Relocation of the catalytic carboxylate group in class A ß-lactamase: the structure and function of the mutant enzyme Glu166->Gln:Asn170->Asp

Celia C.H. Chen and Osnat Herzberg1

Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The hydrolysis of ß-lactam antibiotics by the serine-ß-lactamases proceeds via an acyl–enzyme intermediate. In the class A enzymes, a key catalytic residue, Glu166, activates a water molecule for nucleophilic attack on the acyl–enzyme intermediate. The active site architecture raises the possibility that the location of the catalytic carboxylate group may be shifted while still maintaining close proximity to the hydrolytic water molecule. A double mutant of the Staphylococcus aureus PC1 ß-lactamase, E166Q:N170D, was produced, with the carboxylate group shifted to position 170 of the polypeptide chain. A mutant protein, E166Q, without a carboxylate group and with abolished deacylation, was produced as a control. The kinetics of the two mutant proteins have been analyzed and the crystal structure of the double mutant protein has been determined. The kinetic data confirmed that deacylation was restored in E166Q:N170D ß-lactamase, albeit not to the level of the wild-type enzyme. In addition, the kinetics of the double mutant enzyme follows progressive inactivation, characterized by initial fast rates and final slower rates. The addition of ammonium sulfate increases the size of the initial burst, consistent with stabilization of the active form of the enzyme by salt. The crystal structure reveals that the overall fold of the E166Q:N170D enzyme is similar to that of native ß-lactamase. However, high crystallographic temperature factors are associated with the -loop region and some of the side chains, including Asp170, are partially or completely disordered. The structure provides a rationale for the progressive inactivation of the Asp170-containing mutant, suggesting that the flexible -loop may be readily perturbed by the substrate such that Asp170's carboxylate group is not always poised to facilitate hydrolysis.

Keywords: ß-lactam kinetics/ß-lactamase/site-directed mutagenesis/X-ray structure


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The catalytic mechanism of the serine-ß-lactamases involves acylation and deacylation of the O{gamma} atom of an active site serine residue, resulting in the hydrolysis of the ß-lactam amide bond of ß-lactam antibiotics (Knott-Hunziker et al., 1979Go; Cartwright et al., 1980Go; Cohen and Pratt, 1980Go; Fisher et al., 1981Go). The simplest mechanism that accounts for the acyl–enzyme intermediate is shown in Scheme 1


where the enzyme, substrate and product are represented by E, S and P, respectively; ES denotes the Michaelis complex; and EC denotes the acyl–enzyme 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 acyl–enzyme intermediate (Herzberg and Moult, 1987Go; Herzberg, 1991Go). Mutant enzymes with Glu166 replaced by an uncharged residue are deacylation impaired, while acylation is unperturbed (Adachi et al., 1991Go; Escobar et al., 1991Go). One of these mutant enzymes was used to determine the crystal structure of the acyl–enzyme adduct (Strynadka et al., 1992Go). 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., 1996Go).

Glu166 is located on an -loop, comprising residues 163–178, that forms part of the active site depression (Figure 1Go). 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, 1991Go; Strynadka et al., 1992Go; Jelsch et al., 1993Go), 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., 1991Go). 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., 1997aGo), similar to the kinetics of the protein variant with the entire -loop deleted (Banerjee et al., 1997bGo).



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Fig. 1. The overall fold of ß-lactamase, highlighting the -loop (gold), the three catalytic residues, Ser70, Lys73, Glu166, and the residue targeted for mutation, Asn170 (red). The hydrolytic water molecule is shown as a blue sphere.

 
Usually, the precise locations of catalytic residues are considered crucial to enzyme activity. However, for the class A ß-lactamases, inspection of the structure indicates that the exact location of the negative charge that enhances the nucleophilicity of the hydrolytic water molecule may not be so important, as long as the proximity to the water molecule is preserved. The current study addresses this issue.

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 1Go). 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., 1991Go; Escobar et al., 1991Go). 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 acyl–enzyme with ß-lactam antibiotics, deacylation is restored for the double mutant.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Mutagenesis, expression and protein purification

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 overlap–extension method (Ho et al., 1989Go). 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 {varepsilon}280 value of 19 500 M–1 cm–1 (Carrey and Pain, 1978Go). 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{alpha} 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., 1987Go). The statistics of data processing are shown in Table IGo.


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Table I. Data processing statistics
 
Structure determination and refinement

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, 1992aGo). 12 688 reflections in the resolution range 8.0–2.3 Å for which F >= 2{sigma}(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 163–172 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 = {Sigma}h ||Fo| – |Fc|| / {Sigma}h |Fo|, where |Fo| and |Fc| are the observed and calculated structure factor amplitudes, respectively) and the free R-factor (Brünger, 1992bGo). 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 TURBO–FRODO was used for map inspection and model modification (Roussel and Cambillau, 1989Go). 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{sigma} 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, 1991Go)


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 ({Delta}{varepsilon}500 = 15 900 M– 1cm–1). The hydrolysis of benzylpenicillin was monitored by loss of absorbance at 232 nm ({Delta}{varepsilon}232 = 940 M–1cm–1).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The refined crystal structure

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{sigma}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 2Go shows the electron density at the region of the active site.



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Fig. 2. Stereoscopic representation of the electron density map of the active site of E166Q:N170D ß-lactamase. The coefficients 2FoFc and calculated phases are used. There is discontinuity between residues 166 and 167, and no electron density for Ile169. In addition, no electron density is associated with most of the side chain atoms of Gln166, Ile167, Glu168, Asp170 and Tyr171. These side chains have not been modeled and are not shown. Neither the hydrolytic water molecule or the water molecule that occupies the oxyanion hole could be seen in the electron density map. The map is contoured at 1{sigma} level.

 
The overall fold of the mutant enzyme is similar to that of the native protein except for the mutation sites. The r.m.s.d. between C{alpha} atom positions of the mutant and native structures is 0.3 Å. However, the region of the -loop in the vicinity of the mutated residues exhibits elevated temperature factors and partial disorder (Figure 3Go). No electron density is associated with residue 169, and the map is discontinuous between residues 166 and 167 (Figure 2Go). In addition, the side chains of the following residues are partially or completely disordered: Val163, Arg164, Tyr165, Gln166, Ile167, Glu168, Asp170, Tyr171. The peptide bond between residues 166 and 167 was modeled as a cis bond based on the native structure. However, because of the discontinuous electron density, the peptide bond could be modeled equally well in a trans conformation. Superposition of the active site region of the mutant and native proteins highlights the similarity between most of the residues, and the key side chains that are missing (Figure 4Go). A sulfate ion is located in the active site, close to Ser70 hydroxyl group and to the water molecule that occupies the oxyanion hole site [formed by the main chain nitrogen atoms of Ser70 and Gln237 (Herzberg and Moult, 1987Go)]. Consistent with the high temperature factors and the disorder associated with the mutated residues, the hydrolytic water molecule is not seen in the electron density map.



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Fig. 3. Main chain crystallographic temperature factors along the polypeptide chain of the native and E166Q:N170D ß-lactamases. Native, thick solid line; E166Q:N170D, thin solid line. The numbering is sequential starting from 1 rather than that of Ambler et al. (1991).

 


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Fig. 4. Stereoscopic representation of the superposition of native ß-lactamase and the double mutant enzyme. The native structure is shown in thin solid lines, and the mutant structure is shown in thick solid lines. The side chains of the -loop region in the mutant enzyme are disordered as listed above, and are not included in the model. Note also that the region of the mutations deviates significantly from the native structure (up to 1.4 Å, compared with an r.m.s.d. of 0.3 Å for all C{alpha} atoms).

 
Enzyme kinetics

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, 1991Go).


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., 1976Go). 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., 1992Go; Chen et al., 1993Go), 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., 1994Go). 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, 1985Go). 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., 1994Go; Banerjee et al., 1997aGo,bGo). 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 IIGo). With nitrocefin, a fast burst is observed with stoichiometry of 1 mol product/mol enzyme before reaching the slow steady state (Figure 5aGo). The burst corresponds to the formation of a stable acyl enzyme, assuming the same extinction coefficient for the acyl–enzyme 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., 1991Go; Escobar et al., 1991Go), and for S.aureus mutant enzymes that perturbed the -loop on which Glu166 resides (Herzberg et al., 1991Go; Banerjee et al., 1997aGo), blocked the hydrolytic water molecule site (Zawadzke et al., 1996Go), or eliminated the loop entirely (Banerjee et al., 1997bGo). All these mutant ß-lactamases have been shown to be deacylation impaired, and for two mutants (Banerjee et al., 1997aGo,bGo), the presence of stable acyl–enzyme adducts were also confirmed by mass spectrometry.


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Table II. Hydrolysis rates for the wild-type and mutant ß-lactamases
 




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Fig. 5. Kinetics of mutant ß-lactamases. (a) Progress curve of nitrocefin hydrolysis by E166Q ß-lactamase. The concentration of the enzyme and nitrocefin were 1 and 10 µM, respectively. The stoichiometric burst corresponds to the formation of a stable acyl enzyme. (b) Progress curves of benzylpenicillin hydrolysis by E166Q:N170D ß-lactamase in the presence of varying amounts of (NH4)2SO4. The concentrations of the enzyme and benzylpenicillin were 1.0 µM and 1.1 mM, respectively. Km for the steady-state phase is 15 µM in ammonium sulfate free solution. The concentrations of the ammonium sulfate are given in percent saturation (100% is approximately 4.2 M). (c) Progress curves of nitrocefin hydrolysis by E166Q:N170D ß-lactamase. The concentrations of the enzyme and nitrocefin were 1.0 and 500 µM, respectively. Km for the steady-state phase is 1 µM at ammonium sulfate free solution. Note that nitrocefin is insoluble in concentration of ammonium sulfate above 20% saturation.

 
As formation of a stable acyl enzyme with benzylpenicillin cannot be detected spectroscopically, its presence was tested by incubating the mutant enzyme with twofold excess benzylpenicillin for 100 s, and assaying the mixture for nitrocefin hydrolysis. Neither burst nor any residual activity toward nitrocefin could be detected during the 3 h of monitoring the reaction, consistent with benzylpenicillin forming a stable acyl–enzyme adduct.

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 cGo). The curves fit very well the general integrated equation (Eqn 1). Moreover, the burst amplitude increases with increasing concentration of ammonium sulfate (Table IIGo). 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., 1991Go).

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., 1991Go; Banerjee et al., 1997aGo). 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., 1991Go; Escobar et al., 1991Go), 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., 1997aGo). 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 Glu166–Lys73 ion pair in the first enzyme and Glu166–Asn136 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., 1992Go; Knox et al., 1993Go). 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 3Go, 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., 1991Go; Banerjee et al., 1997aGo). 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, 1992Go; Strynadka et al., 1992Go; Chen et al., 1993Go; Maveyraud et al., 1996Go, 1998Go).

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.


    Acknowledgments
 
We would like to thank Drs John Moult and Soojay Banerjee for useful discussions. This work was supported by NIH grant RO1-AI27175. The coordinates of E166Q:N170D ß-lactamase have been deposited in the Brookhaven Protein Data Bank (entry code 1kgg).


    Notes
 
To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received January 29, 1999; revised April 4, 1999; accepted April 10, 1999.