HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 52, 52724-52729, December 26, 2003

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 278/52/52724    most recent M306059200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Helfand, M. S. Articles by Bonomo, R. A. Search for Related Content PubMed PubMed Citation Articles by Helfand, M. S. Articles by Bonomo, R. A. Social Bookmarking                      What's this?

Understanding Resistance to -Lactams and -Lactamase Inhibitors in the SHV -Lactamase

LESSONS FROM THE MUTAGENESIS OF SER-130^*

Marion S. Helfand, Christopher R. Bethel, Andrea M. Hujer, Kristine M. Hujer, Vernon E. Anderson¶, and Robert A. Bonomo||

From the Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and ^¶Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, June 9, 2003 , and in revised form, October 8, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial resistance to -lactam/-lactamase inhibitor combinations by single amino acid mutations in class A -lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A -lactamases, alterations at Ser-130 confer resistance to inactivation by the -lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 -lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G -lactamase expressed in Escherichia coli. Kinetic analysis of the S130G -lactamase demonstrated a significant elevation in apparent K_m and a reduction in k_cat/K_m for ampicillin. Marked increases in the dissociation constant for the preacylation complex, K_I, of clavulanic acid (SHV-1, 0.14 µM; S130G, 46.5 µM) and tazobactam (SHV-1, 0.07 µM; S130G, 4.2 µM) were observed. In contrast, the k_inacts of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for -lactams and -lactamase inhibitors. These results underscore the mechanistic versatility of class A -lactamases and have implications for the design of novel -lactamase inhibitors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
-Lactamase inhibition is an important strategy to combat the escalating problem of -lactam resistance mediated by bacterial -lactamases (EC 3.5.2.6 [EC] ). Combined with a -lactam antibiotic, -lactamase inhibitors preserve and expand the utility of the partner antibiotic and offer a greater spectrum of microbiological activity. At present, three -lactamase inhibitors are available for clinical use: clavulanic acid, sulbactam, and tazobactam. Combined with a -lactam antibiotic (e.g. amoxicillin/clavulanic acid, ampicillin/sulbactam, ticarcillin/clavulanic acid, and piperacillin/tazobactam), these inhibitors serve as essential therapeutic agents in the treatment of life-threatening infections (1–3) (Fig. 1).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Chemical structures of class A -lactamase inhibitors.

It is particularly noteworthy that, among the inhibitor-resistant class A -lactamases, the S130G substitution in TEM and SHV involves the conserved motif Ser-130/Asp-131/Asn-132, the "SDN" loop. Ser-130 and Asn-132 are hydrogen-bonded to the catalytically important Lys-73 in the active site. This observation challenged our understanding of the role of Gly at the 130 position in inhibitor-resistant variants of the SHV and TEM -lactamases (e.g. SHV-10 and TEM-59) (5, 13). Exploring the consequences of amino acid substitutions on catalytic activity, we show that the apparent affinity of the active site is greatly reduced for substrates and inhibitors. In contrast, S130G and SHV-1 -lactamases are inactivated at nearly the same rate. Our hypothesis, based on a molecular model, is that movement of a catalytic water molecule may potentially serve the same role as the hydroxyl group of Ser-130 in substrate binding and catalysis. These results have implications for the future design of -lactams and -lactamase inhibitors.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains and Plasmids—The chromosomal SHV-1 -lactamase with its native promoter and ribosomal binding site was cloned into the recombinant phagemid vector, pBC SK(–) (Stratagene, La Jolla, CA) as described previously (14). Plasmid DNA was obtained from Epicurean coli^TM XL1 Blue (Stratagene, La Jolla, CA) and Escherichia coli DH10B (Invitrogen) cells using Wizard^TM Purification kits (Promega, Madison, WI).

Mutagenesis—PCR-based site-saturation and site-directed mutagenesis was performed using a QuikChange^TM mutagenesis kit (Stratagene), as reported (9, 15). Two complementary degenerate oligonucleotides (Genosys Biotechnologies, The Woodlands, TX) were first constructed for site-saturation mutagenesis at the Ser-130 position. For site-directed mutagenesis, specific oligonucleotides were designed based upon common codon usage (Genosys Biotechnologies).

DNA Sequencing—We performed DNA sequencing with an A.L.F. Express automated DNA sequencer (Amersham Biosciences) using the Thermo Sequenase^TM fluorescent-labeled primer cycle sequencing kit in a manner similar to methods published previously (8, 9, 15).

Antibiotic Susceptibility—E. coli DH10B expressing the mutated bla_SHV genes were phenotypically characterized by LB agar dilution minimum inhibitory concentrations (MICs)² using a "Steers replicator" that delivered 10⁴ colony forming units/spot. Antibiotics used and their suppliers were described previously (8, 9, 15). Concentrations employed for determining MIC values were in µg/ml. MICs were read after 18–24 h of incubation at 37 °C and performed three times for each antibiotic.

Determination of Steady-state Expression Using an Enzyme-linked Immunosorbent Assay—By using a polyclonal anti-SHV antibody, a quantitative enzyme linked immunosorbent assay (ELISA) method was employed to assess steady-state expression levels (16). The ELISA plates were read at = 492 nm and OD values of the samples were compared with an internal standard curve using the SHV-1 -lactamase.

-Lactamase Purification—SHV-1, S130G, and S130T -lactamases were purified to homogeneity from E. coli DH10B cells according to a method employing preparative isoelectric focusing (8, 9, 15). We assessed the purity of each -lactamase on 5% stacking and 12% resolving SDS-PAGE gels. Gels were stained with Coomassie Brilliant Blue R250 (Fisher) to visualize -lactamases. Protein concentrations were determined with Protein Assay (Bio-Rad) using bovine serum albumin as a standard.

Kinetics—Kinetic constants of the SHV-1, S130G and S130T -lactamases were determined by continuous assays at room temperature, 25 °C, using an Agilent^TM 8453 diode array spectrophotometer. Each experiment was performed in 20 mM phosphate-buffered saline at pH 7.4. Measurements were obtained using ampicillin (₂₃₅ = –900 M^–1cm^–1), piperacillin (₂₃₂ = –5300 M^–1 cm^–1), cephalothin (₂₆₂ = –7660 M^–1 cm^–1), and nitrocefin ( ₄₈₂ = 17400 M^–1 cm^–1) (BD Biosciences). The kinetic experiments and analyses were patterned after the methods of Imtiaz et al. (17) and will be described briefly below.

The kinetic parameters, V_max and K_m, were obtained with non-linear least squares fit of the data (Michaelis-Menten equation) using Enzfitter^TM (Sigma):

(Eq. 1)

A direct competition assay was performed to determine the dissociation constant for the preacylation complex, K_I, of the inhibitors (clavulanic acid and tazobactam). We used a final concentration of 100 µM nitrocefin as the indicator substrate and 7 nM SHV-1 -lactamase or 31 nM S130G -lactamase in these determinations. The data were analyzed according to Equation 2 to account for the affinity of nitrocefin for the SHV-1 -lactamase:

(Eq. 2)

The first-order rate constant for enzyme and inhibitor complex being inactivated, k_inact was measured directly by monitoring the reaction time courses in the presence of inactivators. A fixed concentration of enzyme, nitrocefin, and increasing nM concentrations of inactivator, I, were used in each assay. The k_obs for inactivation was determined graphically as the reciprocal of the ordinate of the intersection of the straight lines obtained from the initial, v₀, and final, v_f, steady-state velocities. The k_obs was calculated from the relationship k_obs = 1/t. Each k_obs was plotted versus I and fit to Equation 3 to determine k_inact:

(Eq. 3)

The second-order rate constant, k_inact /K_I (the rate constant for reaction of free enzyme with free inhibitor to give inactive enzyme) was determined by the fit of k_obs to Equation 3.

The partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation, i.e. the turnover number (t_n = k_cat/k_inact) was obtained in the following manner. First, we incubated increasing amounts of inhibitor (clavulanic acid or tazobactam) with a fixed concentration of SHV-1 or S130G -lactamase in a total volume of 100 µl of 20 mM phosphate-buffered saline, pH 7.4, at room temperature. After 24 h, an aliquot (10 µl) was removed from the mixture and the steady-state velocity was measured and compared with a control sample with no inhibitor added. The proportion of clavulanic acid or tazobactam relative to SHV-1 or S130G that resulted in 90% inactivation after 24 h was used to determine the ratio of inhibitor to enzyme in a 90-min experiment. We reacted 1.6 µM SHV-1 and 25 µM S130G with 60 µM and 1 mM clavulanic acid, respectively, in a final volume of 200 µl. Similarly, we reacted 1.6 µM SHV-1 and 25 µM S130G with 7.75 µM and 125 µM of tazobactam, respectively. From these mixtures, a set aliquot was removed at designated time points, and steady-state velocities were measured.

The contributions of amino acid substitutions to the relative change in Gibbs free energy of the transition state with substrates and inhibitors were determined by using Equations 4 and 5 (18–20):

(Eq. 4)

(Eq. 5)

Molecular Modeling—Insight II^TM software (Accelrys) was used to construct the S130G variant and to perform energy minimizations as described previously (9) based on the SHV-1 crystal structure (Protein Data Bank code 1SHV [PDB] ) (21).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mutagenesis—Site-saturation and site-directed mutagenesis to obtain all 19 amino acid substitutions at the 130 position in SHV-1 was successfully performed, and the most common codons used were selected (8). The DNA sequence of each mutant bla_SHV was first determined in E. coli^TM XL1 Blue cells and confirmed in E. coli DH10B cells.

Antibiotic Susceptibility—The effects of each amino acid substitution at the 130 position in the SHV -lactamase expressed in E. coli DH10B are summarized in Table I (also Fig. 1 and Table I in "Supplemental Material"). Only the S130G and S130T -lactamases expressed in E. coli were ampicillin resistant (MICs 32 µg/ml). Resistance to ampicillin/clavulanic acid was demonstrated by the E. coli DH10B strain with the S130G -lactamase. None of the variant -lactamases expressed in E. coli conferred more resistance than SHV-1 against ampicillin/tazobactam or piperacillin/tazobactam.

View this table: [in this window] [in a new window]   TABLE I MICS (µg/ml) for representative antibiotics against E. coli DH10B with SHV-1, S130G, S130T, and S130X -lactamases

Kinetic Parameters of SHV-1 and S130G -Lactamases—The steady-state kinetic parameters are reported in Table II. Large increases in K_m were demonstrated by S130G -lactamase for nitrocefin (700 µM, 140-fold increase). For the S130G -lactamase the k_cat values were reduced for ampicillin and nitrocefin. The k_cat/K_m ratios clearly show that the S130G mutation markedly reduced catalytic efficiency. Compared with SHV-1 -lactamase, S130G is 3.6% as efficient catalyzing ampicillin and 0.14% as efficient hydrolyzing nitrocefin (Table III).

View this table: [in this window] [in a new window]   TABLE III Relative binding energies of the transition-state species of the slow step in turnover of the given substrate by S130G and SHV-1 -lactamases

View larger version (11K): [in this window] [in a new window]   FIG. 2. Time-dependent inactivation experiments. A, clavulanic acid inactivation of SHV-1 -lactamase () and S130G -lactamase (). B, tazobactam inactivation of SHV-1 -lactamase () and S130G -lactamase ().

View larger version (26K): [in this window] [in a new window]   FIG. 3. Stereo view of the superposition of the energy-minimized active-site structures of SHV-1 wild type (shown colored by element) and the S130G SHV variant (shown in magenta). Key active-site residues are highlighted as well as the catalytic water molecule (CAT H2O). These structures were obtained using the AMBER force field.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In addition to these findings, two important observations of the kinetic behavior of SHV-1 and S130G -lactamases with clavulanic acid and tazobactam deserve emphasis: S130G and SHV-1 -lactamases are inactivated by clavulanic acid at similar rates, and hydrolysis rates are comparable. Analogous results were obtained with tazobactam. These data indicate that the S130G -lactamase, as a clinically important -lactam/-lactamase inhibitor-resistant enzyme, maintains the fundamental hydrolytic mechanism common to all -lactamases (12).

From our model (Fig. 3), we hypothesized that an active-site water molecule may relocate to a region nearer to the Lys-73 residue. The water molecule may also function with Lys-73 in a coordinated proton shuttle and may substitute for the Ser-130 in protonating the -lactam nitrogen (26, 27). In the x-ray structures of the inhibitor resistant TEM-30 (R244S) and TEM-84 (N276D), the relocation of a conserved water plays a crucial role in the binding affinity of inhibitors (10, 12). The molecular modeling studies of the E166N amino acid substitution of TEM-1 -lactamase revealed the movement of a catalytic water molecule was essential in modification of the substrate profile (28). In a similar manner, movement of a water molecule in the active site of S130G may be an important process that preserves catalytic activity.

The mechanism by which S130G is inhibited by clavulanic acid or tazobactam challenges our understanding of the catalytic pathway to inactivation. By using the x-ray structure of SHV-1 inhibited by tazobactam as a guide, we hypothesize that after acylation of S130G by clavulanic acid 1, the reactive imine 2 is formed (Fig. 4). The imine rearranges to form the cis- and trans-enamine 3, 4. This stable intermediate could account for the inhibition observed in our experiments and would occur in both the variant and wild-type enzymes. The absence of the side chain of Ser-130 would deprive this enzyme of the hydroxyl group that acts as the necessary nucleophile required in the final irreversible inactivation step (6, 22). Hence, formation of the cross-linking or bridging species (vinyl ether), and the Ser-130 bound carboxylic acid moiety would not be possible in the S130G -lactamase. However, nucleophilic attack by an active-site water molecule could yield the aldehyde 5 or hydrate aldehyde 6. This also may also lead to inhibition. Lastly, the aldehyde 5 or hydrate aldehyde 6 could undergo hydrolysis and regenerate the active enzyme 7 (22). Alternatively, the imine 2 could be directly hydrolyzed to yield the aldehyde 5 and hydrate aldehyde 6. This pathway is entirely consistent with that proposed for the inhibitor-resistant TEM-33 (M69L) and N276D-substituted enzymes (10, 11). Our kinetic data for tazobactam and clavulanic acid suggest that the rate that each intermediate is formed and the number of branch points differ for each inhibitor. Experiments are in progress attempting to define the predominate pathway for clavulanic acid and tazobactam in inhibitor-resistant SHV enzymes. The design of future -lactam and -lactamase inhibitors should take into account that alterations in the oxyanion hole and free movement of water molecules in the active site will permit more than one pathway to catalysis and inhibition. A deeper appreciation of these alternative mechanisms may avert the unwelcome emergence of even more resistant -lactam bacteria because of excessive -lactam use.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Proposed mechanism of inactivation of S130G -lactamase with clavulanic acid (adapted from Refs. 10, 20, 21).

	FOOTNOTES

The on-line version of this article (available at http://www.jbc.org) contains supplementary Fig. 1.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed: Infectious Disease Section, Louis Stokes Cleveland Dept. of Veterans Affairs Medical Ctr., 10701 East Blvd., Cleveland, OH 44106. Tel.: 216-791-3800 (ext. 4399); Fax: 216-231-3482; E-mail: robert.bonomo{at}med.va.gov.

¹ Amino Acid Sequences for TEM, SHV and OXA Extended-Spectrum and Inhibitor Resistant -Lactamases, available at www.lahey.org/studies/webt.htm.

² The abbreviations used are: MIC, minimum inhibitory concentration; ELISA, enzyme linked immunosorbent assay.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Gorbach, S. L. (1994) Intensive Care Med. 20, S27–S34
2. Fowler, R. A., Flavin, K. E., Barr, J., Weinacker, A. B., Parsonnet, J., and Gould, M. K. (2003) Chest 123, 835–844[Abstract/Free Full Text]
3. Wilson, S. E., and Nord, C. E. (1995) Am. J. Surg. 169, 21S–26S[CrossRef][Medline] [Order article via Infotrieve]
4. Helfand, M. S., and Bonomo, R. A. (2003) Curr. Drug Targets Infect. Disord. 3, 9–23[CrossRef][Medline] [Order article via Infotrieve]
5. Prinarakis, E. E., Miriagou, V., Tzelepi, E., Gazouli, M., and Tzouvelekis, L. S. (1997) Antimicrob. Agents Chemother. 41, 838–840[Abstract]
6. Kuzin, A. P., Nukaga, M., Nukaga, Y., Hujer, A. M., Bonomo, R. A., and Knox, J. R. (2001) Biochemistry 40, 1861–1866[CrossRef][Medline] [Order article via Infotrieve]
7. Vakulenko, S. B., Geryk, B., Kotra, L. P., Mobashery, S., and Lerner, S. A. (1998) Antimicrob. Agents Chemother. 42, 1542–1548[Abstract/Free Full Text]
8. Hujer, A. M., Hujer, K. M., and Bonomo, R. A. (2001) Biochim. Biophys. Acta 1547, 37–50[CrossRef][Medline] [Order article via Infotrieve]
9. Helfand, M. S., Hujer, A. M., Sonnichsen, F. D., and Bonomo, R. A. (2002) J. Biol. Chem. 277, 47719–47723[Abstract/Free Full Text]
10. Swaren, P., Golemi, D., Cabantous, S., Bulychev, A., Maveyraud, L., Mobashery, S., and Samama, J. P. (1999) Biochemistry 38, 9570–9576[CrossRef][Medline] [Order article via Infotrieve]
11. Meroueh, S. O., Roblin, P., Golemi, D., Maveyraud, L., Vakulenko, S. B., Zhang, Y., Samama, J.-P., and Mobashery, S. (2002) J. Am. Chem. Soc. 124, 9422–9430[CrossRef][Medline] [Order article via Infotrieve]
12. Wang, X., Minasov, G., and Shoichet, B. K. (2002) J. Biol. Chem. 277, 32149–32156[Abstract/Free Full Text]
13. Bermudes, H., Jude, F., Chaibi, E. B., Arpin, C., Bebear, C., Labia, R., and Quentin, C. (1999) Antimicrob. Agents Chemother. 43, 1657–1661[Abstract/Free Full Text]
14. Rice, L. B., Carias, L. L., Hujer, A. M., Bonafede, M., Hutton, R., Hoyen, C., and Bonomo, R. A. (2000) Antimicrob. Agents Chemother. 44, 362–367[Abstract/Free Full Text]
15. Hujer, A. M., Hujer, K. M., Helfand, M. S., Anderson, V. E., and Bonomo, R. A. (2002) Antimicrob. Agents Chemother. 46, 3971–3977[Abstract/Free Full Text]
16. Hujer, A. M., Page, M. G., Helfand, M. S., Yeiser, B., and Bonomo, R. A. (2002) J. Clin. Microbiol. 40, 1947–1957[Abstract/Free Full Text]
17. Imtiaz, U., Billings, E. M., Knox, J. R., Manavathu, E. K., Lerner, S. A., and Mobashery, S. (1993) J. Am. Chem. Soc. 115, 4435–4442[CrossRef]
18. Lin, S., Thomas, M., Shlaes, D. M., Rudin, S. D., Knox, J. R., Anderson, V. E., and Bonomo, R. A. (1998) Biochem. J. 333, 395–400
19. Fersht, A. (1984) Enzyme Structure and Mechanism, 2nd edition, W. H. Freeman and Company, New York
20. Zafaralla, G., Manavathu, E. K., Lerner, S. A., and Mobashery, S. (1992) Biochemistry 31, 3847–3852[CrossRef][Medline] [Order article via Infotrieve]
21. Kuzin, A. P., Nukaga, M., Nukaga, Y., Hujer, A. M., Bonomo, R. A., and Knox, J. R. (1999) Biochemistry 38, 5720–5727[CrossRef][Medline] [Order article via Infotrieve]
22. Brown, R. P. A., Aplin, R. T., and Schofield, C. J. (1996) Biochemistry 35, 12421–12432[CrossRef][Medline] [Order article via Infotrieve]
23. Juteau, J. M., Billings, E., Knox, J. R., and Levesque, R. C. (1992) Protein Eng. 5, 693–701[Abstract/Free Full Text]
24. Strynadka, N. C. J., Adachi, H., Jensen, S. E., Johns, K., Sielecki, A., Betzel, C., Sutoh, K., and James, M. N. G. (1992) Nature 359, 700–705[CrossRef][Medline] [Order article via Infotrieve]
25. Jacob, F., Joris, B., Lepage, S., Dusart, J., and Frere, J. M. (1990) Biochem. J. 271, 399–406[Medline] [Order article via Infotrieve]
26. Imtiaz, U., Billings, E. M., Knox, J. R., and Mobashery, S. (1994) Biochemistry 33, 5728–5738[CrossRef][Medline] [Order article via Infotrieve]
27. Galleni, M., Lamotte-Brasseur, J., Raquet, X., Dubus, A., Monnaie, D., Knox, J. R., and Frere, J. M. (1995) Biochem. Pharmacol. 49, 1171–1178[CrossRef][Medline] [Order article via Infotrieve]
28. Guillaume, G., Vanhove, M., Lamotte-Brasseur, J., Ledent, P., Jamin, M., Joris, B., and Frere, J. M. (1997) J. Biol. Chem. 272, 5438–5444[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				P. Pattanaik, C. R. Bethel, A. M. Hujer, K. M. Hujer, A. M. Distler, M. Taracila, V. E. Anderson, T. R. Fritsche, R. N. Jones, S. R. R. Pagadala, et al. Strategic Design of an Effective {beta}-Lactamase Inhibitor: LN-1-255, A 6-ALKYLIDENE-2'-SUBSTITUTED PENICILLIN SULFONE J. Biol. Chem., January 9, 2009; 284(2): 945 - 953. [Abstract] [Full Text] [PDF]

				C. R. Bethel, A. M. Distler, M. W. Ruszczycky, M. P. Carey, P. R. Carey, A. M. Hujer, M. Taracila, M. S. Helfand, J. M. Thomson, M. Kalp, et al. Inhibition of OXA-1 {beta}-Lactamase by Penems Antimicrob. Agents Chemother., September 1, 2008; 52(9): 3135 - 3143. [Abstract] [Full Text] [PDF]

				N. Mendonca, V. Manageiro, F. Robin, M. J. Salgado, E. Ferreira, M. Canica, and R. Bonnet The Lys234Arg Substitution in the Enzyme SHV-72 Is a Determinant for Resistance to Clavulanic Acid Inhibition Antimicrob. Agents Chemother., May 1, 2008; 52(5): 1806 - 1811. [Abstract] [Full Text] [PDF]

				C. R. Bethel, A. M. Hujer, K. M. Hujer, J. M. Thomson, M. W. Ruszczycky, V. E. Anderson, M. Pusztai-Carey, M. Taracila, M. S. Helfand, and R. A. Bonomo Role of Asp104 in the SHV {beta}-Lactamase Antimicrob. Agents Chemother., December 1, 2006; 50(12): 4124 - 4131. [Abstract] [Full Text] [PDF]

				D. Sulton, D. Pagan-Rodriguez, X. Zhou, Y. Liu, A. M. Hujer, C. R. Bethel, M. S. Helfand, J. M. Thomson, V. E. Anderson, J. D. Buynak, et al. Clavulanic Acid Inactivation of SHV-1 and the Inhibitor-resistant S130G SHV-1 {beta}-Lactamase: INSIGHTS INTO THE MECHANISM OF INHIBITION J. Biol. Chem., October 21, 2005; 280(42): 35528 - 35536. [Abstract] [Full Text] [PDF]

				A. M. Hujer, M. Kania, T. Gerken, V. E. Anderson, J. D. Buynak, X. Ge, P. Caspers, M. G. P. Page, L. B. Rice, and R. A. Bonomo Structure-Activity Relationships of Different {beta}-Lactam Antibiotics against a Soluble Form of Enterococcus faecium PBP5, a Type II Bacterial Transpeptidase Antimicrob. Agents Chemother., February 1, 2005; 49(2): 612 - 618. [Abstract] [Full Text] [PDF]

				D. Pagan-Rodriguez, X. Zhou, R. Simmons, C. R. Bethel, A. M. Hujer, M. S. Helfand, Z. Jin, B. Guo, V. E. Anderson, L. M. Ng, et al. Tazobactam Inactivation of SHV-1 and the Inhibitor-resistant Ser130 -> Gly SHV-1 {beta}-Lactamase: INSIGHTS INTO THE MECHANISM OF INHIBITION J. Biol. Chem., May 7, 2004; 279(19): 19494 - 19501. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 278/52/52724    most recent M306059200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Helfand, M. S. Articles by Bonomo, R. A. Search for Related Content PubMed PubMed Citation Articles by Helfand, M. S. Articles by Bonomo, R. A. Social Bookmarking                      What's this?