©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Asparagine to Aspartic Acid Substitution at Position 276 of TEM-35 and TEM-36 Is Involved in the -Lactamase Resistance to Clavulanic Acid (*)

(Received for publication, April 17, 1995; and in revised form, May 24, 1995)

Isabelle Saves(§) (1) Odile Burlet-Schiltz(§) (1) Peter Swarn(§) (2) Fabrice Lefvre (1) Jean-Michel Masson (1) (4)(¶) Jean-Claude Prom (3) Jean-Pierre Samama (2)

From the  (1)Groupe d'Ingnierie des Protines, the (2)Groupe de Cristallographie Biologique, and the (3)Groupe de Spectromtrie de Masse, Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex, France and the (4)Institut National des Sciences Appliques, Complexe Scientifique de Rangueil, 31077 Toulouse Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7) clavulanic acid-resistant beta-lactamases have evolved from TEM-1 beta-lactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276. The substitutions at position 69 have previously been shown to be responsible for the resistance to clavulanic acid, and they are the only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6). However, the N276D substitution has never been found alone in inhibitor-resistant beta-lactamases, and its role in resistance to clavulanic acid was thus unclear. The N276D mutant was constructed, purified, and kinetically characterized. It was shown that the substitution has a direct effect on substrate affinities and leads to slightly decreased catalytic efficiencies and that clavulanic acid becomes a poor substrate of the enzyme. Electrospray mass spectrometry demonstrated the simultaneous presence of free and inhibited enzymes after incubation with clavulanic acid and showed that a cleaved moiety of clavulanic acid leads to the formation of the major inactive complex. The kinetic properties of the N276D mutant could be linked to a salt-bridge interaction of aspartic acid 276 with arginine 244 that alters the electrostatic properties in the substrate binding area.


INTRODUCTION

The production of beta-lactamases (EC 3.5.2.6) is the major mechanism used by Gram-negative bacteria to develop resistance to beta-lactam antibiotics, and TEM-1 is the most currently encountered plasmid-mediated beta-lactamase within this group. This enzyme, which efficiently hydrolyzes penicillins and first and second generation cephalosporins, is not active against third generation cephalosporins. However, it is able to evolve and rapidly confer bacterial resistance to the newer beta-lactam drugs, such as cefotaxime and ceftazidime (Paul et al., 1988; Sougakoff et al., 1989). This new hydrolysis ability results from one to four substitutions in the vicinity of the active site of TEM-1 beta-lactamase (Jacoby and Medeiros, 1991).

To overcome this constant evolution of TEM-1, suicide inhibitors of the enzyme such as clavulanic acid and sulbactam are used clinically in combination with penicillins. Unfortunately, mutations occur leading to TEM-1-derived beta-lactamases that are resistant to these inhibitors, and several inhibitor-resistant TEM beta-lactamases (or IRT (^1)beta-lactamases) have been characterized in hospital strains since 1992 (Thomson and Amyes, 1992). Few amino acid mutations are involved in inhibitor resistance, but they differ from those encountered in extended spectrum beta-lactamases (Table 1): a leucine, an isoleucine, or a valine instead of methionine at position 69; a serine or a cysteine instead of arginine 244; an arginine instead of tryptophane 165; a threonine for a methionine at position 182; and, more recently, an asparagine for an aspartic acid at position 276 (N276D) or an arginine for a leucine at position 275 (Henquell et al., 1995).



The M182T substitution does not affect the level of inhibitor resistance (Blasquez et al., 1993). The R165W mutation results in a small increase of resistance (Lenfant et al., 1993), whereas the major impact of the substitutions at positions 69 and 244 were described by Delaire et al.(1992) and Imtiaz et al.(1993). The role of the N276D substitution encountered in clavulanic acid-resistant TEM-35 and TEM-36 was not clear because this substitution has never been isolated. In order to assess this point, the N276D variant TEM-1 beta-lactamase was constructed by site-directed mutagenesis and studied for its ability to hydrolyze penicillins and cephalosporins and for the inhibitory effect of clavulanic acid.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis

The following oligonucleotides were synthesized on an Applied Biosystem 38A synthesizer. They were phosphorylated as described by Normanly et al.(1986) (N276Am, 5`-GATCTGTCTCTATCGTTCATC-3`; N276D, 5`-GATCTGTCTATCTCGTTCATC-3`). The mutations were introduced by Kunkel's method(1985) on the bla gene, which encodes beta-lactamase TEM-1, in the plasmid pCT-1 (Lenfant et al., 1991). The single-stranded wild type pCT-1 DNA was isolated from strain BW313, after infection by the helper phage M13K07 (Pharmacia). The N276am mutant colonies were selected for their loss of resistance to ampicillin. Missense revertant N276D was obtained in the same manner from the amber-mutated gene, and the mutant colonies were then selected for their resistance to ampicillin. The mutations were confirmed by sequencing by the dideoxy method (Sanger et al., 1977) with the Pharmacia T7 polymerase sequencing kit. S-dCTP was provided by Amersham Corp.

Informational Suppression

This method and the Escherichia coli K12 strains and plasmids have already been described by Lenfant et al.(1990). It allows us to obtain 15 variants of beta-lactamase TEM-1 by introducing an amber mutation at position 276 of the beta-lactamase gene by site-directed mutagenesis and transforming a set of suppressor strains. The variants obtained by suppression were assayed for growth in the presence of various antibiotics at 30 °C. The disc agar diffusion method was used (Lenfant et al., 1990).

beta-Lactamase Expression and Purification

The genes coding for wild type beta-lactamase or the N276D mutant were cloned into plasmid pCT3, a pCT1 derivative with a stronger promoter (Lenfant et al., 1990). All enzymes for genetic engineering were obtained from Pharmacia LKB Biotechnology Inc. E. coli strain XAC-1 (Normanly et al., 1986) was used to produce TEM-1 beta-lactamase and the N276D mutant. This strain was electrotransformed (Dower et al., 1988) with the pCT-3 plasmids.

Bacterial cells were grown at 30 °C on Luria broth supplemented with 0.5% MgSO(4), 0.2% glycerol, and 100 µg/ml ampicillin (Sigma). Cells were harvested by centrifugation during the exponential phase (A = 2) at 5,000 g for 10 min and washed with 200 ml of 200 mM Tris-HCl, pH 8. The cell pellet was resuspended to 60 A with 200 mM Tris-HCl, pH 8. One volume of 1 M sucrose in 200 mM Tris-HCl, pH 8, 0.1 volume of 100 mM EDTA, pH 7.6, and 30 mg of lysozyme were added. The suspension was incubated for 20 min at 4 °C, and 0.2 volume of 0.5 M MgCl(2) was added. The periplasmic fluid was collected by centrifugation at 18,000 g for 20 min and dialyzed against 200 mM Tris-HCl, pH 8, to eliminate sucrose.

This extract was then concentrated and desalted by ultrafiltration using Amicon system 8200 (Amicon GmbH, Witten, Germany) with a PM-10 membrane. The concentrated volume was approximately 5 ml. This suspension was then purified by preparative electrofocusing using a Multiphor II system (Pharmacia, Uppsala, Sweden) on a 4-6.5 pH gradient. The gel portion containing the beta-lactamase was eluted with 20 mM Tris-HCl, pH 7.5, and extensively dialyzed against the same buffer. The protein extract was chromatographed on a Pharmacia Mono Q HR5/5 anion exchanger column. The proteins were eluted with a 0-0.2 M linear NaCl gradient in 20 mM Tris-HCl, pH 7.5, at a flow rate of 0.5 ml/min. Active fractions detected with nitrocephin (Oxoid, Batingstoke, Hampshire, UK) were found to be homogeneous as judged by analytical SDS Phastgel (Pharmacia) and silver staining. Protein concentration was determined according to the Bradford method (Bradford, 1976).

Determination of Kinetic Parameters of Substrate Hydrolysis

The Michaelis-Menten constants k and K were determined at 37 °C in 50 mM sodium phosphate buffer, pH 7. The kinetic measurements were carried out spectrophotometrically. The cleavage of the beta-lactam ring of antibiotics was monitored with an UVIKON 930 (Kontron). The wavelengths for each antibiotic and the corresponding values of Delta calculated according to Samuni(1975) are presented in Table 2. The catalytic efficiency is defined as the ratio k/K.



Determination of Inhibition Kinetic Parameters

These parameters were determined spectrophotometrically at 37 °C in 50 mM sodium phosphate buffer, pH 7. K was determined from competition procedures with a good substrate, amoxicillin. The rate constant of irreversible inactivation by clavulanic acid, k, was determined by incubating the inhibitor in saturating concentration with the enzyme for various times. A large excess of amoxicillin was then added, and the remaining activity was measured. The half-time (t) is the time necessary to inhibit 50% of the enzymatic activity; it allows calculation of the k value: k =ln2/t (Kitz and Wilson, 1962). To measure the turnover (t) of the enzyme for clavulanic acid, the inhibitor and the enzyme were incubated at different molar ratios for 30 min at 37 °C, and the residual activity was measured. The turnover value was deduced from the extrapolated value for 100% inactivation from the plot of the residual activity versus the inhibitor-to-enzyme ratio. The inhibition efficiency is defined as k/K (for details, see Delaire et al., 1992).

Visualization of Free and Complexed Enzymes by Electrospray Mass Spectrometry

Analyses were performed using a TRIO 2000 quadrupole mass spectrometer (VG Biotech, Altrincham, UK) equipped with an electrospray source. Inhibition reactions were carried out at 37 °C by mixing 10 µl of the enzyme solution (1 mg/ml in 10 mM ammonium bicarbonate, pH 7) with 3 µl of the clavulanic acid solution (18 mM in water). After 3 h of incubation, reaction mixtures were directly introduced into the electrospray source via a 10-µl injection loop at 15 µl/min using a 50:48:2 (v/v/v) mixture of acetonitrile:water:formic acid delivered by a syringe pump (140 A Solvent Delivery System, Applied Biosystems, Foster City, CA). Mass spectra were acquired in multichannel analyzer mode at a scan rate of 15 s/scan.

Enzyme Coordinates and Electrostatic Calculations

The atomic coordinates of TEM-1 were from the 1.8-Å resolution refined x-ray structure (Jelsch et al., 1993). The structure includes 199 water molecules and 1 sulfate ion provided by the crystallization conditions. This ion has no biological significance and was discarded from the calculations as well as 33 water molecules that do not exchange hydrogen bonds to protein atoms. All other water molecules were considered as part of the protein and have an average temperature factor of 14.6 Å^2. The N276D mutant was modelled from the structure of the wild type enzyme with the program O (Jones et al., 1991). Polar hydrogen positions were built and energy was minimized using the program X-PLOR (Brnger, 1992). The acyl-enzyme model was constructed according to the description given by Strynadka et al.(1992). In this model, five more water molecules were removed from the active site.

For evaluation of potential fields, we used the DelPhi package (Nicholls and Honig, 1991; Sharp and Nicholls, 1989), which solves the linearized Poisson-Boltzmann equation by a finite difference method. The TEM-1 structure has a length of about 57 Å, which, together with the original DelPhi grid (65 65 65 grid points), gives a resolution of about one grid point/Å. This was considered insufficient, and the grid was extended to 129 129 129 points. Two dummy atoms were introduced to control the position of the molecule within the grid. At a ``perfil'' of 100%, these dummy atoms are located at opposite ends of the diagonal of the cubic grid in such a way that the longest dimension of the protein molecule, including its solvent-accessible surface (computed with a probe radius of 1.4 Å), falls exactly within the grid. The atoms were assigned Connolly radii provided with the DelPhi package. A 2.0-Å-thick ion exclusion layer was added, and the ionic strength of the bulk was set to 145 mM. The protein moiety was assigned a relative dielectric constant of 3.0, whereas that of the bulk was set to 80. Atomic charges were taken from the ``toph19.pro'' file in X-PLOR (Brnger, 1992; Brooks et al., 1983) and distributed over the grid points according to an algorithm described by Gilson et al.(1987). This method gives more accurate potentials at close distances than a traditional distribution over the eight closest grid points. All residues were kept in their normal protonation state at pH 7.8. Independence on the chosen grid origin was verified by repeating the calculations with the geometric center of the protein plus dummy atoms placed at seven different grid points (the grid center itself and its six closest neighbors). A three-step focusing protocol (15, 60, and 100% perfils) was used with the Debye-Hckel potential of the equivalent dipole to the molecular charge distribution as a boundary condition in the initial step (Sharp and Nicholls, 1989). The grid size was 0.52 Å (1.92 grid points/Å) in the last step.

Hardware

The program O was run on an Evans and Sutherland ESV/30-33. DelPhi and X-PLOR were run on a Digital DEC 3000/400 Alpha workstation (96 Mb RAM).


RESULTS

Informational Suppression and Antibiotic Disc Assays

The N276am and N276D mutant bla genes were obtained by site-directed mutagenesis. The N276am pCT-1 plasmid was transformed in the 15 different available strains containing the amber suppressor genes, which introduce 15 different amino acids at position 276. Thus, with only one mutagenesis step, 15 variants of the TEM-1 beta-lactamase were generated. This plasmid was also transformed in the XAC-1 strain, which did not express any suppressor gene as a control. The N276D pCT-3 plasmid was transformed in the XAC-1 strain.

The antibiograms of the exponential cultures of these transformed strains (Table 3) show that all the variant proteins are less active than the wild type TEM-1 beta-lactamase. Substitutions of the asparagine by an arginine, a lysine, a cysteine, an isoleucine, a leucine, a phenylalanine, a proline, or a tyrosine lead to beta-lactamases that are not active enough to confer resistance to penicillin antibiotics such as amoxicillin or ticarcillin. The other substitutions, except for glycine and serine substitutions, confer resistance to penicillins, but the transformed strains are very sensitive to all cephalosporins. This very low activity of the variant enzymes can be explained by a poor suppressional context at the position studied. However, the activity of the N276S and N276G variants is sufficient to confer resistance to cephalosporins, although the efficiency of the serine and glycine suppressors is not very high (Normanly et al., 1990). It thus appears that residue 276 cannot be substituted by most amino acids without a marked decrease of the hydrolytic activity.



The antibiotic disc assays of the N276D mutant show that this variant is as active as the wild type beta-lactamase against penicillins and first and second generation cephalosporins, because growth is not inhibited around the antibiotic discs. The most striking effect of the N276D substitution is the marked resistance to clavulanic acid combinations. The inhibitor is not efficient against the N276D beta-lactamase and does not inhibit growth when it is used in combination with amoxicillin or ticarcillin.

Kinetic Characterization of the N276D Mutant

The N276D mutant was purified to homogeneity and kinetically characterized. The effect of the substitution on the hydrolysis of substrates is not drastic (Table 4) with catalytic efficiencies 11.4-50.4% of those of wild type TEM-1. The K constants for all the substrates are decreased, but the N276D hydrolyzes penicillin substrates at faster rates than the wild type. On the contrary, the hydrolysis rate of cephalosporins is decreased to 19.5-37.2% of that of TEM-1. However, there is no major change in substrate profile on the ground of the catalytic efficiencies, indicating that the N276D mutation does not alter the substrate binding site topology.



As opposed to the behavior with substrates, the effect of the N276D substitution on the inhibitory capacity of clavulanic acid is drastic (Table 5). The main feature is the very poor affinity of the enzyme for this inhibitor, with a 23-fold increased K, leading to an inhibitory efficiency of only 4.3% of that of the wild type enzyme. The decrease in binding constant, combined with a better hydrolysis of clavulanic acid with a turnover of 250, changes the suicide inhibitor into a poor substrate.



ESMS Analysis of the Inhibited Enzymes

ESMS allows the visualization of proteins and their complexes with various substrates or inhibitors and determination of their molecular masses. The wild type enzyme (28,947 Da) and the N276D mutant (28,948 Da) were incubated in the same conditions with clavulanic acid. With an inhibitor-to-enzyme molar ratio of 150, after 3 h of incubation, all the molecules of TEM-1 enzyme are complexed (Fig. 1A), whereas a large amount of the N276D mutant remains free (Fig. 1B) and active against amoxicillin, as shown by spectrophotometric measurement. For TEM-1 and for the N276D mutant, the mass of the major complex observed (29,028 ± 8 Da and 29,025 ± 13 Da for TEM-1 and the N276D mutant, respectively) corresponds to the addition of 80 Da to the mass of the free enzyme. This additional mass is lower than the molecular mass of clavulanic acid (199 Da), indicating that only a cleaved moiety of the inhibitor molecule is covalently bound in the major inactivated enzyme complex. The presence of small peaks at higher masses (Fig. 1) may be due to minor forms of the enzyme-inhibitor complexes, consistent with earlier characterization from isoelectric focusing (Charnas et al., 1978).


Figure 1: Electrospray mass spectra of the reaction of clavulanic acid (inhibitor-to-enzyme ratio of 150) at 37 °C for 3 h with TEM-1 (A) and the N276D mutant (B). The spectra were obtained by applying the MaxEnt deconvolution procedure to raw data. Measurements from at least 6 independent experiments were used to compute mass averages of the major inhibited enzyme complex (Complex). The mass complex is 29,028 ± 8 Da with TEM-1 and 29,025 ± 13 Da with the N276D mutant.



Modeling and Electrostatic Calculations

Modeling of the mutation was straightforward, and energy minimization by X-PLOR (Brnger, 1992) brought the Arg N-2 and the Asp O-1 atoms 2.7 Å from each other. The electrostatic potential maps corresponding to the wild type and to the mutant enzyme were computed using DelPhi, and they showed a decrease of the positive potential in the binding area of the penicillin carboxylate group. This translates into a 4.4 kcal/mol relative decrease of the electrostatic binding energy between this carboxylate group and the enzyme. Desolvation effects were not taken into account in these calculations because they could reasonably be assumed to be of equivalent magnitude in both wild type and N276D enzymes.


DISCUSSION

The IRT-4 (TEM-35) beta-lactamase has been described by Brun et al.(1994). The sequence analysis of this TEM-1-derived beta-lactamase shows two substitutions: a leucine for a methionine at position 69 and an asparagine for an aspartic acid at position 276. The mutation at position 69 has already been shown to induce resistance to clavulanic acid (Zhou et al., 1994), and the natural mutant enzymes TEM-32, TEM-33, and TEM-34 contain an isoleucine, a leucine, or a valine at this position, respectively (Table 1). Zhou et al.(1994) proposed that this substitution was sufficient to explain clavulanic acid resistance of the TEM-35 enzyme and that the N276D substitution had no contribution to this phenomenon. The construction and analysis of the N276D mutant of the TEM-1 beta-lactamase indicate that this is not the case.

The antibiotic disc assays show that, except for the glycine and serine variants, which remain active, all the substitutions obtained by suppression of the amber-mutated codon lead to enzymes with very poor activity, even against penicillins. On the contrary, the decreased activity due to the substitution of the asparagine by an aspartic acid is not significant, because the growth inhibition diameters with all the beta-lactam antibiotics are identical to those obtained with the TEM-1 expressing strain. However, this mutation induces clavulanic acid resistance. It is known that residue 276 is not directly involved in substrate binding and catalysis, but these results emphasize the fact that it indirectly influences the progress of catalysis. Interestingly, as this manuscript was under way, Bonomo et al.(1995) described a N276G mutant in OHIO-1, another class A beta-lactamase that is also highly resistant to clavulanic acid, whereas our TEM-1-derived N276G mutant seems only moderately resistant.

Kinetic and ESMS analyses show that the N276D mutant's main property is its resistance to clavulanic acid inhibition. Imtiaz et al.(1993) suggested that irreversible inactivation involves the capture of the beta-hydroxyl of serine 130 by the iminium group of the acylated intermediate, which results in the covalent binding of only a part of the inhibitor molecule linked on one side to serine 70 and on the other side to serine 130. This hypothesis is consistent with our ESMS results, which demonstrate that only part of the clavulanic acid molecule is bound to the major inactivated enzyme complex. The proposed mechanism involves a deprotonation of Ser-bound clavulanate via a water molecule. This water molecule, which is a proton donor in the stepwise sequence of events of the inactivation process, interacts with the side chain of arginine 244 and with the carboxylate of valine 216 in the wild type enzyme structure. Positional difference of this water molecule as a consequence of the N276D mutation, as well as of the R244S mutation that leads to IRT enzymes, could impair the efficiency of the inactivation process. This would explain why, under identical experimental conditions, free N276D mutant enzyme is observed by ESMS, whereas the wild type enzyme is totally engaged in inactivated complexes. The fact that the same major molecular complex is observed by mass spectrometry for both enzymes also suggests that the inactivation process leading to this species is similar for the wild type and the N276D mutant. From an evolutionary point of view, it is interesting to note that the Gram-positive enzyme PC1 is highly sensitive to clavulanic acid, although it has an aspartic acid at position 276 (as in Bacillus licheniformis and several other Gram-positive enzymes). In the Staphylococcus aureus enzyme, a full clavulanic acid molecule is bound to the active site, thus suggesting another inhibition mechanism (Chen and Herzberg, 1992).

The resistance in the N276D mutant is probably related to the considerable loss of affinity for the inhibitor (23-fold increase in K) and to the increased turnover of clavulanic acid (Table 5), which then behaves as a poor substrate instead of as a suicide inhibitor. In the three-dimensional structure of TEM-1 (Jelsch et al., 1993), asparagine 276 is accessible to bulk solvent and found at hydrogen bond distance to arginine 244 (Fig. 2). According to the structure and to the kinetic data (Table 4), the N276D mutation was not anticipated to induce significant structural changes. Indeed, energy minimization of the modeled mutant enzyme only induces a slight reorientation of the Arg and Asp side chains. The Arg N-2 and Asp O-1 are now 2.7 Å from each other, which strongly suggests that a salt-bridge interaction is formed. The electrostatic potential map computed for the N276D mutant enzyme showed that the altered charge distribution on the Arg and Asp side chains leads to a decrease of the positive potential in the binding site of the substrate carboxylate compared with the wild type enzyme. The electrostatic binding energy provided by this carboxylate, found 2.7 Å from Arg in the x-ray structure of the acyl-enzyme complex (Strynadka et al., 1992), was calculated to be decreased by 4.4 kcal/mol in the N276D protein mutant, a value that corresponds to the strength of this interaction (Hendsch and Tidor, 1994).


Figure 2: Stereo view of the environment of residues 244 and 276 in the wild type (thin lines) and the N276D mutant enzymes (thick lines). The distance between the Arg N-2 and the Asp O-1 atoms is 2.7 Å. Dotted lines indicate short distance interactions (less than 2.9 Å). The water molecule, found at hydrogen bond distance of the Arg N-2 and main chain Val oxygen atoms in the TEM-1 x-ray structure (Jelsch et al., 1993) and involved in the inactivation process (Imtiaz et al., 1993), is shown.



These electrostatic effects in the substrate binding cavity may be responsible for the increased K for all substrates and in turn for the decrease of the catalytic efficiencies. The fact that the decrease of affinity is more marked with penicillins, with a 10-fold increase in K for ticarcillin for instance, than with cephalosporins is consistent with the proposal that penicillins have a stronger interaction with arginine 244 than cephalosporins (Zafaralla et al., 1992).

The decrease of the binding constant of clavulanic acid, assumed to be positioned as a penicillin substrate in the catalytic site (Imtiaz et al., 1993), may also result from the weakened electrostatic interaction between the carboxylate at C-3 of the clavulanic acid molecule and the arginine 244 guanidium group in the acyl-enzyme complex. As a consequence, deacylation could be sufficiently facile to compete with the inactivation reaction and thereby explain the regeneration of N276D. A faster release of the penicilloic acid reaction product would also be in line with the increased k for penicillin substrates.

In conclusion, even though the N276D substitution in TEM beta-lactamases has never been found alone and is always associated with the M69L substitution, known to confer clavulanic acid resistance by itself, it appears that an aspartic acid at position 276 plays a significant role in the resistance to clavulanic acid inhibition. The electrostatic interaction of the aspartic acid with arginine 244 and the possible displacement of the water molecule involved in the inactivation process would be responsible for such effects.


FOOTNOTES

*
This work was supported by the Rgion Midi-Pyrnes and by Grant 930608 from the Institut National de la Sant et de la Recherche Mdicale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Each of these authors contributed equally to this work and should therefore be considered first authors.

To whom correspondence should be addressed. Tel.: 33-61-17-59-59; Fax: 33-61-17-59-94.

^1
The abbreviations used are: IRT, inhibitor resistant TEM; ESMS, electrospray mass spectrometry; am, amber mutant.


ACKNOWLEDGEMENTS

We thank Arlette Savagnac for technical assistance.


REFERENCES

  1. Ambler, R. P. (1980)Philos. Trans. R. Soc. Lond. B. Biol Sci. 289,321-331 [Medline] [Order article via Infotrieve]
  2. Belaaouaj, A., Lapoumeroulie, C., Cania, M. M., Vedel, G., Nvot, P., Krishnamoorthy, R., and Paul, G. (1994)FEMS Microbiol. Lett.120,75-80 [CrossRef][Medline] [Order article via Infotrieve]
  3. Blasquez, J., Baquero, M. R., Canton, R., Alos, I., and Baquero, F.(1993) Antimicrob. Agents Chemother.37,2059-2063 [Abstract]
  4. Bonomo, R. A., Dawes, C. G., Knox, J. R., and Shlaes, D. M.(1995)Biochim. Biophys. Acta1247,121-125 [Medline] [Order article via Infotrieve]
  5. Bradford, M. M. (1976)Anal. Biochem.72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  6. Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., and Karplus, M.(1983)J. Comput. Chem.4,187-217
  7. Brun, T., Pduzzi, J., Cania, M. M., Paul, G., Nvot, P., Barthlmy, M., and Labia, R.(1994)FEMS Microbiol. Lett.120,111-118 [CrossRef][Medline] [Order article via Infotrieve]
  8. Brnger, A. T. (1992) X-PLOR Version 3.1: A System for X-ray Crystallography and NMR, Yale University Press, New Haven, CT
  9. Charnas, R. L., Fisher, J., and Knowles, J. R.(1978)Biochemistry 17,2185-2189 [Medline] [Order article via Infotrieve]
  10. Chen, C. C. H., and Herzberg, O.(1992)J. Mol. Biol.224,1103-1113 [Medline] [Order article via Infotrieve]
  11. Delaire, M., Labia, R., Samama, J. P., and Masson, J. M.(1992)J. Biol. Chem. 267,20600-20606 [Abstract/Free Full Text]
  12. Dower, W. J., Miller, J. F., and Ragsdale, C. W.(1988)Nucleic Acids Res. 16,6127-6143 [Abstract]
  13. Gilson, M. K., Sharp, K. A., and Honig, B.(1987)J. Comput. Chem.9,327-335
  14. Hendsch, Z. S., and Tidor, B.(1994)Protein Sci.3,211-226 [Abstract/Free Full Text]
  15. Henquell, C., Chanal, C., Sirot, D., Labia, R., and Sirot, J.(1995) Antimicrob. Agents Chemother.39,427-437 [Abstract]
  16. Imtiaz, U., Billings, E., Knox, J. R., Manavathu, E. K., Lerner, S. A., and Mobashery, S. (1993)J. Am. Chem. Soc.115,4435-4442
  17. Jacoby, G. A., and Medeiros, A. A.(1991)Antimicrob. Agents Chemother. 35,1697-1704 [Medline] [Order article via Infotrieve]
  18. Jelsch, C., Mourey, L., Masson, J. M., and Samama, J. P.(1993)Proteins Struct. Funct. Genet.16,364-383 [Medline] [Order article via Infotrieve]
  19. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M.(1991)Acta Crystallogr. Sec. A47,110-119 [CrossRef][Medline] [Order article via Infotrieve]
  20. Kitz, P., and Wilson, I. B.(1962)J. Biol. Chem.237,3245-3249 [Free Full Text]
  21. Kunkel, T. A. (1985)Proc. Natl. Acad. Sci. U. S. A.82,482-492
  22. Lenfant, F., Labia, R., and Masson, J. M.(1990)Biochimie (Paris)72,495-503 [Medline] [Order article via Infotrieve]
  23. Lenfant, F., Labia, R., and Masson, J. M.(1991)J. Biol. Chem.266,17187-17194 [Abstract/Free Full Text]
  24. Lenfant, F., Petit, A., Labia, R., Maveyraud, L., Samama, J. P., and Masson, J. M.(1993) Eur. J. Biochem.217,939-946 [Abstract]
  25. Nicholls, A., and Honig, B.(1991)J. Comput. Chem.12,435-445
  26. Normanly, J., Masson, J. M., Kleina, L. G., Abelson, J., and Miller, J. H.(1986) Proc. Natl. Acad. Sci. U. S. A.83,6548-6552 [Abstract]
  27. Normanly, J., Kleina, L. G., Masson, J. M., Abelson, J., and Miller, J. H.(1990) J. Mol. Biol.213,719-726 [Medline] [Order article via Infotrieve]
  28. Paul, G. C., Gerbaud, G., Bure, A., Phillipon, A. M., Pangon, B., and Courvalin, P. (1988)Antimicrob. Agents Chemother.33,1958-1963
  29. Samuni, A.(1975) Anal. Biochem.63,17-26 [Medline] [Order article via Infotrieve]
  30. Sanger, F., Nicklen, S., and Coulson, A. R.(1977)Proc. Natl. Acad. Sci. U. S. A.74,5463-5467 [Abstract]
  31. Sharp, K. A., and Nicholls, A. (1989) DelPhi (v. 3.0) Manual, Columbia University, Dept. of Biochemistry and Molecular Biophysics, New York
  32. Sougakoff, W., Goussard, S., and Courvalin, P.(1989)FEMS Microbiol. Lett. 56,343-348
  33. Strynadka, N. C. J., Adachi, H., Jensen, S. E., Jhons, K., Betzel, C., Sutoh, K., and James, M. N. G.(1992)Nature359,700-705 [CrossRef][Medline] [Order article via Infotrieve]
  34. Thomson, C. J., and Amyes, S. G. B.(1992)FEMS Microbiol. Lett. 91,113-118
  35. Zafaralla, G., Manavathu, E. K., Lerner, S. A., and Mobashery, S.(1992) Biochemistry31,3847-3852 [Medline] [Order article via Infotrieve]
  36. Zhou, X. Y., Bordon, F., Sirot, D., Kitzis, M. D., and Gutmann, L.(1994) Antimicrob. Agents Chemother.38,1085-1089 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.