Abstract
Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the kcat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.
The production of β-lactamases makes a major contribution to β-lactam resistance in gram-negative bacteria (13). During therapy with one of the newer β-lactams, resistance due to derepression of an inducible chromosomally encoded class C β-lactamase (also named AmpC) appears to emerge in 10 to 50% of patients infected with Citrobacter freundii, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa (46). The derepressed β-lactamase production results from mutations in ampD (16, 22, 26, 30, 51). Plasmid-encoded class C β-lactamases have also been described in clinical isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, and Salmonella senftenberg and are a significant cause of β-lactam resistance (8, 10, 13, 27, 29, 37). This increase in the clinical importance of class C β-lactamases and their capacity to confer resistance to expanded-spectrum β-lactam antibiotics such as cefotaxime and ceftazidime and to α-methoxy-β-lactams such as cefoxitin and cefotetan have led to considerable interest in the understanding of their mechanisms of action.
According to a molecular classification scheme (2, 3, 24, 28), the class C β-lactamases, together with class A and D β-lactamases, are active-site serine enzymes that catalyze, via a serine-bound acyl intermediate, the hydrolysis of the β-lactam to an inactive acid. The class C β-lactamases have been placed in the functional group 1 enzymes, which are described as cephalosporinases that are not inhibited by clavulanic acid (13). In addition to the three functional and structural elements (SerXaaXaaLys, TyrXaaAsn, and LysThrGly) that are conserved throughout the class C serine β-lactamases (19), the amino acid sequences of these enzymes exhibit more than 36% identity, and several conserved motifs have been identified (52a).
In the work described here, the class C β-lactamase from E. cloacae P99, for which the three-dimensional structure has been established by X-ray crystallography at 2-Å resolution (36), was used as a model to study the hydrolysis mechanism of class C enzymes. Amino acid residue Ser-289 is located in a critical region at the top of the E. cloacae β-lactamase active site (as viewed in Fig. 2), with its side chain near the C-3 substituent and the C-4 carboxylic acid group of cephalosporins. To elucidate the role of this amino acid residue in the catalytic activities of class C β-lactamases, we undertook an investigation of the effects that mutagenesis had on the kinetics of hydrolysis, and we describe our results in relation to the known crystallographic structure of the wild-type enzyme.
FIG. 2.
MOLSCRIPT drawing (31) of the crystallographic structure of the E. cloacae P99 β-lactamase (36). Ser-289 is located on an exposed loop above the binding site. The positioning of cefoperazone adjacent to the reactive Ser-64 is based on crystallographic observations of aztreonam (43) and phosphonate (35) intermediates at the binding site.
MATERIALS AND METHODS
Chemicals.
Kanamycin, ampicillin, penicillin G, cephalothin, cephaloridine, cefotaxime, cefazolin, cefoperazone, cefaclor, phenylmethylsulfonyl fluoride (PMSF), and DNase I were purchased from Sigma (St. Louis, Mo.). Bovine serum albumin (BSA) was obtained from Pierce (Rockford, Ill.). Ceftazidime was obtained from Glaxo Canada Inc. (Montréal, Québec, Canada). Clavulanic acid, sulbactam, and tazobactam were gifts from SmithKline Beecham Pharma Inc. (Oakville, Ontario, Canada), Pfizer (Groton, Conn.), and Lederle (Carolina, Puerto Rico), respectively. Restriction endonucleases were obtained from New England Biolabs (Mississauga, Ontario, Canada). Nitrocefin was purchased from Oxoid (Nepean, Ontario, Canada).
Bacterial strains and plasmids.
E. coli SNO3 (ampA1 ampC8 pyrB recA rpsL) (41) was obtained from Staffan Normark (Karolinska Institutet, Stockholm, Sweden). E. coli CJ236 [dut ung thi relA; pCJ105 (Cmr)] (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada) (33) was used to prepare single-stranded uracil DNA templates. E. coli MV1190 [Δ(lac-proAB) thi supE (sr1-recA)306::Tn10(Tetr) (F′::traD36 ΔproAB lacIqZ ΔM15)] (Bio-Rad) (39) was used to eliminate the wild-type uracil DNA template and to produce single-stranded DNA. Plasmid pBGS19+ [(Kmr) f1 Ori lacPOZ] was used as a cloning vector (50). Single-stranded DNA production was performed with phage M13K07 (53). All transformed bacteria were grown in tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) and on tryptic soy agar plates (Difco) containing appropriate antibiotics when necessary for plasmid selection.
Production of mutant β-lactamases.
A 1.2-kb DNA fragment containing the ampC gene encoding the class C β-lactamase of E. cloacae P99 was amplified by PCR with Vent DNA polymerase (New England Biolabs) and a lysate of this bacterial strain, the latter of which was prepared by the freezing and thawing method (52). The oligonucleotide primers used for amplification (Ecampc3 [5′-CGCGGGATCCACATCCCCTTGACTCGC-3′] and Ecampc4 [5′-CGCGAAGCTTCAATGTTTTACTGTAGC-3′]) contained a BamHI restriction site and an HindIII restriction site at their respective 5′ ends and were derived from the published E. cloacae P99 ampC sequence (20). The PCR amplification product was cloned into the pBGS19+ vector and was identified as pHUL3-4. Molecular biology techniques were performed as described by Sambrook et al. (45). Site-directed mutagenesis was done by the uracil template protocol (32, 33) by using the Muta-Gene Kit (Bio-Rad) as described by Huletsky et al. (23). Six mutant β-lactamases were constructed by replacing Ser-289 by Ala, Thr, Gln, Arg, Lys, or Cys (see Table 1). Clones containing mutant genes were selected by DNA sequencing by using the dideoxy method (47) and the T7 Sequencing Kit from Pharmacia Biotech (Baie d’Urfé, Québec, Canada). Mutant genes were sequenced entirely.
TABLE 1.
MICs for E. coli SNO3 containing bases encoding wild-type and mutant β-lactamases at position 289
| Plasmid | β-Lactamase substitution | MIC (μg/ml)
|
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Cephaloridine | Cephalothin | Cefotaxime | Ceftazidime | Cefazolin | Cefoperazone | Cefaclor | Penicillin G | Ampicillin | ||
| pBGS19+ (control) | 3.13 | 3.91 | 0.03 | 0.13 | 1.56 | 0.06 | 3.13 | 15.6 | 3.91 | |
| pHUL3-4 | S289a | 400 | 500 | 2 | 0.5 | 800 | 16 | 400 | 250 | 31.2 |
| pHUL3-5 | S289A | 400 | 500 | 2 | 0.5 | 800 | 8 | 200 | 250 | 31.2 |
| pHUL3-6 | S289T | 400 | 500 | 2 | 0.5 | 800 | 16 | 200 | 250 | 31.2 |
| pHUL3-7 | S289Q | 200 | 500 | 2 | 0.25 | 800 | 8 | 400 | 250 | 31.2 |
| pHUL3-8 | S289R | 200 | 250 | 1 | 0.25 | 400 | 4 | 200 | 125 | 31.2 |
| pHUL3-9 | S289K | 50 | 125 | 2 | 0.25 | 200 | 4 | 200 | 250 | 31.2 |
| pHUL3-10 | S289C | 50 | 125 | 1 | 0.25 | 50 | 1 | 100 | 62.5 | 15.6 |
Wild type.
Phenotypic characterization of mutant β-lactamases.
Wild-type and mutant genes were transformed in E. coli SNO3. MICs were determined by a broth dilution method in TSB with 96-well plates (see Table 1). To verify the inhibitory profiles of the mutant enzymes, clavulanic acid, sulbactam, and tazobactam were used in combination with cephaloridine as a substrate (see Table 2).
TABLE 2.
MICsa for E. coli SNO3 containing bases encoding wild-type and mutant β-lactamases at position 289
| Inhibitor(s) | MIC (μg/ml)
|
||||||
|---|---|---|---|---|---|---|---|
| S289b | S289A | S289T | S289Q | S289R | S289K | S289C | |
| Cephaloridine | 400 | 400 | 400 | 400 | 200 | 50 | 100 |
| Cephaloridine + clavulanic acid | 400 | 400 | 400 | 400 | 100 | 25 | 50 |
| Cephaloridine + sulbactam | 3.13 | 3.13 | 3.13 | 3.13 | 3.13 | 3.13 | 3.13 |
| Cephaloridine + tazobactam | 6.25 | 3.13 | 3.13 | 6.25 | 3.13 | 3.13 | 6.25 |
The inhibitory functions of three inhibitors (clavulanic acid at 2 μg/ml, sulbactam at 8 μg/ml, and tazobactam at 4 μg/ml) were tested by using cephaloridine as reference substrate.
Wild type.
β-Lactamase purification.
All β-lactamases were purified to >95% homogeneity as follows. The E. coli SNO3 cells harboring the plasmids coding for the wild-type or mutant AmpC β-lactamases were grown overnight in TSB at 37°C, collected by centrifugation, and resuspended in TEAA buffer (20 mM triethanolamine, 0.5 M NaCl [pH 7.0]). DNase I (100 μg/ml) and 1 mM PMSF were added to the suspension, and the cells were disrupted by three passages through a French pressure cell (18,000 lb/in2). Insoluble material was removed by centrifugation at 229,000 × g for 1 h at 4°C, and the clarified supernatant was loaded on an immobilized phenylboronate gel (MoBiTec, Göttingen, Germany) equilibrated with TEAA buffer (14). The column was then washed with TEAA buffer and the β-lactamases were eluted in borate buffer (0.5 M boric acid, 0.5 M NaCl [pH 7.0]). Fractions containing the β-lactamases were pooled and concentrated by ultrafiltration with a Centriplus-10 device (Amicon, Beverly, Mass.) and were dialyzed overnight against 10 mM potassium phosphate buffer (pH 6.4). The enzymes were then placed on an Econo-Pac High S strong cation-exchange cartridge (Bio-Rad) equilibrated in the same buffer. The enzymes were eluted with a gradient of 0 to 300 mM NaCl in the same buffer. Fractions containing β-lactamase activity were pooled and concentrated as described above. The protein concentration was measured by the Bradford dye-binding procedure (9) by the Bio-Rad protein assay with BSA as a standard. A total of 750 ng of purified β-lactamase was loaded onto a sodium dodecyl sulfate (SDS)–12% polyacrylamide gel. Enzyme homogeneity was demonstrated by the presence of a single band on the silver-stained gel (Bio-Rad). Purified β-lactamases were stored at −20°C in 50% glycerol and 1 mg of BSA per ml.
Evaluation of kinetic parameters.
Hydrolysis of β-lactam antibiotics was determined at 30°C in 50 mM phosphate buffer (pH 7.0) on a Varian Cary 1 spectrophotometer. Dilution of the β-lactamases was performed with phosphate buffer solution containing 0.1 mg of BSA per ml and 5% glycerol. Substrate hydrolysis was monitored by determining the loss of absorbance at 260 nm for cephalothin, 270 nm for cefaclor, 275 nm for cefoperazone, and 295 nm for cephaloridine and by determining the increase in absorbance at 320 nm for cefazolin. The changes in extinction coefficients (Δɛs) were as follows: for cephalothin, Δɛ260 = 7,300 M−1 · cm−1; for cefaclor, Δɛ270 = 6,510 M−1 · cm−1; for cefoperazone, Δɛ275 = 8,640 M−1 · cm−1; for cephaloridine, Δɛ295 = 889 M−1 · cm−1; and for cefazolin, Δɛ320 = 1067 M−1 · cm−1. The steady-state kinetic parameters (Km, kcat, and kcat/Km) were determined from measurements of the initial rates (at least in triplicate) by fitting the Michaelis-Menten equation with the program Leonora (15).
Molecular structures.
Atomic coordinates of the crystallographic structure of the AmpC β-lactamase from E. cloacae P99 at a 2-Å resolution (36) are available from the Protein Data Bank (entry no. 2BLT). The crystallographic structure of a covalent seryl-phosphonate complex of the P99 β-lactamase (35) (entry no. 1BLS) was also used.
The structure of cefoperazone was constructed from cefamandole and piperacillin, the crystal structures of which were obtained from the Cambridge Data Bank (1). The conformation of the ring-opened, seryl-bound form of cefoperazone was obtained from crystallographic structures of acylated intermediates of aztreonam with the C. freundii β-lactamase (43), and of intermediates of cefotaxime and cephalothin with the d-Ala-d-Ala peptidase (34).
RESULTS
Description of mutant β-lactamases.
Six mutant β-lactamases were constructed by site-directed mutagenesis of Ser-289 of the class C β-lactamase of E. cloacae P99. DNA sequencing identified recombinant plasmids with changes at position 289. The wild-type and mutant β-lactamases studied are summarized in Table 1.
Microbiological activity.
The susceptibilities of plasmid-transformed E. coli SNO3 strains producing wild-type and mutant β-lactamases to the selected group of antibiotics were determined by measuring the MICs. As seen in Table 1, production of the wild-type E. cloacae P99 β-lactamase in E. coli SNO3 increased the level of resistance to all β-lactams tested. The susceptibilities of the organisms producing the S289A, S289T, or S289Q mutant to all these β-lactams was not affected compared to that of the wild type-producing organism. The cells with the S289R mutant also had about the same levels of resistance to all β-lactams with the exception of cefoperazone, the MIC of which was decreased fourfold. However, production of the last two mutants, S289K and S289C, in E. coli cells led to increased susceptibilities to many β-lactams. For the organism producing the S289K mutant, the MIC of cephaloridine was reduced eightfold and the MICs of cephalothin, cefazolin, and cefoperazone were reduced fourfold. The same level of decrease in the MIC was observed for the organism producing the S289C mutant for the narrow-spectrum β-lactams cephaloridine and cephalothin. However, the MICs of cefoperazone and cefazolin were further decreased (16-fold), and a 4-fold increased susceptibility to two other β-lactams, cefaclor and penicillin G, was observed. Table 2 presents the MICs of cephaloridine in combination with clavulanic acid, sulbactam, and tazobactam. The wild-type enzyme was not inhibited by clavulanic acid but was well inhibited by sulbactam and tazobactam. The mutant enzymes exhibited the same inhibitory profile as that of wild-type enzyme for the three inhibitors.
Electrophoretic mobilities of purified β-lactamases by SDS-polyacrylamide gel electrophoresis.
To elucidate the role of Ser-289 in the activity of the E. cloacae P99 β-lactamase, the wild-type and mutant enzymes were purified to near homogeneity (≥95% pure). Figure 1 shows the electrophoretic mobilities of the purified wild-type P99 β-lactamase and the six mutants on an SDS-polyacrylamide gel. A single protein band was observed for the seven β-lactamases, and this indicates their purity. The molecular mass was estimated to be 39 kDa for the wild-type P99 β-lactamase and for most of the mutants. However, two mutants, S289R and S289K, exhibited slightly different electrophoretic mobilities and their calculated molecular mass was 36 kDa. Nevertheless, a mass spectrum study (in collaboration with Steve Withers from the University of British Columbia) with wild-type P99 β-lactamase and the S289R mutant confirmed that their molecular masses correspond to their respective amino acid sequences (wild-type P99 β-lactamase, 39,238.9 ± 2.8 Da; S289R β-lactamase, 39,307.6 ± 2.6 Da).
FIG. 1.
Silver-stained SDS-polyacrylamide gel of the purified wild-type and mutant β-lactamases from E. cloacae P99. Lane M, low-molecular-mass marker (in kilodaltons).
Kinetic parameters.
The steady-state parameters for the wild-type and mutant enzymes for cephaloridine, cephalothin, cefoperazone, cefaclor, and cefazolin were determined (Tables 3 to 7, respectively). The results indicated that the S289A, S289T, S289C, and S289Q mutants exhibit catalytic efficiencies almost similar to that of the wild-type P99 enzyme toward most cephalosporins studied with the exception of that of the mutant with the S289C substitution for cefoperazone, which was decreased by more than fivefold. This ratio resulted from a twofold decrease in kcat combined with a twofold increase in Km. The catalytic efficiencies of the S289K and S289R mutants were reduced for most of the cephalosporins studied, especially for cephalothin, cefoperazone, and cephaloridine. The Lys at position 289 decreased by two-, three-, and fivefold the catalytic efficiencies of these three antibiotics, respectively. This effect was mostly due to a decrease in kcat. With Arg at position 289, the catalytic efficiencies for cephalothin and cefoperazone were decreased three- and fourfold, respectively, and these decreases also resulted from a lower kcat. For this last mutant, the highest decrease in kcat/Km (sevenfold) was associated with cephaloridine and was due to both a lowering of the kcat and an increase in the Km.
TABLE 3.
Kinetic parameters of wild-type and mutant β-lactamases for cephaloridine
| β-Lactamase | kcat (s−1) | Km (μM) | kcat/Km (μM−1 · s−1) | Relative valuea |
|---|---|---|---|---|
| S289b | 2,320 ± 130 | 327 ± 9 | 7.1 ± 0.4 | 1.00 |
| S289A | 1,580 ± 60 | 290 ± 13 | 5.4 ± 0.2 | 0.76 |
| S289T | 1,350 ± 80 | 195 ± 6 | 6.9 ± 0.5 | 0.97 |
| S289Q | 1,620 ± 80 | 350 ± 18 | 4.6 ± 0.3 | 0.65 |
| S289R | 680 ± 50 | 681 ± 22 | 1.0 ± 0.1 | 0.14 |
| S289K | 770 ± 50 | 522 ± 19 | 1.5 ± 0.1 | 0.21 |
| S289C | 1,620 ± 80 | 295 ± 11 | 5.5 ± 0.3 | 0.77 |
Ratio of kcat/Km of the mutant β-lactamase to that of the wild-type enzyme.
Wild type.
TABLE 7.
Kinetic parameters of wild-type and mutant β-lactamases for cefazolin
| β-Lactamase | kcat (s−1) | Km (μM) | kcat/Km (μM−1 · s−1) | Relative valuea |
|---|---|---|---|---|
| S289b | 3,790 ± 220 | 1,150 ± 50 | 3.3 ± 0.2 | 1.00 |
| S289A | 3,050 ± 90 | 1,020 ± 40 | 3.0 ± 0.1 | 0.91 |
| S289T | 2,380 ± 150 | 850 ± 40 | 2.8 ± 0.2 | 0.85 |
| S289Q | 4,730 ± 230 | 1,760 ± 70 | 2.7 ± 0.1 | 0.82 |
| S289R | 2,880 ± 200 | 1,770 ± 70 | 1.6 ± 0.1 | 0.49 |
| S289K | 3,840 ± 250 | 2,260 ± 90 | 1.7 ± 0.1 | 0.52 |
| S289C | 2,620 ± 150 | 1,890 ± 110 | 1.4 ± 0.1 | 0.42 |
Ratio of kcat/Km of the mutant β-lactamase to that of the wild-type enzyme.
Wild type.
Structural analysis.
Examination of the crystallographic structure of the P99 β-lactamase shows that Ser-289 is on a loop near the β-lactam binding site between helix H10 and β-strand B2g (as shown in Fig. 2). The side chain is exposed and is directed toward the binding site, and it would be able to make contact with the C-3 substituent of a cephalosporin. The carboxylic acid group at C-4 is about 8 Å from the side chain at position 289 and is partly shielded from it by the C-3 substituent. However, in the serine-bound acyl intermediate (Fig. 3), ring opening is known to permit a rotation of the dihydrothiazine ring (34) which would bring the C-4 carboxylic acid group 3 to 4 Å closer to the side chain at position 289. The side chain of a long amino acid such as Lys or Arg could easily form an electrostatic bond with the carboxylic acid group of the acyl intermediate. In the more slowly hydrolyzed cephalosporins such as cefoperazone, the C-3 group may depart from the intermediate, further opening the carboxylic acid group to interaction with the positive side chain (38).
FIG. 3.
A conformation of the ring-opened serine-bound cefoperazone intermediate based on crystallographic structures of the seryl complexes of aztreonam, cefotaxime, and cephalothin (see Materials and Methods). After rotation of dihydrothiazine ring, Arg-289 is able to form a salt linkage to the carboxylic acid group.
DISCUSSION
In this study, the role of Ser-289 in the β-lactam hydrolysis mechanism of the class C β-lactamase from E. cloacae P99 was investigated by site-directed mutagenesis. Each of the mutants was purified to homogeneity, and precise kinetic analysis was performed.
An alignment of 25 chromosomal and plasmid-mediated class C β-lactamases (52a) revealed that most of these enzymes contain a small and hydrophilic residue at position 289 (Ser, Thr, or Asn) (5, 6, 7, 8, 10, 11, 13, 18, 20, 29, 37, 40, 42, 48, 54). In E. cloacae P99 AmpC, Ser-289 lies at the top of the binding site, where it can interact with the C-3 substituent of cephalosporins (Fig. 2 and 3) (36). The native Ser-289 hydroxyl group could donate to or, in some cases, accept hydrogen bonds from the rather large C-3 substituents of most of the substrates listed here. A similar bonding is possible with threonine, and accordingly, little change in catalytic efficiency is seen in the S289T mutant. This hydrogen bonding would also be possible with a cysteine sulfhydryl group, but the bonding would be much weaker than that of serine or threonine, and therefore, greater changes in catalytic efficiency are seen with the S289C mutant. This consideration of hydrogen bonding fails to explain why the mutant with the alanine mutation exhibits little change in catalytic efficiency. It is worth mentioning that in seven known class C β-lactamases, an apolar Ala or Pro is also found at position 289 (6, 8, 13, 37, 40, 54). Therefore, hydrogen bonding between residue 289 and the C-3 substituent of cephalosporins or with the acyl intermediate would not be essential in the catalytic mechanism of class C β-lactamases.
When examining changes in the kinetic parameters of the substrates, one generally considers Km to reflect molecular interactions in the Michaelis complex and kcat to reflect interactions during the formation and breakdown of the serine-bound acyl intermediate. The ratio of the two, the catalytic efficiency, incorporates both contributions to the overall kinetics. In earlier kinetic studies of the wild-type P99 enzyme, Mazzella and Pratt (38) found that deacylation is the rate-determining step and therefore that kcat and kcat/Km describe the deacylation and acylation steps, respectively. Three types of mutants with mutations at position 289, cysteine, lysine, and arginine, consistently show decreases in kcat/Km for all the cephalosporins studied. For example, cefoperazone hydrolysis by these three mutants exhibited two- to threefold decreases in kcat values, perhaps meaning that interactions of the side chain at position 289 with the acyl intermediate are more important than precatalytic Michaelis interactions.
Given the somewhat greater effects seen in the mutants with lysine and arginine mutations, the positive charge of the side chain must be important. Furthermore, the longer lengths of these basic side chains make possible a strong electrostatic linkage (salt bridge) to the C-4 carboxylic acid group of the dihydrothiazine ring. This linkage is more likely to occur in the acyl intermediate than in the Michaelis complex for two reasons. First, the rotation of the dihydrothiazine ring after acylation (34) moves the carboxylic acid group toward the side chain at position 289. Second, in slowly hydrolyzed broad-spectrum cephalosporins the likely elimination of the C-3 substituent (38) will permit an even more direct interaction with the carboxylic acid group (Fig. 3). A strong linkage could cause a reduction in kcat either by hindering the approach of the hydrolytic water molecule to the seryl ester bond (12) or by slowing the release of the hydrolysis product from the binding site.
Clavulanic acid (44), sulbactam (17), and tazobactam (4, 21) are commonly used inhibitors which are very effective against most bacterial strains that produce class A β-lactamases. The inability of clavulanic acid to inhibit the class C β-lactamase (Table 2) has been discussed in relation to the architecture of the binding site and its differences with respect to class A enzymes (36). On the basis of the earlier modeling of clavulanate binding to the wild-type P99 β-lactamase (36), the relative insensitivities of all the mutants is possibly due to the small size of clavulanate and the absence of strong interactions with the side chain at position 289. The different behaviors of both sulbactam and tazobactam relative to that of clavulanate toward class A β-lactamases have been analyzed (25) and may apply here as well.
These results suggest that Ser-289 is not essential in the binding or hydrolysis mechanism of AmpC β-lactamase and that many amino acid substitutions are possible at this position. However, changes for positively charged amino acids reduce catalytic efficiency and have not been selected during evolution. These findings are supported by the study of Siemers et al. (49), who showed, by using random mutagenesis in the region from positions 286 to 289 of the AmpC β-lactamase from E. cloacae P99, that Ser-289 was not strictly conserved in their active mutants and that two of the four inactive mutants identified in their library contained Arg or Lys at position 289.
TABLE 4.
Kinetic parameters of wild-type and mutant β-lactamases for cephalothin
| β-Lactamase | kcat (s−1) | Km (μM) | kcat/Km (μM−1 · s−1) | Relative valuea |
|---|---|---|---|---|
| S289b | 413 ± 25 | 11.5 ± 0.6 | 36.1 ± 2.7 | 1.00 |
| S289A | 244 ± 6 | 7.5 ± 0.3 | 32.6 ± 1.4 | 0.90 |
| S289T | 209 ± 13 | 4.9 ± 0.2 | 43.0 ± 3.6 | 1.19 |
| S289Q | 243 ± 9 | 8.5 ± 0.3 | 28.0 ± 1.6 | 0.78 |
| S289R | 118 ± 7 | 10.2 ± 0.5 | 11.5 ± 0.9 | 0.32 |
| S289K | 124 ± 7 | 8.3 ± 0.3 | 15.0 ± 1.1 | 0.41 |
| S289C | 124 ± 6 | 8.5 ± 0.4 | 14.6 ± 1.1 | 0.40 |
Ratio of kcat/Km of the mutant β-lactamase to that of the wild-type enzyme.
Wild type.
TABLE 5.
Kinetic parameters of wild-type and mutant β-lactamases for cefoperazone
| β-Lactamase | kcat (s−1) | Km (μM) | kcat/Km (μM−1 · s−1) | Relative valuea |
|---|---|---|---|---|
| S289b | 5.0 ± 0.3 | 6.3 ± 0.2 | 0.79 ± 0.05 | 1.00 |
| S289A | 2.6 ± 0.1 | 3.8 ± 0.2 | 0.70 ± 0.04 | 0.89 |
| S289T | 4.3 ± 0.3 | 8.3 ± 0.3 | 0.52 ± 0.04 | 0.66 |
| S289Q | 3.4 ± 0.1 | 7.1 ± 0.3 | 0.47 ± 0.03 | 0.59 |
| S289R | 1.6 ± 0.1 | 8.6 ± 0.2 | 0.19 ± 0.01 | 0.24 |
| S289K | 2.0 ± 0.1 | 7.8 ± 0.3 | 0.26 ± 0.02 | 0.33 |
| S289C | 2.2 ± 0.1 | 15.1 ± 1.5 | 0.15 ± 0.02 | 0.19 |
Ratio of kcat/Km of the mutant β-lactamase to that of the wild-type enzyme.
Wild type.
TABLE 6.
Kinetic parameters of wild-type and mutant β-lactamases for cefaclor
| β-Lactamase | kcat (s−1) | Km (μM) | kcat/Km (μM−1 · s−1) | Relative valuea |
|---|---|---|---|---|
| S289b | 317 ± 16 | 45 ± 2 | 7.0 ± 0.5 | 1.00 |
| S289A | 274 ± 7 | 46 ± 1 | 6.0 ± 0.2 | 0.85 |
| S289T | 152 ± 10 | 22 ± 1 | 6.7 ± 0.6 | 0.96 |
| S289Q | 266 ± 11 | 37 ± 2 | 7.1 ± 0.4 | 1.01 |
| S289R | 147 ± 10 | 53 ± 2 | 2.8 ± 0.2 | 0.40 |
| S289K | 179 ± 12 | 43 ± 3 | 4.1 ± 0.3 | 0.58 |
| S289C | 120 ± 6 | 32 ± 2 | 3.8 ± 0.3 | 0.54 |
Ratio of kcat/Km of the mutant β-lactamase to that of the wild-type enzyme.
Wild type.
ACKNOWLEDGMENTS
We thank Michèle Dargis for excellent technical assistance. We also thank Steve Withers for mass spectrum analysis.
This work was supported by the Canadian Cystic Fibrosis Foundation and, in part, by Canada’s Networks of Centres of Excellence (CBDN).
REFERENCES
- 1.Allen F H, Davies J E, Galloy J J, Johnson O, Kennard O, Macrae C F, Mitchell E M, Mitchell G F, Smith J M, Watson D G. The development of versions 3 and 4 of the Cambridge Structural Database system. J Chem Inform Comput Sci. 1991;31:187–204. [Google Scholar]
- 2.Ambler R P. The structure of β-lactamases. Philos Trans R Soc London Ser B. 1980;289:321–331. doi: 10.1098/rstb.1980.0049. [DOI] [PubMed] [Google Scholar]
- 3.Ambler R P, Frère J-M, Ghuysen J-M, Joris B, Levesque R C, Tiraby G, Waley S G, Coulson A F W. A standard numbering scheme for the class A β-lactamases. Biochem J. 1991;276:269–272. doi: 10.1042/bj2760269. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Aronoff S C, Jacobs M R, Johenning S, Yamabe S. Comparative activities of the beta-lactamase inhibitors YTR 830, sodium clavulanate, and sulbactam combined with amoxicillin or ampicillin. Antimicrob Agents Chemother. 1984;26:580–582. doi: 10.1128/aac.26.4.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Barnaud G, Arlet G, Danglot C, Philippon A. Cloning and sequencing of the gene encoding the AmpC beta-lactamase of Morganella morganii. FEMS Microbiol Lett. 1997;148:15–20. doi: 10.1111/j.1574-6968.1997.tb10260.x. [DOI] [PubMed] [Google Scholar]
- 6.Bauernfeind A, Stemplinger I, Jungwirth R, Wilhelm R, Chong Y. Comparative characterization of the cephamycinase blaCMY-1 gene and its relationship with other beta-lactamase genes. Antimicrob Agents Chemother. 1996;40:1926–1930. doi: 10.1128/aac.40.8.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bauernfeind A, Stemplinger I, Jungwirth I, Giamarellou H. Characterization of the plasmidic beta-lactamase CMY-2, which is responsible for cephamycin resistance. Antimicrob Agents Chemother. 1996;40:221–224. doi: 10.1128/aac.40.1.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Bauernfeind A, Wagner S, Jungwirth R, Schneider I, Meyer D. A novel class C beta-lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins. Antimicrob Agents Chemother. 1997;41:2041–2046. doi: 10.1128/aac.41.9.2041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- 10.Bradford P A, Urban C, Mariano N, Projan S J, Rahal J J, Bush K. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother. 1997;41:563–569. doi: 10.1128/aac.41.3.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Bret L, Chanal-Claris C, Sirot D, Chaibi E B, Labia R, Sirot J. Chromosomally encoded AmpC-type β-lactamase in a clinical isolate of Proteus mirabilis. Antimicrob Agents Chemother. 1998;42:1110–1114. doi: 10.1128/aac.42.5.1110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Bulychev A, Massova I, Miyashita K, Mobashery S. Nuances of mechanisms and their implications for evolution of the versatile β-lactamase activity: from biosynthetic enzymes to drug resistance factors. J Am Chem Soc. 1997;119:7619–7625. [Google Scholar]
- 13.Bush K, Jacoby G A, Medeiros A A. A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother. 1995;39:1211–1233. doi: 10.1128/aac.39.6.1211. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Cartwright S J, Waley S G. Purification of beta-lactamases by affinity chromatography on phenylboronic acid-agarose. Biochem J. 1984;221:505–512. doi: 10.1042/bj2210505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Cornish-Bowden A. Analysis of enzyme kinetic data. New York, N.Y: Oxford University Press; 1995. [Google Scholar]
- 16.Ehrhardt A F, Sanders C C, Romero J R, Leser S. Sequencing and analysis of four new Enterobacter ampD alleles. Antimicrob Agents Chemother. 1996;40:1953–1956. doi: 10.1128/aac.40.8.1953. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.English A R, Retsema J A, Girard A E, Lynch J E, Barth W E. CP-45,899, a beta-lactamase inhibitor that extends the antibacterial spectrum of beta-lactams: initial bacteriological characterization. Antimicrob Agents Chemother. 1978;14:414–419. doi: 10.1128/aac.14.3.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Feller G, Zekhnini Z, Lamotte-Brasseur J, Gerday C. Enzymes from cold-adapted microorganisms. The class C β-lactamase from the antarctic psychrophile Psychrobacter immobilis A5. Eur J Biochem. 1997;244:186–191. doi: 10.1111/j.1432-1033.1997.00186.x. [DOI] [PubMed] [Google Scholar]
- 19.Frère J-M. Beta-lactamases and bacterial resistance to antibiotics. Mol Microbiol. 1995;16:385–395. doi: 10.1111/j.1365-2958.1995.tb02404.x. [DOI] [PubMed] [Google Scholar]
- 20.Galleni M, Lindberg F, Normark S, Cole S, Honoré N, Joris B, Frère J-M. Sequence and comparative analysis of three Enterobacter cloacae ampC beta-lactamase genes and their products. Biochem J. 1988;250:753–760. doi: 10.1042/bj2500753. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Higashitani F, Hyodo A, Ishida N, Inoue M, Mitsuhashi S. Inhibition of beta-lactamases by tazobactam and in-vitro antibacterial activity of tazobactam combined with piperacillin. J Antimicrob Chemother. 1990;25:567–574. doi: 10.1093/jac/25.4.567. [DOI] [PubMed] [Google Scholar]
- 22.Höltje J-V, Kopp U, Ursinus A, Wiedemann B. The negative regulator of β-lactamase induction AmpD is a N-acetyl-anhydromuramyl-l-alanine amidase. FEMS Microbiol Lett. 1994;122:159–164. doi: 10.1111/j.1574-6968.1994.tb07159.x. [DOI] [PubMed] [Google Scholar]
- 23.Huletsky A, Knox J R, Levesque R C. Role of Ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type β-lactamases probed by site-directed mutagenesis and three-dimensional modeling. J Biol Chem. 1993;268:3690–3697. [PubMed] [Google Scholar]
- 24.Huovinen P, Huovinen S, Jacoby G A. Sequence of PSE-2 beta-lactamase. Antimicrob Agents Chemother. 1988;32:134–136. doi: 10.1128/aac.32.1.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Imtiaz U, Billings E M, Knox J R, Mobashery S. A structure-based analysis of the inhibition of class A β-lactamases by sulbactam. Biochemistry. 1994;33:5728–5738. doi: 10.1021/bi00185a009. [DOI] [PubMed] [Google Scholar]
- 26.Jacobs C, Joris B, Jamin M, Klarsov K, Van Beeumen J, Mengin-Lecreulx D, van Heijenoort J, Park J T, Normark S, Frère J-M. AmpD, essential for both β-lactamase regulation and cell wall recycling, is a novel cytosolic N-acetylmuramyl-l-alanine amidase. Mol Microbiol. 1995;15:553–559. doi: 10.1111/j.1365-2958.1995.tb02268.x. [DOI] [PubMed] [Google Scholar]
- 27.Jacoby G A. Genetics of extended-spectrum beta-lactamases. Eur J Clin Microbiol Infect Dis. 1994;13(Suppl. 1):S2–S11. doi: 10.1007/BF02390679. [DOI] [PubMed] [Google Scholar]
- 28.Jaurin B, Grundström T. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of β-lactamases of the penicillinase type. Proc Natl Acad Sci USA. 1981;78:4897–4901. doi: 10.1073/pnas.78.8.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Koeck J L, Arlet G, Philippon A, Basmaciogullari S, Thien H V, Buisson Y, Cavallo J D. A plasmid-mediated CMY-2 beta-lactamase from an Algerian clinical isolate of Salmonella senftenberg. FEMS Microbiol Lett. 1997;152:255–260. doi: 10.1111/j.1574-6968.1997.tb10436.x. [DOI] [PubMed] [Google Scholar]
- 30.Korfmann G, Sanders C C, Moland E S. Altered phenotypes associated with ampD mutations in Enterobacter cloacae. Antimicrob Agents Chemother. 1991;35:358–364. doi: 10.1128/aac.35.2.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Kraulis P. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr. 1991;24:946–950. [Google Scholar]
- 32.Kunkel T A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 1985;82:488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Kunkel T A, Roberts J D, Zakow R A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- 34.Kuzin A P, Liu H, Kelly J A, Knox J R. Binding of cephalothin and cefotaxime to d-Ala-d-Ala peptidase reveals a functional basis of a natural mutation in a low-affinity penicillin-binding protein and in extended-spectrum β-lactamases. Biochemistry. 1995;34:9532–9540. doi: 10.1021/bi00029a030. [DOI] [PubMed] [Google Scholar]
- 35.Lobkovsky E, Billings E M, Moews P C, Rahil J, Pratt R F, Knox J R. Crystallographic structure of a phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase: mechanistic interpretation of a β-lactamase transition state analog. Biochemistry. 1994;33:6762–6772. doi: 10.1021/bi00188a004. [DOI] [PubMed] [Google Scholar]
- 36.Lobkovsky E, Moews P C, Liu H, Zhao H, Frère J-M, Knox J R. Evolution of an enzyme activity: crystallographic structure at 2 Å resolution of the cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. Proc Natl Acad Sci USA. 1993;90:11257–11261. doi: 10.1073/pnas.90.23.11257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Marchese A, Arlet G, Schito G C, Lagrange P H, Philippon A. Characterization of FOX-3, an AmpC-type plasmid-mediated β-lactamase from an italian isolate of Klebsiella oxytoca. Antimicrob Agents Chemother. 1998;42:464–467. doi: 10.1128/aac.42.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Mazzella L J, Pratt R F. Effect of the 3′-leaving group on turnover of cephem antibiotics by a class C β-lactamase. Biochem J. 1989;259:255–260. doi: 10.1042/bj2590255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.McClary J A, Witney F, Geisselsoder J. Efficient site-directed in vitro mutagenesis using phagemid vectors. BioTechniques. 1989;3:282–289. [PubMed] [Google Scholar]
- 40.Nomura K, Yoshida T. Nucleotide sequence of the Serratia marcescens SR50 chromosomal ampC beta-lactamase gene. FEMS Microbiol Lett. 1990;70:295–299. doi: 10.1111/j.1574-6968.1990.tb13992.x. [DOI] [PubMed] [Google Scholar]
- 41.Normark S, Burman L G. Resistance of Escherichia coli to penicillins: fine structure mapping and dominance of chromosomal beta-lactamase mutations. J Bacteriol. 1977;132:1–7. doi: 10.1128/jb.132.1.1-7.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Nukaga M, Haruta S, Tanimoto K, Kogure K, Taniguchi K, Tamaki M, Sawai T. Molecular evolution of a class C beta-lactamase extending its substrate specificity. J Biol Chem. 1995;270:5729–5735. doi: 10.1074/jbc.270.11.5729. [DOI] [PubMed] [Google Scholar]
- 43.Oefner C, D’Arcy A, Daly J J, Gubernator K, Charnas R L, Heinze I, Hubschwerlen C, Winkler F K. Refined crystal structure of β-lactamase from Citrobacter freundii indicates a mechanism for β-lactam hydrolysis. Nature. 1990;343:284–288. doi: 10.1038/343284a0. [DOI] [PubMed] [Google Scholar]
- 44.Reading C, Cole M. Clavulanic acid: a β-lactamase-inhibiting β-lactam from Streptomyces clavuligerus. Antimicrob Agents Chemother. 1977;11:852–857. doi: 10.1128/aac.11.5.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989. pp. 1.21–1.101. [Google Scholar]
- 46.Sanders C C. Chromosomal cephalosporinases responsible for multiple resistance to newer β-lactam antibiotics. Annu Rev Microbiol. 1987;41:573–593. doi: 10.1146/annurev.mi.41.100187.003041. [DOI] [PubMed] [Google Scholar]
- 47.Sanger F, Nicklen S, Coulson A R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977;74:5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Seoane A, Francia M V, Garcia Lobo J M. Nucleotide sequence of the ampC-ampR region from the chromosome of Yersinia enterocolitica. Antimicrob Agents Chemother. 1992;36:1049–1052. doi: 10.1128/aac.36.5.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Siemers N O, Yelton D E, Bajorath J, Senter P D. Modifying the specificity and activity of the Enterobacter cloacae P99 β-lactamase by mutagenesis within an M13 phage vector. Biochemistry. 1996;35:2104–2111. doi: 10.1021/bi9514166. [DOI] [PubMed] [Google Scholar]
- 50.Spratt B G, Hedge P J, te Heesen S, Edelman A, Broome-Smith J K. Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene. 1986;41:337–342. doi: 10.1016/0378-1119(86)90117-4. [DOI] [PubMed] [Google Scholar]
- 51.Stapleton P, Shannon K, Phillips I. DNA sequence differences of ampD mutants of Citrobacter freundii. Antimicrob Agents Chemother. 1995;39:2494–2498. doi: 10.1128/aac.39.11.2494. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Starnbach M N, Falkow S, Tompkins L S. Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization. J Clin Microbiol. 1989;27:1257–1261. doi: 10.1128/jcm.27.6.1257-1261.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52a.Trépanier, S., and A. Huletsky. Personal communication.
- 53.Vieira J, Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
- 54.Walsh T R, Hall L, MacGowan A P, Bennett P M. Sequence analysis of two chromosomally mediated inducible β-lactamases from Aeromonas sobria, strain 163a, one a class D penicillinase, the other an AmpC cephalosporinase. J Antimicrob Chemother. 1995;36:41–52. doi: 10.1093/jac/36.1.41. [DOI] [PubMed] [Google Scholar]