Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA
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Abstract |
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Keywords: ß-lactam kinetics/ß-lactamase/site-directed mutagenesis/X-ray structure
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Introduction |
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where the enzyme, substrate and product are represented by E, S and P, respectively; ES denotes the Michaelis complex; and EC denotes the acylenzyme complex. Progress curves for the hydrolysis of simple ß-lactam antibiotics such as benzylpenicillin are linear.
The class A ß-lactamases utilize an invariant glutamic acid residue, Glu166 in the consensus numbering scheme of Ambler et al. (1991), to activate a proximal water molecule for nucleophilic attack on the acylenzyme intermediate (Herzberg and Moult, 1987; Herzberg, 1991
). Mutant enzymes with Glu166 replaced by an uncharged residue are deacylation impaired, while acylation is unperturbed (Adachi et al., 1991
; Escobar et al., 1991
). One of these mutant enzymes was used to determine the crystal structure of the acylenzyme adduct (Strynadka et al., 1992
). The identity of the hydrolytic water molecule was confirmed by structural and kinetic studies of the mutant enzyme N170Q, with an extended side chain that blocks access to the water molecule and impairs deacylation (Zawadzke et al., 1996
).
Glu166 is located on an -loop, comprising residues 163178, that forms part of the active site depression (Figure 1
). In the ß-lactamase from Staphylococcus aureus PC1, the packing of the loop against the rest of the molecule is imperfect, with several internal water molecules separating it from the core. In addition, the peptide bond between the catalytic residue, Glu166, and the following residue, Ile167, is a cis bond, an energetically unfavorable conformation. In crystal structures of other class A ß-lactamases the core is well packed and residue 167 is a proline (Knox and Moews, 1991
; Strynadka et al., 1992
; Jelsch et al., 1993
), a residue that quite frequently forms a cis peptide bond. The imperfect features of the S.aureus ß-lactamase
-loop suggest that its conformation may be readily perturbed. This hypothesis was tested by producing mutant enzymes with removed interactions that are important to the loop's structural integrity. Such mutants exhibited impaired deacylation, while the rate of the acylation step was not much altered. The first example was that of the P54 mutant ß-lactamase in which the replacement D179N eliminates a salt bridge with Arg164, invariant in all class A ß-lactamases. The crystal structure of the mutant enzyme revealed conformational disorder of the
-loop (Herzberg et al., 1991
). Larger effect on catalysis has been observed with the mutant enzyme N136A, which removes a key interaction between this side chain's amido group and the main chain atoms of Glu166. In this case, there was no detectable deacylation (Banerjee et al., 1997a
), similar to the kinetics of the protein variant with the entire
-loop deleted (Banerjee et al., 1997b
).
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Inspection of the refined native structure of S.aureus ß-lactamase (Herzberg, 1991; Protein Data Bank (PDB) entry code 3BLM) reveals that Asn170 interacts with both Glu166 and the hydrolytic water molecule (Figure 1). Asn170 is an invariant residue in all known class A ß-lactamases. Nevertheless, substitution of the asparagine by an aspartic acid side chain is a conservative change in terms of the space these residues occupy. To test the hypothesis that it is possible to shift the location of a catalytic residue, the double mutant E166Q:N170D was produced. While retaining the overall polar character of the active site, the location of the negatively charged group is shifted. Another mutant protein, E166Q, lacking the carboxylate group was produced as a control. Single mutations of this type are known to preserve acylation but abolish deacylation as was previously reported for ß-lactamase from Escherichia coli and B.licheniformis (Adachi et al., 1991
; Escobar et al., 1991
). This was demonstrated recently in more detail by Guillaume et al. (1997) with the E.coli mutant enzyme E166N. For good substrates the deacylation rate was 109-fold lower than that of the wild-type enzyme, whereas the reduction in the combined binding and acylation rate was 200-fold at most. The functional and structural consequences of the double mutation E166Q:N170D are reported here, showing that in contrast to E166Q ß-lactamase which form a stable acylenzyme with ß-lactam antibiotics, deacylation is restored for the double mutant.
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Materials and methods |
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The two mutant ß-lactamases from S.aureus PC1 were cloned, expressed in E.coli TG1, and purified following a protocol similar to that used by Zawadzke et al. (1995), with modifications described in Zawadzke et al. (1996). The pKK233-2-derived plasmid, pTS32, which exploits an IPTG-inducible trc promoter, was used to produce the mutant proteins. pTS32 contains the S.aureus ß-lactamase gene (blaZ). It also contains a Tn9 chloramphenicol acetyl transferase gene, inserted into the TEM blaZ gene to prevent expression of the E.coli ß-lactamase, and as a selectable marker of mutagenized genes. A N-terminal methionine has been added to the engineered gene for expression. The leader peptide of ß-lactamase was not included, hence the first residue following the initiator methionine is Lys31. Mutations were introduced by the four-primer overlapextension method (Ho et al., 1989). The altered blaZ genes were sequenced to confirm that the desired substitutions were obtained, and that no spurious changes had occurred.
The concentration of the purified protein was estimated from the absorbance of solutions at 280 nm by using a 280 value of 19 500 M1 cm1 (Carrey and Pain, 1978
). For storage, the proteins were kept at 4°C in solutions containing 60% saturated ammonium sulfate.
Crystallization and X-ray data collection
Single crystals of E166Q:N170D were obtained at room temperature by vapor diffusion in hanging drops, using similar conditions to those used to obtain crystals of the native protein, with some modification. The protein drops were equilibrated against reservoir solutions containing 89% saturated ammonium sulfate, 0.5% PEG 2000, and buffered at pH 8 by 0.1 M sodium bicarbonate. The hanging drops contained equal volumes of 10 mg/ml enzyme and reservoir solutions. The crystals belong to space group I222 and are isomorphous with the native protein crystals. The unit cell dimensions are a, 54.0; b, 94.8; c, 138.6 Å. There is one molecule in the asymmetric unit. Attempts to crystallize E166Q ß-lactamase were unsuccessful.
X-Ray intensity data were collected at room temperature on a Siemens area detector mounted on a Siemens three-circle goniostat. Monochromated Cu-K X-rays were generated by a Siemens rotating anode. Data to 2.3 Å resolution were collected from a single crystal. The data were processed with the XENGEN package (Howard et al., 1987
). The statistics of data processing are shown in Table I
.
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The starting model for refinement was that of the native S.aureus PC1 ß-lactamase (PDB entry code 3BLM), except that the amino acid residues at positions 166 and 170 were truncated to alanine. The structure factors were scaled to absolute values with the computer program ORESTES written by W.E.Thiessen and H.A.Levy. The structure was refined with the program X-PLOR (Brünger, 1992a). 12 688 reflections in the resolution range 8.02.3 Å for which F
2
(F) were used. The simulated annealing protocol between 3000 and 300 K was first employed, followed by positional refinement and temperature factor refinement cycles. After simulated annealing, it became clear that the
-loop exhibits considerable disorder. Accordingly, residues 163172 and 179 were omitted from the model and rebuilt gradually as the refinement progressed. The progress of the refinement was evaluated by the improvement in the quality of the electron density maps and the reduced values of the conventional R factor (R =
h ||Fo| |Fc|| /
h |Fo|, where |Fo| and |Fc| are the observed and calculated structure factor amplitudes, respectively) and the free R-factor (Brünger, 1992b
). During the last cycle of positional and temperature factor refinement, the data reserved for free R calculations were added to the refined set.
The interactive graphics program TURBOFRODO was used for map inspection and model modification (Roussel and Cambillau, 1989). Two types of electron density maps with the coefficients 2|Fo| |Fc| and |Fo| |Fc|, and with calculated phases were inspected simultaneously. Solvent molecules were added once the R-value was lower than 0.200. These were assigned in the |Fo| |Fc| difference Fourier maps with a 3
cut-off level for inclusion in the model. Solvent molecules that refined with crystallographic temperature factors larger than 80 were deleted.
Enzyme kinetics
Kinetic measurements were made on a Hewlett Packard 8452A diode array spectrophotometer. Progress curves that exhibited substrate-induced inactivation were analyzed by fitting the data to the general integrated equation (Waley, 1991)
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where P is the ratio of the concentration of the product to enzyme at time t, vi is the initial velocity, vs is the steady-state velocity (analogous to kcat), and k is the rate constant characterizing the change. Assays were performed at 25°C in solutions containing 0.1 M potassium phosphate buffer at pH 6.8. The dependence of the kinetics on ammonium sulfate concentration was also evaluated.
Nitrocefin was purchased from Unipath (Ogdensburg, NY). Benzylpenicillin was purchased from Sigma (St Louis, MO). Hydrolysis of the chromogenic cephalosporin, nitrocefin, was monitored by the increase in absorbance at 500 nm (500 = 15 900 M 1cm1). The hydrolysis of benzylpenicillin was monitored by loss of absorbance at 232 nm (
232 = 940 M1cm1).
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Results and discussion |
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The final model of E166Q:N170D ß-lactamase includes 257 amino acid residues. The first methionine residue that was added for expression in E.coli is not seen in the electron density map. The model includes a total of 67 water molecules and a sulfate ion. The final crystallographic R-factor for all data between 8.0 and 2.3 Å resolution for which F 2
F is 0.187. The R-free value, prior to adding the reserved reflections to the refined set at the final stage of refinement, was 0.263 and the R-factor for the working set was 0.178. The root mean square deviations (r.m.s.d.) from ideal bond length and bond angle values of the standard geometry, compiled by Engh and Huber (1991), were 0.018 Å and 1.9°, respectively. Figure 2
shows the electron density at the region of the active site.
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Two kinetic schemes are considered in this study. The first follows the simple acylation and deacylation steps as shown in Scheme 1. The second mechanism, illustrated by Scheme 2, is termed the branched pathway and is consistent with substrate-induced progressive inactivation (Waley, 1991).
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Here, the initial acyl enzyme, EC, is converted to a second form of an acyl enzyme, EC*, which does not turn over (or turns over more slowly). For this mechanism, the progress curve exhibits an initial exponential phase followed by a slower steady-state linear phase, since a less active form of the enzyme accumulates. Branching at ES may also result in the accumulation of the less active enzyme. Differentiating between the two branching alternatives is beyond the scope of this work.
Progressive inactivation was first observed in class A ß-lactamase more than 20 years ago, when the kinetics with substrates carrying bulky side chain substituents on the ß-lactam ring were characterized (Citri et al., 1976). The slow phase of the biphasic kinetics was suppressed when non-inhibitory antibodies were added to the enzyme. This was interpreted as an indication that the slow phase corresponds to induced conformational changes of the enzyme, which may be prevented in the presence of the antibodies. Crystallographic binding studies show that the ß-lactam side chain substituent is oriented toward the
-loop (Strynadka et al., 1992
; Chen et al., 1993
), therefore accommodation of a bulky group may lead to conformational perturbations of the loop. By analogy, if the conformational integrity of the
-loop is compromised by other means, for example by site-directed mutagenesis, progressive inactivation may occur even when a penicillin without a bulky side chain, such as benzylpenicillin, is used.
For a progressive inactivation mechanism, product formation can be described by the general integrated equation (Eqn 1). Extrapolation of the steady-state line to time zero provides the amplitude of the burst. Usually, the initial burst of product is greater than the concentration of the enzyme. When k3 < k4, the burst amplitude can be 1 mol product/mol enzyme, as was demonstrated previously for E166C ß-lactamase from B.licheniformis (Escobar et al., 1994). On the other hand, a burst amplitude of 1 can also occur with the mechanism described in Scheme 1, when k2 > k3.. For ß-lactamase, ammonium sulfate is known to stabilize the native form of the enzyme (Mitchinson and Pain, 1985
). Thus, carrying the assay in solution containing ammonium sulfate assisted in distinguishing between the two mechanisms when the burst amplitude was 1 (Escobar et al., 1994
; Banerjee et al., 1997a
,b
). For kinetics that follow Scheme 2, increased salt concentration led to increased amplitude of the burst, whereas for the simple mechanism described in Scheme 1 the burst amplitude remained 1.
Fast small bursts cannot be reliably measured with benzylpenicillin because its hydrolysis is monitored at 232 nm, where absorption of the protein is much stronger than that of the antibiotics. Fortunately, the chromogenic substrate, nitrocefin, exhibits large absorption changes upon hydrolysis at a wavelength where the protein does not absorb light. This enables reliable detection of small bursts.
The control mutant enzyme, E166Q, exhibits almost no hydrolytic rates with both benzylpenicillin and nitrocefin, when compared with the wild-type ß-lactamase. Benzylpenicillin and nitrocefin are hydrolyzed by this mutant ß-lactamase at 3x104- and 7000-fold slower rates than by the wild type enzyme, respectively (Table II). With nitrocefin, a fast burst is observed with stoichiometry of 1 mol product/mol enzyme before reaching the slow steady state (Figure 5a
). The burst corresponds to the formation of a stable acyl enzyme, assuming the same extinction coefficient for the acylenzyme as for the hydrolyzed nitrocefin. The burst amplitude remained 1 when the assay was performed in solution containing 15% saturated ammonium sulfate (~0.6 M). As explained above, this rules out the possibility that the kinetics of the control mutant follows the branched pathway mechanism (Scheme 2). The kinetics of E166Q ß-lactamase were consistent with those obtained for several other Glu166 mutants from E.coli and B.licheniformis (Adachi et al., 1991
; Escobar et al., 1991
), and for S.aureus mutant enzymes that perturbed the
-loop on which Glu166 resides (Herzberg et al., 1991
; Banerjee et al., 1997a
), blocked the hydrolytic water molecule site (Zawadzke et al., 1996
), or eliminated the loop entirely (Banerjee et al., 1997b
). All these mutant ß-lactamases have been shown to be deacylation impaired, and for two mutants (Banerjee et al., 1997a
,b
), the presence of stable acylenzyme adducts were also confirmed by mass spectrometry.
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While the single mutation, E166Q, abolishes deacylation activity, adding the second mutation, N170D, restores deacylation, although not to the level of the wild-type enzyme. Neither are the kinetics with benzylpenicillin and nitrocefin linear, in contrast with the wild-type enzyme. Rather, the E166Q:N170D ß-lactamase exhibits hydrolysis with progressive inactivation (Figure 5b and c). The curves fit very well the general integrated equation (Eqn 1). Moreover, the burst amplitude increases with increasing concentration of ammonium sulfate (Table II
). The presence of ammonium sulfate increases also the initial and steady-state rates. Although a significant trend, the changes are not large. They are consistent with the 1.5-fold increase in kcat measured for benzylpenicillin hydrolysis by the native enzyme in the presence of 60% saturated ammonium sulfate (Herzberg et al., 1991
).
When assessing the rates of hydrolysis of the E166Q:N170N ß-lactamase, it is reasonable to consider the initial rate values (vi), and compare these with the catalytic rates of the wild-type ß-lactamase, because they represent the capacity to hydrolyze substrates prior to the onset of the inactivation, whether due to conformational transition or due to other processes. Thus, E166Q:N170D ß-lactamase is approximately 110- and 6-fold less efficient in hydrolyzing benzylpenicillin and nitrocefin, respectively, compared with the wild-type enzyme. These are significant rates, which demonstrate that some flexibility is possible in the location of the catalytic carboxylate group in the active site. Additional engineering, perhaps exploiting the selection power of the ß-lactamase system, may further improve the efficacy of this new deacylation apparatus.
Structural basis for enzyme activity
The conformational integrity of the -loop in the class A ß-lactamases is crucial for hydrolysis because Glu166, the residue that enhances the nucleophilicity of the hydrolytic water molecule, resides on the loop. In addition, Glu166 participates in an ion pair interaction with the catalytic residue, Lys73, an interaction that links the
-loop to the main body of the molecule. For the enzyme from S.aureus, with a marginally stable
-loop, elimination of any interaction that helps keep the loop intact leads to conformational disorder and defective deacylation (Herzberg et al., 1991
; Banerjee et al., 1997a
). This is demonstrated with the mutant enzymes produced for this study.
The ion pair interaction is eliminated in the single mutant E166Q. Similarly to mutants of ß-lactamase from E.coli and B.licheniformis, which lack a carboxylate group at position 166 (Adachi et al., 1991; Escobar et al., 1991
), this is a deacylation-impaired enzyme. Attempts to crystallize the S.aureus E166Q ß-lactamase failed, as was the case with the S.aureus ß-lactamase mutant, N136A (Banerjee et al., 1997a
). In fact, these are the only two mutant ß-lactamases from S.aureus that so far failed to crystallize. Substitution of Asn136 by an alanine eliminates a key interaction between the
-loop and the main protein body, and also lead to a deacylation impaired enzyme. Although structural information is unavailable for either of the E166Q and N136A enzymes, we speculate that elimination of the Glu166Lys73 ion pair in the first enzyme and Glu166Asn136 interaction in the latter enzyme leads to conformational disorder of the entire
-loop, which in turn prevents crystal formation.
In contrast to E166Q S.aureus ß-lactamase, crystals of the E166N mutant enzyme from E.coli and the E166A mutant enzyme from B.licheniformis were obtained, showing that for those enzymes, the structural integrity of the -loop is maintained (Strynadka et al., 1992
; Knox et al., 1993
). The
-loop of the E.coli and B.licheniformis ß-lactamases is well packed with no internal solvent molecules separating it from the protein core and with a proline cis peptide after Glu166. Thus, the elimination of the negative charge in these proteins is tolerated and the
-loop remains intact.
The structure/activity relationship of the E166Q:N170D ß-lactamase, the main subject of the current study, may now be addressed. Initially, Asp170 catalyzes ß-lactam hydrolysis at appreciable rate, but the activity deteriorates with time. The addition of ammonium sulfate delays inactivation. As shown in Figure 3, the crystal structure of the double mutant enzyme exhibits high temperature factors and partial disorder in the region of the
-loop. Previous work indicated that the
-loop of the S.aureus ß-lactamase may be readily perturbed (Herzberg et al., 1991
; Banerjee et al., 1997a
). Thus, a plausible rationale for of the increase in burst amplitude with salt concentration is that the interaction with substrate induces a conformational transition that leads to a less active form of the enzyme, and that the salt stabilizes the active form. Note also that the crystals have been obtained at a very high concentration of ammonium sulfate (89% saturation) and that packing constraints may further assist with preserving the conformational integrity of the
-loop. Evaluation of the structural disorder in solution at low salt concentration is beyond the scope of the current study; however, it is reasonable to assume that such disorder would be more pronounced in solution than in the crystal.
Other mechanisms that may be consistent with progressive inhibition should be considered. For example, product inhibition, or a branched mechanism producing an adduct with two bound substrate molecules. Both possibilities seem unlikely. Product inhibition has never been observed with this ß-lactamase, and the crystal structure of the mutant enzyme is very similar to the native enzyme. Thus there is no reason to expect tight product binding to the mutant. Similarly, binding of a second substrate molecule was never observed. Moreover, the available structural information indicates that the dimension of active site depression is approximately the size of a single substrate/inhibitor molecule and a second molecule would not be accommodated (Chen and Herzberg, 1992; Strynadka et al., 1992
; Chen et al., 1993
; Maveyraud et al., 1996
, 1998
).
What are the structural features that may be correlated with the kinetic properties? The location of the carboxylate group in the active site depression has been moved to a position nearly 7Å away from the amino group of Lys73. Without the close ion pair interaction, the -loop is destabilized, but apparently to a lesser extent compared with when the carboxylate group is eliminated (as in the E166Q mutant enzyme). Even within the constraints of the crystal, the loop is partially disordered so that the carboxylate group of Asp170 spends only part of the time in a position appropriate for activation of the hydrolytic water molecule. Moreover, substrate binding may perturb the already flexible loop further, leading to the progressive inactivation seen with both benzylpenicillin and nitrocefin.
In summary, this study shows that there is some flexibility in the design of the deacylation apparatus of the class A ß-lactamase, which allows the position of the essential carboxylate group to be moved to residue 170. For the S.aureus enzyme, the double mutation E166Q:N170D enhances the mobility of the -loop, resulting in reduced hydrolytic rates compared with the wild-type enzyme. It remains to be seen what would be the enzymatic properties of the same double mutant when engineered into ß-lactamase from E.coli or B.licheniformis, enzymes with a more stable
-loop. Also, optimization of this new apparatus within the S.aureus enzyme may be investigated.
The two classes of serine ß-lactamases, A and C, employ two different deacylation machineries, one with the carboxylate group of Glu166 and the second with a tyrosine residue at a completely different position. We have seen now that there is a third possibility. Moreover, given the diversity and rapid evolution of ß-lactamase activity, one is tempted to speculate that such an alternative mechanism of bacterial resistance to ß-lactam antibiotics may exist or evolve in nature, although it has not been yet identified.
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Acknowledgments |
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Notes |
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References |
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Received January 29, 1999; revised April 4, 1999; accepted April 10, 1999.