β-lactamase (263 residues, MW 28,909.8 (12Sutcliffe J.G. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3731-3741Crossref Scopus (599) Google Scholar)) was extracted from a recombinant high level overexpressing Escherichia coli strain (13Rasmussen B.A. Gluzman Y. Tally F.P. Mol. Microbiol. 1991; 5: 1211-1219Crossref PubMed Scopus (29) Google Scholar) and was purified to near homogeneity using Sephadex G-75 column chromatography. Sequencing grade endoproteinases, Glu-C and trypsin (modified), were purchased from Roche Molecular Biochemicals. Ambler's numbering system for the amino acid sequence of PC1 (14Ambler R.P. Coulson A.F.W. Frere J.-M. Ghuysen J.-M. Joris B. Forsman M. Levesque R.C. Tiraby G. Waley S.G. Biochemistry J. 1991; 276: 269-272Crossref PubMed Scopus (854) Google Scholar) was used throughout the text. The free acid form of TZB (MW 300) was synthesized at Wyeth-Ayerst Research (Pearl River, NY). TZB was prepared as a 2 mm stock solution in HPLC grade water unless otherwise indicated. For intact protein analysis, enzyme stock solutions (100 μm) of PC1 and TEM-1 were prepared in HPLC grade water. Samples of TZB reacted with PC1 and TEM-1, respectively, were prepared by incubating the enzyme with inhibitor at inhibitor/enzyme molar ratios of 10:1 and 100:1, respectively, in H2O for 60 min at 25 °C. Greater than 90% inhibition of PC1 and TEM-1 by TZB, respectively, was confirmed by testing for enzyme activity using the chromogenic substrate nitrocefin and monitoring at 495 nm using a Beckman Model DU7400 spectrophotometer (15O'Callanghan C.H. Morris A. Kirby S. Single A.H. Antimicrob. Agents Chemother. 1972; 1: 283-288Crossref PubMed Scopus (1477) Google Scholar). To verify the specificity of the reaction of TZB with PC1 and TEM-1, the enzymes were denatured using 2% formic acid in H2O: acetonitrile (H2O:ACN, 1:1 v/v) for 10 min prior to incubation with TZB. The endoproteinase Glu-C was used to cleave the peptide bonds C-terminally at glutamic acid residues. PC1 and TZB were dissolved in 20 mm ammonium carbonate, pH 7.8. TZB and PC1 were mixed (inhibitor:enzyme, 10:1, m/m) and incubated for 60 min at 25 °C. PC1 hydrolytic activity was inhibited to greater than 90% by TZB. This was confirmed by testing for residual enzyme activity using nitrocefin (as above). The TZB-PC1 reaction mixture and PC1 control (without TZB) were each individually digested with Glu-C at a molar ratio of 40:1 (PC1:Glu-C) for 4 h at 25 °C. Digestion with trypsin was performed using sequencing grade, modified trypsin protease. PC1 and TZB were dissolved in 100 mm Tris/HCl, pH 8.5, at concentrations of 50 and 2000 μm, respectively. TZB and PC1 were mixed (inhibitor:enzyme, 10:1, m/m) and incubated for 60 min at 25 °C. Inactivation of enzymatic activity by TZB was confirmed (as above). The TZB-PC1 reaction mixture and PC1 control (without TZB) were each individually digested with trypsin at a molar ratio of 40:1 (PC1:trypsin) for 10 h at 37 °C. Electrospray ionization mass spectra were obtained in the positive and negative ion modes with a Micromass Quattro triple quadrupole mass spectrometer and a Platform II single quadrupole mass spectrometer. The samples were prepared at ∼10 pmol/μl in 3% acetic acid in H2O:ACN (1:1, v/v). Samples were flow injected (2–5 μl) into the source of the mass spectrometer at a rate of 10 μl/min utilizing a carrier solvent of H2O:ACN (1:1, v/v). Protein data were acquired over a wide scan mass range of m/z ∼500–1600 with 16 points/m/z or over a narrow scan mass range ofm/z 800–1000 at 64 data points/m/z, with a scan duration of 15–25 s. The cone voltage was set to 50 V. Nitrogen was used as the nebulizing and drying gas with flow rates of 0.2 and 5 liters/min, respectively. Ten to twenty spectra were averaged, smoothed, baseline subtracted, and transformed using the Micromass Maximum Entropy program. The mass spectrometer was calibrated with sodium iodide. HPLC/ESI/MS/MS spectra were obtained with the Micromass Quattro triple quadrupole mass spectrometer using a collison energy of 50 eV with argon as the collision gas at 1.5 × 10−3millibar. FAB mass spectra were obtained in the negative ion mode using a high performance Finnigan-MAT 95 magnetic sector mass spectrometer equipped with a xenon fast atom gun. The matrix used was a 3:1 (w/w) mixture of dithiothreitol and dithioerithritol. Low resolution FAB data were acquired by magnetic scanning at 15 s/decade. Exact mass data were acquired by peak matching at a resolution of 6000 (10% peak height). The FAB negative ion calibration utilized the following peptides: for wide mass range (electrostatic calibration) AGSE (361.1359 [M − H]1−) and AGFL (405.2138 [M − H]1−) and for narrow mass range (peak matching) LV (229.1552 [M − H]1−). A Hewlett-Packard Model 1100 HPLC pump and DAD detector, set to record UV-visible spectra over the 200–600 nm range, were employed in all HPLC/ESI/MS experiments. A solvent gradient of ACN:H2O from 5:95 to 95:5 (v/v) containing 0.05% trifluoroacetic acid was applied over 60 min at 0.20 ml/min through a Vydac 218TP5215 2.1 × 150 mm C18 column. An eluent split ratio of 3:1 was maintained to deliver 50 μl/min to the ion source of a Micromass Q-TOF mass spectrometer. Nitrogen was used as the nebulizing and drying gas. Mass spectra were acquired over a range of m/z 300–2000 at 1 s/scan using electrospray ionization in the positive ion mode with a cone voltage of 45 V. Ten microliters of Glu-C or trypsin-digested PC1 and TZB-PC1 were injected. HPLC/ESI/MS/MS was performed under the same chromatographic conditions and with the same mass spectrometer as in the HPLC/ESI/MS experiments. MS/MS spectra were acquired using an automated MS/MS mode over a range of m/z 50 to 1500 and 32 eV collision energy. Helium was used as the collision gas. The notation used for analysis of the MS/MS data followed the proposal of Roepstorff and Fohlman (16Roepstorff P. Fohlman J. Biomed. Mass Spectrom. 1984; 11: 601Crossref PubMed Scopus (2386) Google Scholar). For full-length protein studies, β-lactamases and TZB were dissolved in HPLC grade water. Note, the pH of the reaction mixture was 4.5. Both PC1 and TEM-1 enzymes maintained their activities in water as well as in a control phosphate buffer (50 mm NaH2PO4, pH 4.6) during the incubation period. Using water instead of a buffer system provided better mass spectral resolution during ESI/MS analyses. The acid form of TZB was utilized for reactions involving ESI/MS analyses to avoid the formation of salt adducts, which were observed when using the sodium salt form of TZB. The ESI mass spectra were obtained in the positive ion mode under narrow scan high resolution conditions, and the data were transformed with a maximum entropy algorithm. Fig. 1 depicts the transformed ESI mass spectra for native PC1 (Fig. 1 a, MW 28,797) and the TZB-PC1 reaction products (Fig. 1 b). For Fig. 1 b, in addition to the native PC1 species (Peak A, MW 28,802), four new components were observed corresponding to the mass of PC1 plus mass increments of ∼52, ∼70, ∼88, and ∼300 Da (Peaks B, C, D, and E, respectively). Fig. 2 depicts the transformed ESI mass spectra of native TEM-1 (Fig. 2 a, MW 28,912) and TZB-TEM-1 reaction products (Fig. 2 b). For Fig.2 b, in addition to native TEM-1 (Peak A, MW 28,911), three new components were observed corresponding to the mass of TEM-1 plus mass increments of ∼52, ∼70, and ∼88 Da (Peaks B, C, and D, respectively). Table I summarizes the observed masses and structural assignments for the products generated following the reaction of TZB with PC1 and TEM-1, respectively. The relative abundances of the new products (illustrated in Figs.1 b and 2 b, respectively) indicate the extent of formation of the reaction intermediates under the experimental conditions used.Figure 2Transformed maximum entropy enhanced ESI/MS spectra for TEM-1 and TZB-TEM-1. a, native TEM-1 (MW 28,912). b, TZB-TEM-1 reaction products: Peak A, native TEM-1 (MW 28,911); Peaks B, C, and D, TZB-modified TEM-1 with mass increments of ∼52, ∼70, and ∼88 Da, respectively.View Large Image Figure ViewerDownload (PPT)Table IMeasured masses for the reaction of PC1 and TEM-1 with TZB and proposed structural assignmentsEnzymeComponentMeasured MWMass increment relative to enzyme MWPredicted mass increment (Da)Proposed structure (Scheme FS3)PC1 (Fig.1 b)A28802.0 ± 1.51-aPredicted mass 28,794.3 (10,11).TZB:PC1 = 10:1 M/MB28852.6 ± 1.350.6 ± 2.8527, 11C28871.5 ± 1.569.5 ± 3.0708, 10D28887.2 ± 1.885.2 ± 3.3889E29101.1 ± 4.2299.1 ± 5.73002, 3, 4, 5TEM-1 (Fig. 2 b)A28911.9 ± 2.11-bPredicted mass 28,909.8 (12).TZB:TEM-1 = 100:1 M/MB28962.5 ± 1.450.6 ± 3.5527, 11C28981.2 ± 1.669.3 ± 3.7708, 10D28999.2 ± 1.787.3 ± 3.88891-a Predicted mass 28,794.3 (10Ambler R.P. Biochem. J. 1975; 151: 197-218Crossref PubMed Scopus (58) Google Scholar,11McLaughlin J.R. Murray C.J. Rabinowitz J.C. J. Biol. Chem. 1981; 256: 11272-11282PubMed Google Scholar).1-b Predicted mass 28,909.8 (12Sutcliffe J.G. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3731-3741Crossref Scopus (599) Google Scholar). Open table in a new tab Following PC1 inactivation by TZB, three low mass components were observed in both the positive and negative ion electrospray modes. These three components have MWs of 248, 264, and 280, respectively. These masses were also observed by FAB mass spectrometry in the negative ion mode. Exact mass measurements of the observed [M − H]1− ions for the three components were m/z 247.0502, 263.0458, and 279.0398 with corresponding elemental compositions C7H11O4N4S (Δ = 0.0 mDa), C7H11O5N4S (Δ = 0.7 mDa), and C7H11O6N4S (Δ = −0.2 mDa), respectively. These results together with electrospray MS/MS data in the positive and negative ion modes allowed the determination of three proposed structures corresponding to TZB reaction products 6, 12, and 13 (17, also see "Discussion"). The formation of these ions was independent of the pH used (pH 3–7). When denatured PC1 and TEM-1 (prepared by exposure to 2% formic acid) were each reacted with TZB, no new mass components were observed by ESI/MS. Only the masses corresponding to the MWs of PC1 and TEM-1 were observed, indicating that the enzymatically active forms are required for the specificity of the reactions. Fig. 3 illustrates the UV chromatograms for the HPLC/ESI/MS data for Glu-C-digested PC1 (Fig.3 a) and TZB-PC1 (Fig. 3 b). The identities of the numbered peaks are listed in Table II. HPLC/ESI/MS data for the Glu-C-digested PC1 Peak 2, eluting at 17.4 min, exhibited ions at m/z 634.2 [M + 3H]3+and 950.6 [M + 2H]2+. The transformed data gave a molecular weight of 1899.6, which matched the predicted molecular weight of 1898.9 for a peptide composed of amino acid residues 125–141, containing Ser-130. The HPLC/ESI/MS data for the Glu-C-digested TZB-PC1 (Fig. 3 b), in addition to Peak 2 with MW 1899.9, also had a corresponding peak identified as Peak 2A co-eluting with Peak 3 at 17.7 min. Peak 2A exhibited an ion atm/z 985.6 [M + 2H]2+ with a molecular weight of 1969.2. This MW corresponds to a mass increment of 70 Da relative to the mass of the PC1 peptide containing amino acid residues 125–141.Table IIAssignments for HPLC/ESI/MS analysis of PC1 and TZB-PC1 adducts digested with endoproteinase Glu-CPeakRetention timeMeasured MWPredicted MWAmino acid residuesmin116.81496.81495.9142–154 2 2-aAlso studied by HPLC/ESI/MS/MS.17.41899.61898.9125–141 2A 2-aAlso studied by HPLC/ESI/MS/MS.17.71969.21968.9125–141 + 70 Da317.71390.21389.7155–166419.72122.52121.1 38–56519.91038.81038.5282–290625.13038.93037.7 83–1102-bAmino acids corresponding to positions 85 and 86 were omitted according to McLaughlin et al. (11).725.51590.81589.9111–124825.73714.33712.0169–202926.02773.32772.5 58–82 2-cAmino acid corresponding to position 58 were omitted according to McLaughlin et al.(11). 9A26.02824.22824.7 58–82 + 52 Da26.02843.22842.5 58–82 + 70 Da26.02860.92860.0 58–82 + 88 Da9B26.22754.82754.5 58–82 − 18 Da1030.78801.78797.6203–2812-a Also studied by HPLC/ESI/MS/MS.2-b Amino acids corresponding to positions 85 and 86 were omitted according to McLaughlin et al. (11McLaughlin J.R. Murray C.J. Rabinowitz J.C. J. Biol. Chem. 1981; 256: 11272-11282PubMed Google Scholar).2-c Amino acid corresponding to position 58 were omitted according to McLaughlin et al.(11McLaughlin J.R. Murray C.J. Rabinowitz J.C. J. Biol. Chem. 1981; 256: 11272-11282PubMed Google Scholar). Open table in a new tab HPLC/ESI/MS data for Glu-C-digested PC1 showed that Peak 9 (Fig. 3,a and b), eluting at 26.0 min, exhibited ions atm/z 925.7 [M + 3H]3+ and at m/z1387.7 [M + 2H]2+. The transformed HPLC/ESI/MS spectrum indicated the molecular weight of this peptide to be 2773.3 (Fig.4 a). This observed MW closely matches the predicted molecular weight of 2773.5 for a peptide containing amino acid residues 58–82 (Table II). The HPLC/ESI/MS data for the Glu-C-digested TZB-PC1 adducts showed three additional components co-eluting at 26 min (Peak 9A). The transformed spectrum of multiply charged ions produced by these components gave MWs of 2824, 2843, and 2862 (Fig. 4 b and Table II), which corresponded to 52, 70, and 88 Da mass increments relative to the mass of the PC1 peptide composed of amino acid residues 58–82. Peak 9B, eluting at 26.2 min, exhibited ions at m/z 919.7 [M + 3H]3+ and 1378.8 [M + 2H]2+. In the transformed spectrum, Peak 9B has a molecular weight of 2755, corresponding to the dehydrated (18 Da loss) peptide composed of amino acid residues 58–82. The above HPLC/ESI/MS analysis of Glu-C-digested TZB-PC1 showed that TZB fragments were bound to peptides composed of residues 58–82, containing Ser-70, and residues 125–141, containing Ser-130. HPLC/ESI/MS data for trypsin-digested PC1 and TZB-PC1 are summarized in Table III. HPLC/ESI/MS data for trypsin digested PC1 and TZB-PC1 showed that Peak 9 (Table III), eluting at 13.7 min, consisted of an ion at m/z 874.4 [M + H]1+, which corresponded to a peptide composed of amino acid residues 66–73. HPLC/ESI/MS data for trypsin digested TZB-PC1 showed that Peak 9A (Table III), eluting at 14.1 min, consisted of an ion at m/z 944.4 [M + H]1+. The difference of 70 Da between the masses of Peaks 9 and 9A indicated that a 70-Da fragment of TZB was bound to one of the amino acid residues 66–73. Peak 9A (Table III) was not observed in the HPLC/ESI/MS data for trypsin-digested PC1.Table IIIAssignments for HPLC/ESI/MS analysis of PC1 and TZB-PC1 digested with trypsinPeakRetention timeMeasured MWPredicted MWAmino acid residuesmin13.4474.5474.3189–19223.7465.4465.3112–11535.1765.4765.460–6546.5463.4463.3285–28858.7614.5614.4193–19869.2615.5615.4141–14679.8609.5609.360–649.8609.5609.493–97811.6760.5760.4278–284 9 3-aAlso studied by HPLC/ESI/MS/MS.13.7873.4873.466–73 9A 3-aAlso studied by HPLC/ESI/MS/MS.14.1943.4943.466–73 + 70 Da1014.2987.5987.531–38 or 32 –391114.7993.4993.5179–1881215.0732.5732.4216–2221315.7588.5588.4116–1201420.51051.41051.5245–2531523.51417.41417.7165–1751632.11219.51219.7206–2153-a Also studied by HPLC/ESI/MS/MS. Open table in a new tab The peptides corresponding to Peak 2 (Table II and Fig. 3 a) and Peak 2A (Table II and Fig. 3 b) from PC1 and TZB-PC1, respectively, following digestion with Glu-C, were analyzed using HPLC/ESI/MS/MS. The sequences of these two peptides were confirmed to be that of peptides composed of amino acid residues 125–141. Fig.5 a shows the MS/MS spectrum of the ion at m/z 950.6 [M + 2H]2+ corresponding to Peak 2 (Table II and Fig. 3 a) from PC1. The masses of the Y" series ions from Y"2 through Y"13 were consistent with that of a peptide composed of residues 125–141 (see Scheme 1 in Fig. 5 a, inset). The MS/MS spectrum of the ion at m/z 985.6 [M + 2H]2+, corresponding to Peak 2A (Table II and Fig. 3 b) of the modified peptide from TZB-PC1, is shown in Fig. 5 b. This MS/MS spectrum has a similar fragmentation pattern to that of the peptide composed of amino acid residues 125–141. The same Y" series of ions from Y"2 through Y"11 were observed in both spectra. An additional fragment ion at m/z 1417 (Y"12 + 70) was observed for the Peak 2A peptide, indicating that Ser-130 was the attachment point of the 70-Da TZB fragment. The peptides corresponding to Peak 9 (Table III) and Peak 9A (Table III) from PC1 and TZB-PC1, respectively, following digestion with trypsin, were analyzed by HPLC/ESI/MS/MS. The sequences of these peptides were confirmed to be amino acid residues 66–73. The residue modified by TZB was also determined. Fig. 6 compares the MS/MS spectra for Peak 9 (Fig. 6 a) and Peak 9A (Fig.6 b). The masses of the Y" series ions from Y"1through Y"7 in both spectra are consistent with the amino acid sequence of residues 66–73 (see Scheme 2 in Fig. 6 a, inset). The TZB moiety of the modified peptide is readily lost in the collisionally activated dissociation process, resulting in a predominant peak at m/z 874, a 70-Da lower mass component than the parent ion (m/z 944). The TZB-PC1 adduct is labile under collisionally activated dissociation conditions. The collisionally activated dissociation favors the loss of the TZB moiety prior to the Y" cleavages, hence the abundance of the Y" + 70 adducts are weak and sometimes absent. Similar observations withO-glycosylated peptides were previously reported (18Mirgorodskaya E. Roepstorff P. Zubarev R.A. Anal. Chem. 1999; 77: 4431-4436Crossref Scopus (348) Google Scholar). Two fragment ions, Y"4 + 70 (m/z 492) and Y"5 + 70 (m/z 563), were observed only in the spectrum for the TZB-modified peptide (Fig. 6 b). Their existence suggests that Ser-70 is the site of modification by the TZB fragment. Interestingly, a series of fragment ions were observed (marked with asolid circle (●) in Fig. 6 b) corresponding to dehydrated Y" ions. One fragment ion was observed at m/z856. This represented an 18-Da mass loss from the peptide composed of residues 66–73 at m/z 874 (Fig. 6 b). This finding is in agreement with the observation of a dehydrated peptide composed of residues 58–82 in the Glu-C-digested PC1-TZB complex (Peak 9B, Table II, and Fig. 4 b). In addition, four more fragment ions with an 18-Da mass loss for the Y" series were observed (Fig.6 b) at m/z 709 compared with Y"7(m/z 727); at m/z 638 compared with Y"6 (m/z 656); at m/z 475 compared with Y"5 (m/z 493); and at m/z 404 compared with Y"4 (m/z 422). The 18-Da mass loss fragments were only observed with the higher mass fragment ions containing Ser-70 but not with the lower mass fragment ions lacking Ser-70, namely Y"1, Y"2, and Y"3. The presence of these ions in the TZB-treated peptide sample and the absence in the untreated sample suggested that their appearance is linked to the presence of the 70-Da fragment from TZB. One possibility is that during the HPLC/ESI/MS/MS procedure, the TZB adduct is cleaved from the peptide as an 88-Da fragment that includes an OH group from the attached serine. If this is true, it would lend further support for the attachment of the TZB adduct to Ser-70. ESI/MS technology has proven to be a powerful tool in analyzing the molecular weights of biomolecules. This soft ionization technique has been applied to the detection of covalent β-lactamase substrate and β-lactamase-clavulanate complexes (6Brown R.P. A Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar, 19Aplin, R. T., Robinson, C. V., Schofield, C. J., and Waley, S. G. (1993) J. Chem. Soc. Chem. Commun.121–123Google Scholar, 20Saves I. Burlet-Schiltz O. Maveyraud L. Samama J.-P. Prome J.-C. Masson J.-M. Biochememistry. 1995; 34: 11660-11667Crossref PubMed Scopus (51) Google Scholar, 21Payne D.J. Bateson J.H. Gasson B. Proctor D. Krushi T. Farmer T.J. 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). Here we present an analysis of the inactivation mechanism of the potent penicillanic sulfone β-lactamase inhibitor, tazobactam, on the class A β-lactamases, PC1 and TEM-1, using various applications of mass spectrometry. ESI/MS was used to identify the masses of the TZB fragments following reaction with the enzymes. HPLC/ESI/MS confirmed the findings of ESI/MS and detected the TZB-modified PC1 peptides composed of amino acid residues 58–82, 66–73, and 125–141. In place of HPLC purification and chemical amino acid sequence analysis, HPLC/ESI/MS/MS was used to provide supportive evidence for the TZB-reactive PC1 serine residues, Ser-70 and Ser-130. The data presented here are the first complete study of the mechanism of inactivation by a penicillanic sulfone inhibitor with class A β-lactamases and are the first based solely on mass spectrometry techniques. A scheme depicting the process of inactivation of class A β-lactamases by TZB is proposed (Scheme FS3) and is found to be consistent with the mass spectrometry data (22Yang, Y., Tabei, K., Siegel, M. M., Lin, Y.-I., Rasmussen, B. A., and Shlaes, D. M. (1997) in Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Toronto, Canada, September, 28-October 1, 1997, C-210, p.82, American Society for Microbiology, Washington, D. C.Google Scholar). The increased masses observed by ESI/MS of ∼52, ∼70, and ∼88 Da for TZB-PC1 were also identified by HPLC/ESI/MS performed on the proteolytic digested TZB-PC1 samples. The ∼52-Da increase corresponded to the formation of proposed intermediate 11,the propiolylated enzyme that could have formed from decomposition of intermediates 4, 5, and 7. The mass shift of ∼70 Da corresponded to intermediates 8 and10. HPLC/ESI/MS/MS analysis indicated that ∼70 Da modifications occurred at Ser-70, 8, and Ser-130,10. The ∼88-Da increment corresponded to the hydrated aldehyde 9. Using ESI/MS and FAB/MS, components with molecular masses of 248, 264, and 280 Da were identified as products of the reaction of TZB with PC1. Exact masses for these reaction products enabled the determination of their molecular formula and proposed structures based upon ESI/MS/MS and FAB/MS (17Tabei K. Siegel M.M. Yang Y. Lin Y-I. Huang N. Rasmussen B.A. Shlaes D. Proceedings of the American Society for Mass Spectrometry and Allied Topics, 46th ASMS Conference, Orlando, FL, May 31-June 4, 1998. American Society for Microbiology, Washington, D. C.1998: 186Google Scholar). The observed ESI/MS, ESI/MS/MS, and FAB/MS data were consistent with the release of a sulfinic acid 6 (MW 248), which underwent further oxidation to form the products of sulfonic acids 12 (MW 264) and 13 (MW 280) (17Tabei K. Siegel M.M. Yang Y. Lin Y-I. Huang N. Rasmussen B.A. Shlaes D. Proceedings of the American Society for Mass Spectrometry and Allied Topics, 46th ASMS Conference, Orlando, FL, May 31-June 4, 1998. American Society for Microbiology, Washington, D. C.1998: 186Google Scholar). These results are consistent with the rapid rearrangement of the TZB adduct 2 to intermediates 3, 4, and5, followed by the loss of 6 from imine3 or enamines 4 and 5. Among the components observed in the full-length protein by ESI/MS analysis of TZB-PC1 was a minor component with a mass increment of ∼300 Da relative to native PC1. This ∼300-Da mass increment component corresponded to some or all of the proposed reaction intermediates 2, 3, 4, and5. This is consistent with the proposed mechanistic scheme whereby TZB and the enzyme initially form the acyl-enzyme complex2, which is identical to that formed with the β-lactam substrates of these enzymes. This 300-Da adduct was not observed in ESI/MS analysis of the TZB-TEM-1 reaction product, indicating the rapid loss of compound 6 and formation of products 7, 8, and 9. After acylation, the TZB-PC1 adduct2 undergoes β-elimination resulting in the opening of the sulfone containing the five-membered ring to form the imine acyl-enzyme complex 3. Imine 3 tautomerizes to the enamines4 and 5. Previous UV spectrophotometric studies of TZB-PC1 (7Bush K. Macalintal C. Rasmussen B.A. Lee V. Yang Y. Antimicrob. Agents Chemother. 1993; 37: 851-858Crossref PubMed Scopus (235) Google Scholar) are consistent with the formation of imine and enamine forms of the ring-opened β-lactamase inhibitor. The observed TZB-PC1 adduct with a 52-Da mass increment 11, the propiolylated enzyme, can be rationalized to arise from 4 and5. In addition, rearrangement of compound 7 also forms compound 11. Following the initial acylation, interaction of Ser-130 with intermediates 3, 4,or 5 and the displacement of compound 6 form a cross-linked vinyl ether 7. Hydrolysis of compound7 produces structure 10. Hydrolysis of the imine and enamine acyl enzyme complexes 3, 4, or5 and elimination of 6 lead to structure8. Hydrolysis of compound 7 also forms compound8. Further hydrolysis of compound 8 gives compound 9. Hydrolysis of the inhibitor adducts8, 9, 10, and 11 regenerate active enzymes. Most of the observed reaction products of CA-inactivated TEM-2 (6Brown R.P. A Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar) were also identified in TZB inactivation products of class A β-lactamases, except the short lived imine and enamine intermediates. The binding residues, Ser-70 and Ser-130, proposed by HPLC/ESI/MS/MS data for TZB-PC1, are identical to the binding residues in TEM-2 for CA. In the CA-TEM-2 study, the component that exhibited a 52-Da mass increment was detected only in the full-length protein. This is likely the same 52-Da adduct represented by structure 7. However, no 52-Da adducts were reported following proteolysis and HPLC/ESI/MS. In this study, a 52-Da adduct was identified for the Glu-C-digested TZB-PC1 peptide composed of amino acid residues 58–82. This component is most likely represented by structure 11 in Scheme FS3. The presence of the vinyl ether cross-linked with both Ser-70 and Ser-130, 7, as an intermediate in β-lactamase inactivation by TZB, was not identified by endoproteolysis followed by HPLC/ESI/MS. The involvement of Ser-130 in the inactivation of class A β-lactamases by CA was first suggested by Imtiaz et al. (9Imtiaz U. Billings E.M. Knox J.R. Manavathu E., K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar). It was proposed that Ser-130 facilitates the collapse of the tetrahetral intermediate by hydrogen bonding to the β-lactam amine. In support of this, Brownet al. (6Brown R.P. A Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar) reported a 70-Da adduct attached to the TEM-2 peptide composed of amino acid residues 121–146. The 70-Da TZB fragment, attached to the peptide composed of PC1 amino acid residues 125–141, was identified after Glu-C digestion of TZB-PC1. Attachment of the fragment to Ser-130 was supported by the analysis of the HPLC/ESI/MS/MS data. Thus, Ser-130 is also directly involved in β-lactamase inactivation by sulfone inhibitors. In summary, our findings indicate that although clavams and sulfones are structurally distinct, the inactivation process of class A β-lactamases by CA and TZB are very similar, especially after the formation of the acylimine inhibitor-enzyme complex.

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Fourceftazidime-resistantEscherichiacolistrainswereisolatedfromelderlynursinghomepatientsinaNew York hospital during 1993. Strains MCQ-2, MCQ-3, and MCQ-4 were determined to be identical by pulsed- field gel electrophoresis and plasmid profiles, whereas strain MCQ-1 was unique. Strain MCQ-1 was deter- minedtoproduceaTEM-10 b-lactamase.StrainsMCQ-2,MCQ-3,andMCQ-4werealsonotedtoberesistant to cefotaxime. These three strains produced two b-lactamases with pIs of 5.4 (TEM-1) and 7.6. b-Lactamase assays revealed that the pI 7.6 enzyme hydrolyzed cefotaxime faster (at a relative hydrolysis rate of 30% compared with that of benzylpenicillin) than either ceftazidime or aztreonam (relative hydrolysis rates of 13 and3.3%,respectively).NucleotidesequencingofthegeneencodingthepI7.6 b-lactamasefromstrainMCQ-3 revealedablaSHV-typegenedifferingfromthegeneencodingSHV-1atfournucleotideswhichresultedinamino acid substitutions: phenylalanine for isoleucine at position 8, serine for arginine at position 43, serine for glycine at position 238, and lysine for glutamate at position 240. This novel SHV-type extended-spectrum b-lactamase is designated SHV-7.