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Several class A, inhibitor-resistant -lactamases have been described. The enzymes comprise the 2br group of the Bush-Jacoby-Medeiros classification scheme (8). Most of the 2br -lactamases have evolved from TEM-1 by point mutations that result in amino acid substitutions, including that of Met-69 (Ambler et al.'s numbering scheme [1]) to Ile, Leu, or Val (11). Two inhibitor-resistant Met-69Ile laboratory mutant enzymes of OHIO-1 -lactamase, one of which is also a Gly-238Ser mutant enzyme, have been described (5, 6). The hydrolytic efficiency of the extended-spectrum double mutant was reduced (6). In a complex TEM mutant enzyme (TEM-50) it appears that Met-69Leu weakens the extended-spectrum characteristics conferred by Lys for Glu-104 and Ser for Gly-238 substitutions (21). The above-described findings, considered together with data relevant to the three-dimensional structure of the TEM and SHV -lactamases (12-14), suggest that some mutations that confer extended-spectrum activity may have an effect other than additive when combined with replacement of Met-69. In this study we examined the effect of substitution of Ile or Val for Met-69 in the SHV-5 extended-spectrum -lactamase (ESBL).

A 1.4-kbp SmaI-ClaI fragment of plasmid pAFF2 carrying the bla_SHV-5 gene (3) was cloned into pBCSK(+) (Stratagene, La Jolla, Calif.). The resulting plasmid, pNF2 (bla_SHV-5), was used to transform Escherichia coli DH5 competent cells. Cesium chloride-purified pNF2 was used for site-specific mutagenesis and sequencing. SHV-5 mutant enzymes were obtained by a PCR-based method (17). Two pairs of primers were used for each substitution. One primer of each pair contained a single base mismatch to direct mutagenesis to codon 69 of the mature peptide (ATC for Met-69Ile and GTG for Met-69Val). The mutagenic primers were 22 nucleotides long, and the mismatched base was close to the center of the sequence. They were prepared in an Applied Biosystems (Foster City, Calif.) DNA synthesizer. To confirm the lack of any other changes, the DNA sequences of the mutated genes were determined by the dideoxy chain termination method with a Sequenase 2.0 kit (United States Biochemical Corp., Cleveland, Ohio).

MICs of -lactam antibiotics were determined by a microdilution method with Mueller-Hinton broth (16). The screening for clones producing SHV-5 mutant -lactamases was performed by the disk diffusion method with Mueller-Hinton agar.

For -lactamase preparations, the E. coli DH5 clones producing SHV-5 -lactamase and the Met-69Ile and Met-69Val mutant enzymes were grown overnight in tryptone-soy broth. -Lactamases were released after ultrasonic treatment of cell suspensions. The extracts were clarified by ultracentrifugation, desalted with Econo-Pac 10DG columns (Bio-Rad Laboratories, Richmond, Calif.), and concentrated by ultrafiltration with Centriprep-10 filters (Amicon, Witten, Germany). The protein contents of the preparations were determined with a Bio-Rad protein assay kit. -Lactamase activities were quantitated with nitrocefin and expressed as nanomoles of substrate hydrolyzed per minute per milligram of protein. Hydrolysis of penicillin G, ampicillin, cephalothin, and cefotaxime was moniored spectrophotometrically (2, 18) at 37°C in phosphate buffer (50 mM, pH 7) on a temperature-controlled Hitachi model 3000 double-beam spectrophotometer with a 1.0-cm-path-length cuvette. At least five concentrations of each substrate were used. The maximum rate of hydrolysis (V_max) and K_m values, calculated from initial hydrolysis rates, were determined from Lineweaver-Burk plots by means of four assays. The V_max values were expressed relative to that of penicillin G, which was set at 100. The wavelengths of maximal absorption differences and extinction coefficients used were as described previously (2). Inhibition profiles of -lactamases were determined with clavulanate, tazobactam, and sulbactam (22). The reporter substrate was nitrocefin at a concentration of 100 µM. Before the addition of nitrocefin, the inhibitor was preincubated with the enzyme for 5 min at 37°C. The amount of each enzyme was normalized to give approximately 150 µM nitrocefin hydrolyzed per min. The 50% inhibitory concentrations (IC₅₀s) were determined from plots of the inhibitor concentration versus percent inhibition.

The MICs of -lactams for the E. coli strains are shown in Table 1. The E. coli strains that expressed the Met-69Ile and Met-69Val mutant -lactamases were fourfold less resistant to ampicillin and piperacillin, as well as to cefepime and cefpirome, than the SHV-5-producing strain. Eight- to 32-fold reductions in the MICs of the remaining oxyimino--lactams tested were also noticed. Changes in susceptibilities to penicillin-inhibitor combinations were not observed.

View this table: [in this window] [in a new window]   TABLE 1.   MICs of -lactams for E. coli DH5 clones producing SHV-5 -lactamase or the Met-69Ile or Met-69Val mutant enzyme

The SHV-5 and the two mutant enzymes were expressed under isogenic conditions that permitted a direct comparison of the activities of different -lactamases (19). Therefore, the observed reductions in the MICs of the -lactams suggested that substitution of Ile or Val for Met-69 impaired the hydrolytic efficiency of SHV-5. As shown in Table 2, the mutant enzymes hydrolyzed ampicillin, cephalothin, and cefotaxime at efficiencies lower than that of SHV-5 -lactamase. The substitutions caused 7- to 10-fold decreases in affinities for cefotaxime, but the relative hydrolysis rates were affected to lesser extents. Three- to fourfold reductions in the relative rates of ampicillin hydrolysis were also observed. Significant differences between the two mutant enzymes were not found, except for the fact that the affinity of the Met-69Ile mutant -lactamase for cephalothin was higher than that of the Met-69Val mutant -lactamase.

View this table: [in this window] [in a new window]   TABLE 2.   Kinetic data for SHV-5 -lactamase and Met-69Ile and Met-69Val mutant enzymesa

Determination of IC₅₀s of clavulanic acid, sulbactam, and tazobactam showed that the changes of Met-69 rendered SHV-5 -lactamase less susceptible to the inhibitory activity of the above-described compounds. Clavulanic acid was affected to a greater extent than penam sulfones. The Met-69Ile mutant enzyme was slightly more resistant (1.5-fold) to clavulanic acid and sulbactam than the Met-69Val mutant enzyme. The IC₅₀s of tazobactam were similar for the two mutant -lactamases (Table 3). Notwithstanding their reduced susceptibilities to inhibitors, the mutant enzymes did not confer resistance to penicillin-inhibitor combinations (Table 1). This result may be due to the simultaneous weakening of the penicillinase activity.

View this table: [in this window] [in a new window]   TABLE 3.   Inhibition profiles of the SHV-5 and mutant -lactamases

The data presented above show that replacement of Met-69 by Ile or Val in the SHV-5 -lactamase is not beneficial for the enzyme, in that it causes a decrease in its hydrolytic activity towards expanded-spectrum cephalosporins and penicillins. The fold increase in the resistance level to mechanism-based inhibitors was lower than that conferred by the same substitutions in TEM-1 (4, 23) and OHIO-1 penicillinases (5) and unable to compensate for the partial loss of penicillinase activity in the susceptibility tests. In fact, the IC₅₀ of clavulanate for the Met-69Val mutant enzyme SHV-5 was similar to that of TEM-1 -lactamase, which was tested in parallel (data not shown). The substitution of Ile for Met-69 reduced the catalytic efficiencies for penicillins of an extended-spectrum G238S OHIO-1 mutant obtained by site-directed mutagenesis (6). As with SHV-5, the Met-69Leu substitution in the TEM-50 -lactamase conferred low-level resistance to inhibitors at the expense of its hydrolytic activities towards most -lactams, including oxyimino-cephalosporins and aztreonam (21). A significant decrease in cephalosporinase activity has also been observed in the naturally occurring SHV-10 -lactamase, which is a derivative of an SHV-5-type -lactamase in which the catalytically critical Ser-130 residue has been replaced by a glycine (20).

The above-described data may be explained on the basis of results of previous studies which indicated a structural role for Met-69 in the formation of the active-site cavity of TEM and SHV -lactamases. SHV-5 is a Gly-238Ser-Glu-240Lys mutant of the SHV-1 enzyme. The steric contact of the side chain of Ser-238 with that of the opposing Met-69 amino acid displaces the 3 strand and expands SHV-5's catalytic cavity. This, in turn, allows the accommodation of cephalosporins with bulky acyl-amido substituents and assists the bonding of hydrogen with the backbone groups of Ala-237 (13). Electrostatic interaction of Lys-240 with carboxylate found in the oxyimino substituents of ceftazidime and aztreonam enhances activity towards the latter -lactams (12). The widened active-site cavity also facilitates attractive interactions with clavulanic acid and penam sulfones, and thus ESBLs are usually more susceptible to -lactamase inhibitors than the parent TEM-1 or SHV-1 enzymes (15). It has been suggested that replacement of Met-69 by another hydrophobic amino acid in TEM-1 modifies the orientations of small -lactams like clavulanate in such a way as to decrease binding (10). The decrease in susceptibilities to inhibitors after replacing Met-69 with Ile or Val in the SHV-5 -lactamase may be due to a similar mechanism. It must also be noted that the effects of these substitutions on the inhibitor susceptibility status of SHV-5 were more pronounced for clavulanate than for penam sulfones. This finding is in line with the results of a previous work reporting that tazobactam is a potent inactivator of various inhibitor-resistant, class A -lactamases (7). The deformation of the active-site cavity and the possible displacement of the reactive Ser-70 residue relative to the residues that contribute to substrate binding may be responsible for the reduction in the catalytic efficiencies of the Met-69Ile and Met-69Val mutant enzymes of SHV-5. This mechanism may be analogous to that proposed to explain the decreased activity of ESBLs against penicillins and older cephalosporins (9, 13).

Susceptibility testing showed that the constructed SHV-5 mutant enzymes did not provide any particular advantage to the E. coli strain used here. However, their expression under different conditions in clinical enterobacteria (e.g., in combination with common plasmid-mediated penicillinases or in bacteria with decreased outer-membrane permeability) would produce different resistance phenotypes. Hence, such mutant extended-spectrum -lactamases may also arise in vivo.