The DK4 β-lactamase gene and its product.
K. pneumoniae DK4 exhibited a high level of resistance to extended-spectrum cephalosporins and a moderate level of resistance to carbapenens, i.e., imipenen. The resistance was conjugally transferred to E. coli 1037 Rifr. The β-lactam resistance was due to a β-lactamase with a pI of 8.6 and was mediated by an R plasmid termed RDK4. The activity of the DK4 β-lactamase was completely inhibited by 10 mM EDTA. The DK4 β-lactamase gene in a 9.7-kb DNA fragment was cloned into a plasmid vector, pHSG398, and the recombinant plasmid was termed pDK4-1. From the 9.7-kb DNA fragment, four shorter DNA fragments (of 3.9, 2.6, 2.0, and 1.2 kb) containing the β-lactamase gene were prepared, and each fragment was cloned into the plasmid vector (Fig. ). The recombinant plasmids obtained were termed pDK4-2, pDK4-3, pDK4-4, and pDK4-5, respectively. The complete nucleotide sequence of the 1.2-kb fragment inserted into pDK4-5 was determined. The sequenced region contained an open reading frame, and an amino acid sequence comprising 246 amino acids was deduced. Through determination of the N-terminal amino acid sequence of the purified enzyme protein, a signal peptide with 18 amino acids and a mature enzyme with 228 amino acids were detected.
Restriction maps of inserts of the clone and subclones carrying the target β-lactamase gene.
Alignment of the mature enzyme with known metallo-β-lactamase amino acid sequences indicated that the DK4 β-lactamase has 38.4% homology to the enzymes of Bacillus cereus
), 35.4% homology to the enzyme of Bacteroides fragilis
), 32.3% homology to the enzyme of Aeromonas hydrophila
), and 19.5% homology to the enzyme of Stenotrophomonas maltophilia
). On the other hand, the amino acid sequence of the DK4 β-lactamase is identical to that of a metallo β-lactamase termed IMP-1 and produced by S. marcescens
). The IMP-1 β-lactamase gene (blaIMP
) was previously reported to be located on the chromosome of S. marcescens
. Our detection of an identical β-lactamase gene incorporated in an R plasmid suggests the insertion of this gene into the chromosome of S. marcescens
Dose effect of DK4 β-lactamase on resistance to β-lactam antibiotics in E. coli cells.
During the preparation of the subcloned β-lactamase gene, we observed that the β-lactamase activity in the E. coli AS226-51 cells was inversely proportional to the size of the DNA fragment inserted into the vector plasmid (Table ). This phenomenon may be due to the difference in copy number of the plasmid in the host cells because all the β-lactamase genes were expressed by their own promoters, and the pDK4 series can be used to provide an understanding of the dose effect of metallo-β-lactamase production with respect to the level of resistance of the bacterial cells to β-lactams. The β-lactamase activity of K. pneumoniae DK4 was 0.12 U per mg of bacterial protein, as determined with cephalothin as a substrate. When RDK4 was transferred to E. coli AS226-51 cells, the enzyme activity was 0.098 U per mg of bacterial protein, about 80% of that for the original strain. For the series of E. coli subclones, the MICs for the cells increased up to 8 times and the level of enzyme production increased up to 13 times. The results in Table show quantitatively that the metallo-β-lactamase contributed to the resistance of the bacteria to all β-lactams except the monobactam aztreonam.
TABLE 1 Dose effect of DK4 β-lactamase on resistance of bacteria to typicalβ-lactams Characteristics of DK4 β-lactamase and its kinetic parameters.
The DK4 β-lactamase was extracted from the E. coli
cells carrying pDK4-5 and was completely purified. From 18 liters of the bacterial culture, 18 mg of the purified enzyme protein was obtained, and its purity was confirmed by SDS-PAGE. The presence or absence of 0.1 mM zinc sulfate throughout the purification processes did not affect the recovery of active enzyme, indicating a tight binding of zinc to the enzyme. The purified enzyme showed a high degree of thermostability, as the enzyme retained its full activity even after 30 min of incubation at 60°C in 50 mM MOPS buffer (pH 7.0) containing 0.1 mM zinc sulfate. The activity was assayed within 30 s of the enzyme reaction at 30°C. The effect of pH on the activity of the enzyme against cephalothin at between pH 5.0 and 8.0 was measured in the following buffers: 50 mM MES buffer with 0.5 M NaCl and 1 mM zinc sulfate (pH 5.0 to 6.5) and 50 mM MOPS buffer with 0.5 M NaCl (pH 7.0 to 80). The β-lactamase activity increased concomitantly with pH, such that at pHs 7.0, 6.0, and 5.0 the activities were 75, 49, and 32%, respectively, of that at pH 8.0. The enzyme was found to be unstable at a pH lower than 6.0, and this phenomenon may be attributable to the presence of 2-(N
-morpholino)ethanesulfonic acid (MES) (8
). Stability at lower pH could be protected by the presence of 1 mM zinc sulfate. We failed to measure activity at a pH higher than 8.0 because of alkaline hydrolysis of the substrate in the reaction mixture.
The zinc content in the purified enzyme molecule was determined by means of atomic absorption spectrophotometry. The results indicated that the DK4 β-lactamase contains 2.0 zinc atoms per mature enzyme protein. Further testing indicated that enzyme activity was not influenced by the addition of 5 mM zinc sulfate to the reaction mixture, and for complete inactivation of the enzyme, an EDTA concentration greater than 1 mM was required. The 50% inhibitory dose of EDTA was 0.62 mM.
During the UV spectrophotometric assay for enzyme activity, we observed that the molar extinction coefficient decreased upon the addition of zinc sulfate. In the case of cephalothin as the substrate, the coefficient was changed from 7,200 to 6,300 M−1 cm−1 by the addition of 1 mM zinc sulfate. This effect of zinc could be negated in the presence of 0.5 M NaCl. This phenomenon may be attributable to an ionic interaction between the zinc cation and the cleaved β-lactam ring, and it may lead to an erroneous conclusion that the activity of the metallo-β-lactamase is inhibited by a high concentration of zinc.
The kinetic parameters of the purified DK4 β-lactamase for nine β-lactams were determined in MOPS buffer (pH 7.0) at 30°C, and the results are summarized in Table . The DK4 β-lactamase has a broad substrate specificity, including penicillins, cephalosporins, cephamycin, and carbapenen, similar to those of known metallo-β-lactamases. When the kinetic parameters of the DK4 β-lactamase for typical β-lactams were compared with those for IMP-1 from a more recent report (13
), some differences in the parameters, especially in the kcat
value for ampicillin and Km
values for cephalosporins including cefoxitin, were observed. The kcat
value of the DK4 enzyme for ampicillin was about 1/16 of that of the IMP-1 enzyme. The Km
values of the IMP-1 enzyme for the cephalosporins were 3 to 46 times greater than those of the DK4 enzyme. We confirmed that the kinetic data in this paper were reproducible under the conditions used in the study.
TABLE 2 Kinetic parameters of DK4 β-lactamase forβ-lactams
The effects of the serine β-lactamase inhibitors and a renal membrane dipeptidase inhibitor (cilastatin) on the DK4 β-lactamase were examined. The activity of the DK4 enzyme was not affected by 10 mM sulbactam, 10 mM clavulanic acid, or 1 mM aztreonam; and it had undetectable hydrolytic activity against the three β-lactams. Cilastatin, which is known to be an inhibitor of dipeptidase, showed weak inhibitory activity against the DK4 β-lactamase. The 50% inhibitory concentration of cilastatin was about 3 mM, and this value is about 104
times the 50% inhibitory concentration of cilastatin for dipeptidase (12
The apo-DK4 β-lactamase and its restoration.
A DK4 β-lactamase that was missing the two zincs was prepared by 3 days of dialysis of the EDTA-treated enzyme against zinc-free MOPS buffer. The enzyme, termed apo-DK4, was completely lacking enzyme activity, and its activity was estimated to be less than 0.004% of that of the holoenzyme. The activity of the apo-DK4 enzyme was restored to about 30% of its original activity by the addition of 1 mM zinc sulfate to the enzyme solution. This restoration was hindered in the presence of a sulfhydryl reagent, methylmethane thiosulfonate, suggesting the contribution of a cysteine to zinc binding. The reactivated enzyme had about the same Km value for cephalothin as that of the native enzyme, and it was therefore thought that about 70% of the enzyme molecules were irreversibly denatured during production of the apoenzyme state.
Functions of His28, His86, His88, His149, His210, and Cys168 as zinc ligands.
The alignment of the amino acid sequences of the metallo-β-lactamases indicated that histidines at positions 86, 88, 149, and 210 were conserved in all the enzymes, and a histidine at position 28 was found in some metallo-β-lactamases. All of these histidine residues except His28 were estimated to be located at positions close to the zincs by reference to a three-dimensional structure of a metallo-β-lactamase from B. fragilis
). Cys168 was also presumed to be situated in the vicinity of the zinc. To establish the functions of these residues in the DK4 β-lactamase, these five histidines and the cysteine were individually replaced by an alanine by site-directed mutagenesis, and the mutant genes on the vector plasmid were transformed into E. coli
The mutant β-lactamases were purified in the same way as the wild-type enzyme. All the mutant enzymes except the enzyme with the H28A mutation showed decreased activity following dialysis against zinc-free MOPS buffer. Therefore, purification of these mutant enzymes was carried out in the presence of 0.1 mM zinc sulfate.
The wild-type and His mutant β-lactamases purified were dialyzed in zinc-free MOPS buffer (pH 7.0) containing Chelex 100 resin. The wild-type and the mutant with the H28A mutation retained their activities even after the dialysis, and the 2 mol of zinc per mol of enzyme was detected in the dialyzed enzymes (Table ). This result agreed with the metal/enzyme ratio for the wild-type enzyme reported by Laraki et al. (13
TABLE 3 Zinc contents of DK4 metallo-β-lactamase and itsmutantsa
On the other hand, the enzymes with the H86A, H88A, H149A, and C168A mutations showed significantly decreased activity, and this residual activity remained constant 24 h after the dialysis. The zinc content after exhaustive dialysis of the enzymes with H86A, H88A, H149A and C168A mutation was about half that of the wild-type enzyme, suggesting that all the enzymes except that with the H28A mutation had one zinc atom per one enzyme molecule (Table ). In the case of the mutant with the H210A mutation, activity was completely absent following dialysis, and the zinc content was estimated to be 0.5 mol per enzyme mol. These data suggest a significant distortion of the active site in the mutant with the H210A mutation.
Cys168 is the only cysteine residue in the DK4 β-lactamase. Its replacement by alanine resulted in a significant lowering of activity, as measured after enzyme purification, and the missing activity was only slightly restored even by 1 mM zinc (Table ). This result is consistent with that for the mutant of the B. cereus
β-lactamase with the C168A mutation (19
). On the basis of the fact that serine is situated at position 168 of a metallo-β-lactamase from S. maltophilla
), a mutant with the C168S mutation was prepared from the DK4 β-lactamase. The zinc content of the enzyme with the C168S mutation was 1.07 mol after dialysis at pH 7.0, but the zinc content was increased to 1.85 mol by dialysis at pH 9.5. This observation suggested that a negative charge at position 168 is necessary for retention of the second zinc atom. This assumption was confirmed by the fact that a mutant with a C168D mutation that we prepared had a zinc content of 2.04 mol after dialysis at pH 7.0 (Table ).
TABLE 4 Kinetic parameters of mutant metallo-β-lactamases for cephalothin in the presence or absence of 1 mM zincsulfatea
The mutant enzymes with the H86A and C168A mutations had detectable activity even after dialysis, and their Michaelis-Menten constants for cephalothin were significantly greater than that of the wild-type enzyme (Table ). Residual activity in the mutant enzymes was essentially missing following treatment with 1 mM EDTA. This observation indicated that a little activity may still be retained in the enzyme lacking one of the two zincs.
The enzymes with the alanine substitutions showed increased specific activity with an increase in the zinc concentration in the reaction medium. The maximum activity was achieved with 1 mM zinc sulfate, in the presence of which the enzyme probably retains two zincs at the active site. The kinetic parameters of the dialyzed enzymes for cephalothin in the presence or absence of 1 mM zinc sulfate are summarized in Table .
Three histidines at positions 86, 88, and 149 and the cysteine at position 168 are thought to be the residues that function as zinc ligands. Alteration of the kinetic parameters of the His mutant enzymes by the addition of 1 mM zinc sulfate was most remarkable in the cases of the enzymes with the H86A, H88A and H149A mutations; however, the activity of the enzyme with the C168A mutation was only slightly increased in the presence of 1 mM zinc sulfate. The difference in the effect of zinc between the mutants with the histidine mutations and the mutant with the cysteine mutation may indicate a difference in the zinc atom associated with ligands. The mutant enzyme with the C168D mutation retained two zinc atoms, but its activity could not be detected in the absence of 1 mM zinc sulfate, suggesting that the zincs combine irregularly or weakly to the active site. On the other hand, the mutant enzyme with the C168D mutation exhibited significantly higher kcat and Km values than the wild-type enzyme in the reaction medium with 1 mM zinc sulfate, and its kcat/Km value was restored up to 70% of that for the wild-type enzyme. It can be presumed that the kinetic properties of a metallo-β-lactamase are dependent on the situation of the zincs in the active site.
In order to compare the structural stabilities of the wild type and the mutants with His mutations, their thermal stabilities were examined. After various incubation times in 50 mM MOPS buffer containing 0.1 mM zinc sulfate at 50°C, aliquots of the enzyme solution were withdrawn and the residual activity was determined. The β-lactamases with the H86A, H88A, and H149A mutations lost nearly all of their activities within 20 min of incubation. On the other hand, the wild-type β-lactamase retained its activity, and the β-lactamase with the H28A mutation had about 60% of its original activity even after 60 min (data not shown). These observations suggest a close relationship between structural stability and appropriate binding of the zinc atoms in the active site.
Lim et al. (14
) claimed that His28 of the B. cereus
metallo-β-lactamase is essential for enzyme activity on the basis of the observation that E. coli
cells with the H28Y mutant gene showed high levels of susceptibility to ampicillin and cephalosporin C (14
). In the case of the DK4 β-lactamase, we could not observe significant differences in the enzymatic properties and zinc contents between the wild type and the mutant with the H28A mutation. It may be concluded that His28 of the DK4 enzyme is not a functional residue.
Function of Asp90 as a general base in the enzyme reaction.
Aspartic acid at position 90 is one of the conserved residues in known metallo-β-lactamases. Concha et al. (6
) claimed that Asp90 of a metallo-β-lactamase from B. fragilis
is one of the zinc ligands. The B. fragilis
β-lactamase with the D90V mutation, in fact, had a lower zinc content than the wild-type enzyme (7
). We observed that the kcat
value of the DK4 β-lactamase increased with an increase in the pH from 5.0 to 8.0, suggesting the existence of a general base in the reaction. A candidate for the general base was Asp90, which was assumed to be localized to the active-site area. In order to confirm this assumption, Asp90 was replaced by asparagine.
The purified β-lactamase with the D90N mutation was extensively dialyzed against MOPS buffer without zinc, and its zinc content was determined to be 2.29 mol of zinc per mol of enzyme. Kinetic parameters of the β-lactamase with the D90N mutation for cephalothin were determined at pH 7.0 and compared with those of the wild-type enzyme (Table ). It was also noted that varying the zinc concentration in the reaction mixture did not affect the parameters of either the wild-type or the mutant β-lactamase. The β-lactamase with the D90N mutation showed an approximately 1,000 times lower kcat value for cephalothin than that of the wild-type β-lactamase; however, no difference in Km values was detected.
TABLE 5 Kinetic parameters of the β-lactamase with the D90N mutation for cephalothin and its zinccontent
value was determined from pH 5.0 to 8.0 by using cephalothin as the substrate. Figure shows the effect of pH on the kcat
value, which is expressed as the percentage of the kcat
determined at pH 8.0. The β-lactamase with the D90N mutation exhibited a lower value than the wild-type enzyme from pH 5.5 to 8.0. This result suggests that Asp90 acts as a general base in the enzyme reaction and is consistent with the results obtained with the B. cereus
569/H/9 β-lactamase (3
FIG. 2 Effect of pH on the relative kcat values of the wild-type and the D90N β-lactamases. The following buffers were used for the assay; 50 mM MES buffers containing 0.5 M NaCl and 1 mM zinc sulfate (pH 5.0 to 6.5) and 50 mM MOPS buffer containing (more ...)