1 Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Pierre et Marie Curie (Paris VI), Faculté de Médecine Pitié-Salpêtrière and Faculté de Médecine Broussais-Hôtel Dieu, F-75634 Paris cedex 13 and 2 Laboratoire de Minéralogie-Cristallographie, CNRS URA 09, Université Pierre et Marie Curie (Paris VI), F-75252 Paris cedex 05, France
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Abstract |
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Keywords: ß-lactamase/expanded-spectrum cephalosporins/homology modelling/PER-1/serine enzyme
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Introduction |
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Recently, we have undertaken biochemical studies in order to elucidate the molecular basis of PER-1 activity against expanded-spectrum cephalosporins (Bouthors et al., 1998). Molecular modelling and site-directed mutagenesis were used to investigate in this enzyme the role played by the amino acid residues corresponding to those found at positions 104, 164, 238 and 240 in the TEM-type ESBLs. In brief, two residues, Asn104 and Ala164, were shown to be important for the activity of PER-1. Asn104, which corresponds to the lysine residue found at the same position in various TEM-type ESBLs, would be connected to the key catalytic residue Glu166 via a hydrogen bond network, whereas Ala164, which corresponds to a highly conserved arginine in the
-loop of class A ß-lactamases described so far (Ambler et al., 1991
), could play an important structural role. By contrast, modification of the serine residue found at position 238 in PER-1, which is an amino acid found specifically in a large number of TEM-type ESBLs (Bush and Jacoby, 1997
), resulted in no significant modification of the activity of PER-1 against expanded-spectrum cephalosporins. Similarly, Gly240 in PER-1 was shown to have no essential role in the substrate profile of the enzyme. Finally, the catalytic residue Glu166, found in all class A ß-lactamases, appeared to be essential to the ß-lactamase activity of PER-1. However, an unexpected residual activity against CAZ and AZT was observed for a mutant in which Glu166 was replaced by Ala, suggesting that other residues in PER-1 could contribute to the high activity of the enzyme against expanded-spectrum cephalosporins.
In this work, we investigated other amino acid residues found either within or at the vicinity of the PER-1 active site: Ala164, His170, Ala171 and Asn179 which are located within the putative -loop of PER-1, Thr237 which is found at the end of the ß3 strand and is likely to participate in the formation of the oxyanion hole (Herzberg and Moult, 1987
; Strynadka et al., 1992
), Arg220 which is located in a position similar to that of Arg244 found on strand ß4 in TEM-1 and which could contribute to the stabilization of the oxyanion pocket via hydrogen bonding interactions with strand ß3 (Moews et al., 1990
; Jacob-Dubuisson et al., 1991
) and Lys242 found in the loop connecting strands ß3 and ß4. All these residues were modified by site-directed mutagenesis and the kinetic properties of the resulting mutants were characterized. By using homology modelling and molecular dynamics simulations, we have attempted to interpret at the structural level the kinetic data obtained for some of the
-loop mutants.
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Materials and methods |
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Antibiotic powders were provided by the following manufacturers: penicillin G, Laboratoires de Thérapeutique Moderne (Suresnes, France); ampicillin, cephalothin and kanamycin, Sigma Chemical (St Louis, MO, USA); cefotaxime, Laboratoires Roussel (Paris, France); nitrocefin and ceftazidime, Glaxo (Paris, France); and aztreonam, Bristol Myers Squibb (Paris-La Défense, France).
The restriction enzymes used in this study were obtained from Boehringer Mannheim (Meylan, France) and T4 DNA ligase from Promega (Madison, WI, USA). [33P]dCTP was purchased from Isotopchim (Ganagobie, France).
Escherichia coli strains, plasmids and growth conditions
E.coli CJ236 (Kunkel et al., 1987) and MV1190 (McClary et al., 1989
) were used as hosts for phages in site-directed mutagenesis experiments. E.coli JM109 (Promega) was used for DNA cloning experiments and for expression of blaPER-1 and the corresponding mutant genes.
The recombinant plasmid pRAZ1, encoding blaPER-1, has been described by Nordmann et al. (1993). Bacteriophage M13mp19 (Messing, 1983) was used as a vector in site-directed mutagenesis experiments. Plasmid pK19 (kanamycin®) (Pridmore, 1987
) was used in cloning experiments.
E.coli MV1190 and JM109 were grown at 37°C in LuriañBertani (LB) (Difco, Detroit, MI, USA) and brain-heart infusion (BHI) (Difco), respectively. Solid media were obtained by the addition of 2% Bacto-Agar (Difco). Kanamycin (25 µg/ml) and ampicillin (100 µg/ml) were added when necessary. Competent E.coli cells were prepared and transformed as described by Chung et al. (1989).
Nucleic acid techniques
Plasmid DNA was purified using either the alkaline lysis for mini-preparations (Birnboim and Doly, 1979) or the Qiagen plasmid kit for maxi-preparations (Qiagen, Hilden, Germany). Isolation of single-stranded DNA and other standard DNA manipulations were carried out according to Sambrook et al. (1989). Double- and single-stranded DNA sequencing were carried out by the dideoxynucleotide chain termination method (Sanger et al., 1977
) using the T7 Sequencing kit (Pharmacia Biotech, Saint Quentin en Yvelines, France).
Site-directed mutagenesis
Site-directed mutagenesis experiments were performed as described previously (Bouthors et al., 1998). In brief, the blaPER-1 gene was excised from pRAZ1 (1.3 kb) and introduced into M13mp19 RF. Site-directed mutagenesis was performed using the uracil template procedure of Kunkel et al. (1987). The sequences of the synthetic phosphorylated oligonucleotides (Eurogentec, Liège, Belgium) used to introduce the different mutations in the blaPER-1 gene are listed in Table I
. After mutagenesis, each mutant gene was cloned into plasmid pK19 and the recombinant plasmids thus obtained were introduced by transformation into E.coli JM109. The mutant genes were all sequenced in their entirety and on both strands.
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The wild-type and mutant enzymes were purified from 1 l cultures by a two-step procedure based on an anion-exchange column followed by a gel filtration, as described previously (Bouthors et al., 1998). The mutant ß-lactamases displaying significant activity were detected using the chromogenic cephalosporin nitrocefin (O'Callaghan et al., 1972
), while the almost inactive enzymes A164R, N179D and A164R+N179D were identified by electrophoresis on 12% SDSpolyacrylamide gels (Laemmli, 1970
), with the wild-type PER-1 ß-lactamase as a molecular mass reference. In order to avoid concerns about enzyme stability, kinetic studies were performed shortly after purification. The purity of the different enzymes was assessed by Coomassie Blue staining of SDSpolyacrylamide gels after electrophoresis. Protein concentration was determined by measuring the absorbance at 280 nm (Lorber and Giegé, 1992
) with an
value of 34 850 M1.cm1 (Bouthors et al., 1998
). For mutants exhibiting more than one protein band on SDSPAGE analysis, the intensity of the ß-lactamase band was measured with a computerized densitometer (Densylab, Bioprobe) and the enzyme concentration was determined with reference to a standard BSA scale analyzed in the same conditions.
Isoelectric focusing
Isoelectric focusing was performed with a LKB Multiphor apparatus with pH 3.59.5 PAG plates (Pharmacia Biotech). Gels were focused at 30 W for 90 min at 10°C. ß-Lactamase activity was revealed by staining with the nitrocefin assay.
Determination of the kinetic parameters of the wild-type and mutant enzymes
Kinetic assays were performed spectrophotometrically in 0.1 M sodium phosphate buffer (pH 7.0) at 30°C on a Uvikon 940 spectrophotometer. The wavelengths and the extinction coefficients used were as follows: penicillin G, 232 nm, = 1100 M1.cm1; cephalothin, 262 nm,
= 7960 M1.cm1; cefotaxime, 260 nm,
= 6710 M1.cm1; ceftazidime, 260 nm,
= 8660 M1.cm1; and aztreonam, 318 nm,
= 650 M1.cm1. For each antibiotic, initial rates were measured at six different substrate concentrations. Kinetic parameters were determined by fitting the MichaelisMenten equation to the experimental data using the regression analysis program LEONORA written by Cornish-Bowden (1995). The values for kcat and Km were estimated using a non-linear least-sqares regression method with dynamic weights (Cornish-Bowden, 1995
).
Molecular modelling
The refined theoretical three-dimensional structures of PER-1 and the mutant enzymes were constructed by homology modelling using the computer program Swiss-Model (Peitsch, 1996), as described previously (Bouthors et al., 1998
). The models were then subjected to 5000 steps of energy minimization using the Powell minimizer of X-PLOR (Brunger, 1988
). The
-loop region in the resulting minimized structures was subjected to molecular dynamic simulations in vacuum. The molecular dynamics were initially performed on the 150190 region of PER-1 containing the
-loop (residues 161179) and the two
-helix regions enclosing the loop (residues 150160 and 180 190, respectively). The results obtained from this large segment indicated that the two
-helix regions enclosing the loop were very stable (r.m.s.d. = 0.2 Å). Therefore, the molecular dynamic simulations were subsequently confined to the region encompassing residues 160180, using the following simulation procedure: the target temperature started at 0 K to reach the final temperature, 300 K, within 18 ps. After 30 ps of stabilization at 300 K, the molecular dynamic phase lasted 100 ps at 300 K, with a time step of 0.001 ps and a dielectric constant (
) of 4.0. The conformations trapped at 300 K were visualized by using the VMD (Visual Molecular Dynamics) program (Humphrey et al., 1996
). The mean of the conformations, which was subjected to 500 steps of energy minimization, was used in structure comparison.
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Results |
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Site-directed mutagenesis was used to replace the amino acid residues located at positions 164, 170, 171, 179 and 237 in PER-1 by those found at the same positions in TEM-1 having no significant activity against expanded-spectrum cephalosporins. Thus, the single mutants A164R, H170N, A171E, N179D and T237A, and also two double mutants, A164R+A171E and A164R+N179D, were constructed. In addition, Lys242 in PER-1, which could be the counterpart of the lysine residue found at position 240 in various TEM-ESBLs (Bush and Jacoby, 1997), was replaced by a glutamic acid residue, as found in TEM-1 at position 240 (Sutcliffe, 1978
). Finally, Arg220 in PER-1, which is equivalent to Arg244 located on the ß4 strand in TEM-1, was replaced by a leucine. SDSPAGE analysis of crude extracts showed that all the mutant ß-lactamases except three, were expressed in normal amounts (data not shown). Indeed, when compared with the wild-type enzyme, the A164R, N179D and A164R+N179D mutants were expressed at very low levels and the various purification attempts carried out in order to determine the kinetic features of the three mutants remained unsuccessful. Production and purification of the other enzymes, which were all active, were performed as described previously (Bouthors et al., 1998
).
Isoelectric focusing, carried out on the purified enzymes, indicated that three mutants, A164R+A171E, H170N and T237A, displayed pI values indistinguishable from that of the wild-type protein (pI = 5.4). Conversely, the pI values found for the R220L, A171E and K242E mutants were shifted towards more acidic values (pI = 5.2, 5.0 and 4.9, respectively) (data not shown). Finally, the isoelectric points of the three mutants A164R, N179D and A164R+N179D could not be determined since the corresponding crude extracts contained no significant ß-lactamase activity.
Kinetic analysis
The steady-state kinetic parameters kcat and Km for penicillin G, cephalothin, cefotaxime (CTX), ceftazidime (CAZ) and aztreonam (AZT) were determined from the purified active ß-lactamases. The values obtained are shown in Table II.
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Mutants of residues located in the -loop.
Four positions were investigated at the level of the
-loop region of the protein: 164, 170, 171 and 179. Four single mutants (A164R, H170N, A171E and N179D) and two double mutants (A164R + A171E and A164R + N179D) were analyzed, as described below.
Mutants A164R, A171E and A164R+A171E.
As observed previously (Bouthors et al., 1998), no significant enzymatic activity was detected with the A164R mutant. By contrast, the substitution of the alanine residue found at position 171 in PER-1 by a glutamate resulted in no significant modifications of the kcat and Km values, when compared with the wild-type enzyme (Table II
). Similarly, the double mutant A164R+ A171E yielded an active enzyme which showed kcat and Km values similar to those of PER-1, but the kcat/Km ratios for CTX, CAZ and AZT were increased by at least an order of magnitude (Table II
).
Mutants N179D and A164R+N179D.
Position 179 is well conserved in class A ß-lactamases, where an aspartate residue is generally found (Table III). The mutation Asn179
Asp in PER-1, either in the N179D mutant or in the double mutant A164R + N179D, resulted in a complete loss of activity and the corresponding enzymes could not be purified.
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Mutants of residues located in the /ß domain.
Three positions were studied in the
/ß domain: position 220, position 237 on strand ß3 and position 242 on the loop connecting ß3 and ß4 (Bouthors et al., 1998
).
Mutant R220L.
An arginine is found at position 220 in PER-1 (Table III). This residue might be the equivalent of Arg244 in the TEM enzymes, as previously suggested (Matagne and Frère, 1995
). Replacement of Arg220 by a leucine yielded a mutant (R220L) displaying no significant modifications of the kinetic parameters for penicillin G and cephalothin. For the other drugs (CTX, CAZ and AZT), a general increase in apparent affinity was observed. In addition, a significant decrease in kcat for CTX was noticed (3.2-fold) (Table II
).
Mutant T237A.
As in the ESBLs TEM-5 and TEM-24 (Sougakoff et al., 1989; Chanal et al., 1992
), position 237, which contibutes to the oxyanion pocket and corresponds to an alanine in TEM-1, is occupied by a threonine in PER-1 (Table III
). The replacement of Thr237 by Ala yielded an enzyme which exhibited a higher apparent affinity for most of the substrates tested, particularly for CTX and AZT (Km values lowered by 40- and 10-fold, respectively). By contrast, specific and divergent variations of kcat were observed for CAZ (6-fold increase) and CTX (4-fold decrease), but, overall, the kcat/Km ratio for all the substrates tested was markedly increased.
Mutant K242E.
Lysine 242, which would be located in PER-1 on a large loop connecting strands ß3 and ß4 (Bouthors et al., 1998), could be the counterpart of Lys240 found in various ESBLs displaying a high activity against CAZ and AZT (Bush and Jacoby, 1997
) (Table III
). This residue was replaced by a glutamic acid, which is the residue found at position 240 in TEM-1 (Table III
). As shown in Table II
, the steady-state kinetic parameters determined from the K242E mutant were nearly identical with those measured from PER-1.
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Discussion |
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In PER-1, an alanine residue is found at position 164 instead of the highly conserved arginine identified in the other class A ß-lactamases (Ambler et al., 1991). As reported above and as observed previously (Bouthors et al., 1998
), replacement of Ala164 by Arg in PER-1 resulted in a mutant protein which could not be detected on SDSPAGE analysis and which displayed no detectable ß-lactamase activity. In order to explain such a result, theoretical three-dimensional models of the class A ß-lactamase PER-1 and the corresponding mutant A164R were constructed and compared with each other (Figure 1A
). Despite the relatively low degree of identity found at the amino acid level between PER-1 and the other class A ß-lactamases, homology modelling was used to generate the model structures of PER-1 and the A164R enzyme because it is now well established that class A ß-lactamases form a super family of enzymes that are all characterized by a very similar structural organization, particularly at the level of the active site (Joris et al., 1991). Molecular dynamic simulations were then performed from the models in order to assess the extent of the conformational modifications that could occur in the
-loop region of the mutant by comparison with that of the wild-type enzyme. Based on the results obtained, the
-loop region in PER-1 appears to be characterized by fairly high flexibility (data not shown). Such a result could be related to the fact that the PER-1
-loop is not stabilized by several ionic-bonding interactions, thus contrasting with the four salt bridges found in TEM-1 between the
-loop residues Arg161 and Asp163, Arg164 and Glu171, Arg164 and Asp179, and Asp176 and Arg178 (Jelsch et al., 1993
). Therefore, it is likely that the Ala164
Arg substitution induces in PER-1 significant conformational modifications at the level of the
-loop. Accordingly, the topology of the main-chain atoms between residues 171179 is significantly different in the A164R mutant, when compared with PER-1 (r.m.s.d. = 0.6 Å) (Figure 1A
). In the the wild-type enzyme model, the
-loop conformation is generally wider than in the mutant structure, the side chain of Asp172 being oriented outwards the loop. By contrast, in the A164R enzyme, the bulky side chain of Arg164 would point inwards the
-loop and, due to a putative salt bridge bonding interaction, the Asp172 side chain would be reoriented towards that of Arg164 (Figure 1A
). Such a salt bridge cannot be established without a significant conformational modification of the 172 region (Figure 1A
), which accounts for the instability and the loss of activity of the mutant enzyme.
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Two other amino acids (Asn179 and His170) were investigated in the -loop of PER-1. The asparagine residue, found at position 179 in PER-1 as in the ESBL SHV-8 (Rasheed et al., 1997
) (Table III
), was initially thought to play a specific role in the activity of PER-1 against expanded-spectrum cephalosporins. Unexpectedly, replacement of Asn179 by an aspartate, which is a residue conserved in a large number of class A ß-lactamases, was highly deleterious for the overall ß-lactamase activity of the two PER mutants N179D and A164R+N179D (Table II
). It must be pointed out that the interaction between residues 164 and 179, which links the two ends of the
-loop region in class A ß-lactamases, is important for a suitable positioning of the key catalytic residue Glu166 (Knox, 1995
; Matagne et al., 1998
). Therefore, it is tempting to speculate that the presence of an aspartate residue at position 179 in the inactive mutants N179D and A164R+N179D could alter significantly the position of Glu166 and, thereby, the ß-lactamase activity.
The histidine found at position 170 in PER-1 corresponds to a highly conserved asparagine residue in the other class A ß-lactamases (Ambler et al., 1991) (Table III
). Unexpectedly, the kinetic parameters exhibited by the H170N mutant were similar to those obtained from PER-1, except for a 5.5-fold increase in the kcat value for CTX with a concomitant decrease in the apparent affinity for this antibiotic. Palzkill et al. (1994) have reported that the replacement of the highly conserved Asn170 by a histidine in TEM-1 yielded an active enzyme showing unmodified catalytic constants. Taken altogether, these data suggest that His170 is not a key residue for the substrate profile of PER-1 and one can hypothesize that this residue was present in the ancestor of the PER-1 ß-lactamase and has been conserved during the evolution process leading to PER-1.
Three positions in PER-1 were investigated in the region of the /ß domain forming one of the two edges of the active site. Residue 237, located on the ß3 strand, belongs to the so-called oxyanion pocket and is involved in the binding of ß-lactams (Ghuysen, 1994
; Matagne et al., 1998
). In PER-1, a threonine is found at position 237 (Figure 1C
), which is located between the KTG triad and Ser238. Strikingly, it has been previously reported that various TEM-type ESBLs harbour a A237T substitution (Bush and Jacoby, 1997
). Moreover, another hydroxylated residue (a serine) is found naturally at position 237 in the class A ß-lactamase from Proteus vulgaris which displays a high catalytic activity against CTX and it has been shown that the substitution Ser237
Ala in this enzyme leads to a decrease in the catalytic efficiency against this drug (Tamaki et al., 1994
). Therefore, the decrease in kcat observed for CTX with the T237A mutant of PER-1 confirms that Thr237 is important for the catalytic activity of PER-1 towards this drug. However, the general increase in kcat/Km observed for the T237A mutant of PER-1 against CTX, CAZ and AZT, which is due to a general increase in apparent affinity towards cephalosporins, was rather unexpected (Table II
). Nonetheless, these results were confirmed by modifying the arginine found at position 220 in PER-1. Indeed, according to the hypothetical model of PER-1 shown in Figure 1C
, the side chain of Arg220 would point towards the active site cavity and could be hydrogen-bonded to that of Thr237. As a consequence of this structural organization, it is likely that both residues contribute to adjusting the topology of the oxyanion pocket, as previously suggested for other class A ß-lactamases (Matagne and Frère, 1995
). In accordance with such a model, the replacement of Arg220 by Leu in PER-1, which leads to the loss of the hydrogen-bonding interactions between residues 220 and 237, yielded a mutant enzyme (R220L) showing kinetic properties similar to those exhibited by the T237A mutant, i.e. a significant decrease in the catalytic activity against CTX associated with a better apparent affinity for expanded-spectrum cephalosporins and AZT (see Table II
).
Finally, we also studied the lysine residue found at position 242 at the end of the ß3 strand in PER-1 (Figure 1C), which might be the counterpart of Lys240 found in various TEM-type ESBLs (Bush and Jacoby, 1997
). The replacement of Lys242 in PER-1 by a glutamic acid residue, which is the residue found at position 240 in TEM-1 (Table III
), yielded a mutant enzyme with kinetic properties very similar to those of PER-1. This result indicates that Lys242 does not play in PER-1 a role equivalent to that of the lysine found at position 240 in the TEM-type ESBLs.
In conclusion, PER-1 is a class A ESBL which illustrates well the fact that enzymes showing a high level of divergence in their amino acid sequences can share very similar substrate profiles. Furthermore, our results indicate that, in contrast to the TEM-type ESBLs, the PER-1 activity towards expanded-spectrum cephalosporins does not stem from the presence in the active site of a limited number of residues having a specific role in the hydrolysis of these drugs. The X-ray structure determination of PER-1, which is in progress, will aid further understanding of the structureactivity relationships of this peculiar class A ß-lactamase.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed. E-mail: sougakof{at}lmcp.jussieu.fr
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Received July 23, 1998; revised January 4, 1999; accepted January 21, 1999.