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Antimicrobial Agents and Chemotherapy, December 2006, p. 4124-4131, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00848-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role of Asp104 in the SHV ß-Lactamase{triangledown}

Christopher R. Bethel,1 Andrea M. Hujer,1 Kristine M. Hujer,1 Jodi M. Thomson,2 Mark W. Ruszczycky,3 Vernon E. Anderson,3 Marianne Pusztai-Carey,3 Magdalena Taracila,1 Marion S. Helfand,1,3 and Robert A. Bonomo1,2*

Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio,1 Departments of Pharmacology,2 Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio3

Received 12 July 2006/ Returned for modification 9 August 2006/ Accepted 7 September 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Among the TEM-type extended-spectrum ß-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV ß-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in ß-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys ß-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser ß-lactamases. Correspondingly, the overall catalytic efficiency (kcat/Km) of hydrolysis for cefotaxime was increased from 0.60 ± 0.07 µM–1 s–1 (mean ± standard deviation) for Gly238Ser to 1.70 ± 0.01 µM–1 s–1 for the Asp104Lys and Gly238Ser ß-lactamase (threefold increase). We also showed that (i) k3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser ß-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 ß-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The global emergence of class A extended-spectrum ß-lactamase (ESBL)-producing bacteria resistant to oxyimino-cephalosporin antibiotics has seriously compromised the ability of clinicians to treat infections caused by Escherichia coli and Klebsiella pneumoniae (21, 22). Studies examining the molecular basis of the ESBL phenotype have shown that one or two critical amino acid substitutions enhance the catalytic activity of ß-lactamases against extended-spectrum cephalosporins (4, 24). The foremost amino acid substitution in the TEM and SHV ß-lactamases that improves the hydrolysis of oxyimino-cephalosporins is Gly238Ser (Ambler numbering scheme; http://www.lahey.org/studies/webt.asp) (1). Atomic resolution studies of clinically important TEM-type ESBLs and SHV-2 with the Gly238Ser substitution reveal that movement of the b3 ß-strand widens the dimensions of the active site, allowing for the binding of extended-spectrum cephalosporins (2, 19, 34, 35).

In certain TEM-type ESBL variants (e.g., TEM-3, -9, -26, and -52), an amino acid substitution is also observed at Ambler position 104 (Glu104Lys). Previous investigations have shown that this mutation is important in the development of the ESBL phenotype in TEM (16, 23, 24, 29, 30). A comparison of class A ß-lactamases reveals that Ambler position 104 is not a strictly conserved residue (1, 6, 8, 28). In the SHV-1 ß-lactamase, Asp instead of Glu is present at Ambler position 104. Moreover, the atomic structure of the SHV-1 ß-lactamase shows that the width of the substrate binding cavity, as defined by residues 104 to 105 and 130 to 132 to the ß-strand spanning residues 235 to 238, is wider than TEM-1 by 0.7 to 1.2 Å (15). Kuzin et al. also observed that Asp104 is solvated and that its backbone CO group is hydrogen bonded to Asn132 in the SHV-1 ß-lactamase (15).

To examine the role of Asp104 in SHV, we performed site saturation mutagenesis at this position. Our intent was (i) to investigate the amino acid properties at Ambler position 104 that affect resistance to penicillins and (ii) to see whether any other substitutions in this background increase resistance to oxyimino-cephalosporins. We extended these observations to also include a study of the effects of substitutions at position 104 in an SHV-2 (Gly238Ser) background.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and plasmids. blaSHV-1 was first cloned into phagemid vector pBC SK(–) (Stratagene, La Jolla, CA) from a clinical strain of K. pneumoniae 15571 and maintained in E. coli DH10B cells (Invitrogen, Carlsbad, CA) as previously described (25). This E. coli strain was used for MIC determinations and purification of SHV-1 ß-lactamase. Epicurean Coli XL1-Blue cells (Stratagene) were used in mutagenesis experiments. For protein expression of SHV-1, Asp104Lys, Gly238Ser, and the doubly substituted Asp104Lys Gly238Ser ß-lactamases, the pET 24a(+) plasmid containing these constructs was transformed into E. coli BL21(DE3) (Stratagene). The plasmid pET-24a(+) was obtained from Invitrogen.

Mutagenesis to create a blaSHVAsp104Xaa library. Site-saturation mutagenesis was performed at the Asp104 position in the blaSHV-1 gene in pBC SK(–) phagemid using a QuikChange II site-directed mutagenesis kit (Stratagene) according to a previously established protocol (9, 10, 12). Degenerate oligonucleotide primers D104N-1 and D104N-2 were purchased from Sigma-Genosys Biotechnologies (The Woodlands, TX) (Table 1).


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  		TABLE 1. PCR amplification and sequencing primers used in the experiments

 
Mutagenic PCR was performed on 50 ng of blaSHV-1 DNA template according to the supplied protocol using high-fidelity Pfu Turbo DNA polymerase and 125 ng of each degenerate primer. The cycling parameters for mutagenesis were 95°C (30 s), 55°C (1 min), and 68°C (10 min) for a total of 14 cycles. Epicurean Coli XL1-Blue cells were transformed with the mutagenic DNA to repair nicks. Transformants were selected on plates with 20 µg/ml chloramphenicol (Sigma, St. Louis, MO) and screened by DNA sequencing to identify each mutation and to select the most common blaSHV codons. Plasmid DNA containing the blaSHV gene encoding for each amino acid was then transformed into E. coli DH10B.

The blaSHVAsp104Cys variant was constructed by site-directed mutagenesis using primers D104C-1 and D104C-2 (Table 1) according to a previously published method (12).

Mutagenesis to create a blaSHVAsp104Xaa, Gly238Ser library. Plasmids were purified from E. coli DH10B cells containing the full complement of mutations (blaSHVAsp104Xaa). Site-directed mutagenesis was performed on these DNA templates using mutagenic oligonucleotides (primers G238S-1 and G238S-2) designed to engineer the Gly238Ser substitution as previously described by Hujer et al. (Table 1) (12).

Cloning into protein expression vectors. blaSHV-1 was amplified with primers (SHVNDE and SHVBAMH) containing an NdeI site at the 5' end and a BamHI site at the 3' end (Table 1) (14). This amplicon was directionally cloned into pET-24a(+) and transformed into E. coli DH10B. The blaSHVAsp104Lys, blaSHVGly238Ser, and blaSHVAsp104Lys Gly238Ser genes were then engineered by site-directed mutagenesis as described above in the blaSHV-1-pET-24a(+) plasmid using primers listed in Table 1 (D104K-1, D104K-2, G238S-1, and G238S-2).

DNA sequencing. We performed DNA sequencing using an ALF Express automated DNA sequencer (Amersham Pharmacia Biotech, Piscataway, NJ) with a Thermo Sequenase fluorescent-labeled primer cycle sequencing kit according to the protocol provided. The primers used for sequencing included commercially available universal and reverse primers in conjunction with customized sequencing primers internal to the blaSHV-1 gene. Table 1 lists the sequencing primers.

Antibiotic susceptibility. E. coli DH10B expressing the mutant blaSHV genes in pBC SK(–) (blaSHVAsp104Xaa and blaSHVAsp104Xaa Gly238Ser libraries) were phenotypically characterized by Luria-Bertani (LB) agar dilution MICs. The MICs for various antibiotics were determined using a Steers replicator that delivered 10 µl of an overnight culture of cells containing 104 CFU/spot. Ampicillin, cephalothin, ceftazidime, cefotaxime, and chloramphenicol were obtained from Sigma Chemical Co. The concentrations used for determining MICs were in µg/ml.

ß-Lactamase purification. The SHV-1 ß-lactamase was purified to homogeneity using preparative isoelectric focusing (13). E. coli BL21(DE3) cells grown in LB broth containing 50 µg/ml kanamycin (Sigma) and harboring the pET-24a(+) expression vector with the insert blaSHVAsp104Lys, blaSHVGly238Ser, or blaSHVAsp104Lys Gly238Ser were grown with agitation at 37°C until an optical density at 600 nm of 0.8 was reached. After the addition of 0.2 mM isopropyl-ß-D-thiogalactopyranoside (IPTG; Sigma), the cultures were grown for an additional 3 h. Following ß-lactamase induction, the cells were pelleted by centrifugation and frozen. Lysis was achieved with the addition of lysozyme and EDTA, and the ß-lactamases were purified using preparative isoelectric focusing as previously described (13). Purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ß-lactamase concentration was determined with Bio-Rad's protein assay (Hercules, CA) using bovine serum albumin (Sigma) as a standard. The Gly238Ser, Asp104Lys, and the doubly substituted Asp104Lys Gly238Ser ß-lactamases required further purification by size exclusion chromatography, using a Waters high-performance liquid chromatography system as described by Helfand et al. (11).

Kinetics. ß-Lactamase enzymes follow a covalent intermediate reaction as summarized by the following:

Formula
wherein E represents the ß-lactamase, S is the substrate (ß-lactam), E:S is the Michaelis complex, E-S is the acyl enzyme, P is the product, k1 and k–1 represent the on and off rates, k2 is the acylation rate constant, and k3 is the deacylation rate constant. The steady-state kinetic parameters, Vmax and Km, were obtained with nonlinear least-squares fit of the data (Michaelis-Menten equation) using Enzfitter (Biosoft Corporation, Ferguson, MO) (24, 31): v = Vmax[S/(Km + S)] and Km = [k3/(k2 + k3)] x Ks, where Ks = (k–1 + k2)/k+1. The determination of the dissociation constant for the preacylation complex (Ks, KD) and the calculation of the microscopic rate constants (k2 and k3) were performed based upon previous published methods (24) using the following equations:

Formula 1(1)
and

Formula 1
As discussed previously, a decrease in Km represents a decrease either in Ks (i.e., better affinity) or in the k3/k2 ratio. A large Km value indicates poor affinity (large Ks).

The contribution of amino acid substitutions to the free energy changes was calculated based on catalytic efficiency (17): {Delta}{Delta}G = –RT ln [(kcat/Km of SHV variant)/(kcat/Km of SHV-1)]. This equation (R is the universal gas constant [8.31 J/kmol], and T is in degrees Kelvin) calculates the difference in binding energy between SHV-1 and the variant ß-lactamase in going from free ß-lactamase and substrate to the transition state.

The kinetic parameters of the SHV-1, Gly238Ser, Asp104Lys, and the doubly substituted Asp104Lys Gly238Ser ß-lactamases were determined by continuous assays at room temperature using an Agilent 8453 diode array spectrophotometer. Unless otherwise indicated, each determination was performed in 20 mM phosphate-buffered saline at pH 7.4. Measurements were obtained using ampicillin ({Delta}{varepsilon}235 = –900 M–1 cm–1), cefotaxime ({Delta}{varepsilon}264 = –7,250 M–1 cm–1), cephalothin ({Delta}{varepsilon}262 = –7,660 M–1 cm–1), and nitrocefin ({Delta}{varepsilon}482 = 17,400 M–1 cm–1) (Becton Dickinson, Cockeysville, MD).

Solution 1H NMR spectra of cefotaxime. The nuclear magnetic resonance (NMR) of cefotaxime in 40 mM sodium phosphate buffer at pH 7.5, 8.5, and 9.0 was determined using 1H NMR available at the core facility at Case Western Reserve University. Buffers were prepared in H2O and replaced with an equivalent volume of D2O after dehydration in a Speed Vac. Due to the isotope effect on the ionization of weak acids, the pDs (pH in D2O) of the resulting buffers are approximately 0.5 units higher than the pHs of the corresponding H2O buffers (3, 26, 27). Cefotaxime was dissolved in the D2O buffers at a concentration of 10 mM. Sodium salt (0.2 µmol) of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid was added to 800 µl of the resulting solution as a chemical shift standard, and 1H NMR spectra were obtained using a 600-MHz NMR.

Molecular representations. The crystal structure coordinates of the SHV-2 (1N9B) ß-lactamase were used to generate a representation of the doubly substituted Asp104Lys Gly238Ser variant (19). First, the SHV-2 model was energy minimized with an Insight II, version 2005, molecular modeling system (Accelrys, San Diego, CA) using the Biopolymer and Discover_3 modules (SGI Octane Workstation IRIX 6.5.28). The minimization was done using the constant valence force field, including hydrogens and waters at a pH of 7.0, and a conjugate gradient method (Polak-Ribiere) to find the final minimum. The energy-minimized structure of the SHV-2 ß-lactamase was then used to generate the Asp104Lys variant. This was energy minimized again, and the lowest energy conformer of 104Lys was chosen. A nonbond cutoff distance of 8 Å was chosen for these operations. The structures of TOHO-1 (1IYS), TEM-52 (1HTZ), TEM-64 (1JWZ), and PER-1 (1E25) were obtained from the Protein Data Bank (http://www.rcsb.org).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antibiotic susceptibility. (i) Effect of single amino acid substitutions at Ambler position 104. As seen in Table 2, only Asp104Asn conferred resistance to ampicillin equal to that of the wild type (WT) (16,384 µg/ml). For the other mutant ß-lactamases, there was a remarkable variation in ampicillin MICs (256 to 8,192 µg/ml). Many of the same substitutions (Asp104Thr, Asp104Tyr, and Asp104Leu) that drastically reduced ampicillin resistance (MIC = 256 µg/ml) also reduced cephalothin resistance (MIC = 4 µg/ml).


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  		TABLE 2. MICs for the Asp104Xaa and G238S variants and the DH10B strain

 
It is of particular interest that Asp104Arg and Asp104Lys variants expressed in E. coli DH10B increased resistance against cefotaxime (0.06 to 0.25 µg/ml). This result is intriguing since clinical variants of the SHV-1 ß-lactamase have not yet been described with this substitution (http://www.lahey.org/studies/inc_webt.asp). Compared to that of cefotaxime resistance, the increase in ceftazidime resistance was not as large (MIC range, 1 to 2 µg/ml).

(ii) Effect of mutations at Ambler position 104 in a Gly238Ser (SHV-2) background. Summarized in Table 3 are the MICs of the doubly substituted 104Xaa Gly238Ser variants expressed in E. coli. As a group, the doubly substituted ß-lactamases were slightly more resistant to ampicillin than the singly substituted variants at position 104 (MICs of 16 of the 19 variants were ≥ 2,000 µg/ml). Specifically, Gly238Ser restored resistance to ampicillin for the Asp104Thr, Asp104Tyr, and Asp104Leu mutations.


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  		TABLE 3. MICs for the Asp104Xaa Gly238Ser variants and the DH10B strain

 
With regard to oxyimino-cephalosporin MICs, the Asp104Arg and Asp104Lys variants of SHV-2 ß-lactamase conferred significantly higher levels of resistance than the parent SHV-2 enzyme (32 µg/ml for cefotaxime and ceftazidime versus 8 and 4 µg/ml conferred by the SHV-2 enzyme, respectively).

Kinetic parameters. Because of the effects observed on cefotaxime MICs and their clinical importance, our attention focused on investigating the role of Asp104Lys in the SHV-1 and Gly238Ser (SHV-2) backgrounds. Thus, the SHV-1, Gly238Ser, Asp104Lys, and the doubly substituted Asp104Lys Gly238Ser enzymes were purified to homogeneity.

The kinetic parameters for turnover of ampicillin, cephalothin, nitrocefin, and cefotaxime for the SHV-1 ß-lactamase and each variant are summarized in Table 4. In general, there was agreement between MICs and steady-state kinetics. The Asp104Lys-substituted ß-lactamase is slightly less catalytically efficient at hydrolyzing ampicillin and cephalothin than the WT enzyme (25% reduction in kcat/Km of Asp104Lys versus that of the WT for ampicillin and 20% reduction for cephalothin). The overall effect on kcat/Km is small.


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  		TABLE 4. Steady-state kinetic parametersa

 
A prominent difference in the behavior of Asp104Lys versus SHV-1 was noted when cefotaxime was studied as a substrate. We were not able to determine the kcat of cefotaxime for the SHV-1 ß-lactamase (very large Ks). In contrast, the Asp104Lys substitution introduced in SHV-1 improved the ability of the enzyme to achieve saturation and turnover for cefotaxime (Km =198 ± 55 µM; kcat = 1.7 ± 3 s–1 [mean ± standard deviation]). Comparing the Gly238Ser to the doubly substituted Asp104Lys Gly238Ser SHV ß-lactamase, we also observed that the Asp104Lys substitution in an SHV-2 background decreased Km and increased kcat for cefotaxime. The combined effect translates into a threefold increase in kcat/Km (0.60 ± 0.07 to 1.7 ± 0.01 µM–1 s–1). This result is consistent with the cefotaxime MICs that show an increase from 0.06 (SHV-1) to 8.0 (Gly238Ser) to 32 (Asp104Lys Gly238Ser) µg/ml.

We next determined the dissociation constant for the preacylation complex, Ks (or KD), and the k2 (acylation) and k3 (deacylation) rates for Asp104Lys. In a direct competition experiment using nitrocefin, we measured a KD of 1,104 ± 155 µM for cefotaxime. This result was anticipated (31). We then showed that k2 (10 s–1) is greater than k3 (2 s–1) and that k3 is similar to the kcat (1.7 ± 0.3 s–1) for cefotaxime hydrolysis by Asp104Lys. Thus, the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys is deacylation.

Lastly, we wondered whether the protonation state of Lys could play a role in the determination of Km for cefotaxime. To measure the relative contribution of the charge on Lys at position 104 in cefotaxime hydrolysis, we compared the activities of the doubly substituted Asp104Lys Gly238Ser and Gly238Ser ß-lactamases at three different pH values (40 mM sodium phosphate buffer at pH 7.5, 8.5, and 9.0). In this buffer range (ionic strength varied less than 15%), we observed that as the pH increased, the Km and turnover of cefotaxime by Asp104Lys Gly238Ser progressively approached that of the Gly238Ser ß-lactamase (Table 5). In order to show that cefotaxime was not altered by the changes in pH, we determined the 1H NMR spectra at each pH and observed that cefotaxime was not deprotonated (data not shown).


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  		TABLE 5. Kinetics of cefotaxime hydrolysis as a function of pHa

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The striking observation that emerges from our analysis is that Ambler position 104 plays a significant role in substrate recognition, discrimination, and turnover in the SHV ß-lactamase. This is intriguing since position 104 is not strictly conserved in class A ß-lactamases: many variations in amino acid sequence exist at this position (e.g., Glu-Asp-Asn-Ala-Thr-Val-Phe-Tyr-Pro, as reviewed in reference 8). We chose a wide range of ampicillin concentrations in our MIC testing (0 to 16,384 µg/ml) to discriminate the effect of changing amino acids at position 104. In the majority (11/19 or 58%) of the variants, we observed an unanticipated fourfold or greater decrease in ampicillin MICs. Similar results were seen when the first-generation cephalosporin, cephalothin, was tested. This extensive variation in ampicillin and cephalothin MICs reveals that substitutions at position 104 are not freely tolerated. Amino acid properties that universally explain this significant difference are not obvious. Considerations of size, charge, and hydrophobicity do not yield consistent predictions.

Because of its scientific and clinical importance among ESBLs, we first chose to define the role of Asp104 by examining interactions with extended-spectrum cephalosporins (i.e., cefotaxime). For SHV ß-lactamases expressed in E. coli, resistance to cefotaxime increases when Lys (or Arg) is introduced at position 104. This effect is even greater when Asp104Lys is present in SHV-2. Our kinetic analysis of SHV-2 showed that substituting Lys for Asp lowers the Km for cefotaxime. To understand the basis for this observation, we next examined the hydrolysis of cefotaxime by the doubly substituted Asp104Lys Gly238Ser and Gly238Ser ß-lactamases as a function of pH. Choosing a pH range (pH 7.5 to 9.0), we observed a two-thirds decrease in the catalytic efficiency of hydrolysis of cefotaxime by Asp104Lys Gly238Ser with increasing pH (from 1.8 ± 0.3 to 0.5 ± 0.03 µM–1 s–1). This change was driven largely by a decrease in cefotaxime Km; the Km values of Gly238Ser (24 ± 3.0 µM) and the doubly substituted Asp104Lys Gly238Ser (26 ± 1.0 µM) ß-lactamases at pH 9.0 are essentially equivalent. We interpret these findings to mean that with an increase in pH, the NH3+ of Asp104Lys loses a proton, substrate binding decreases, and the Asp104Lys Gly238Ser ß-lactamase behaves like the Gly238Ser ß-lactamase. In order to verify that deprotonation of the substrate (cefotaxime) is not responsible for this difference, we obtained the 1H NMR spectra of cefotaxime at different pH values and showed that the protons on cefotaxime were not changed as a result of altering the pH from 7.5 to 9.0. The pH range chosen is below the pKa of the lysine {varepsilon}-amino group (pKa = 10.53); however, Golemi-Kotra et al. have shown that the pKa of Lys73 in the TEM ß-lactamase is attenuated to 8.0 to 8.5 (7, 18). It is our interpretation that the pKa of Lys104 may also be lowered in SHV. We realize that these results should be interpreted cautiously, especially when using phosphate buffer. Nevertheless, at the diluting buffer concentrations used, ionic strength is low and varies minimally.

We then measured the microscopic rate constants (k2 and k3) for the turnover of cefotaxime by Asp104Lys. We determined that the deacylation rate constant, k3, was the rate-limiting step for hydrolysis. This analysis was reminiscent of the hydrolysis of cefotaxime by the TEM {Omega}-loop variants, Arg164Asn and Arg164Ser (31). The bulky C-7 side chain which undergoes adverse steric interactions with {Omega}-loop residues and displaces the hydrolytic water molecule defines the stability of extended-spectrum cephalosporins against class A ß-lactamases such as TEM and SHV (31). Substituting Lys for Asp at position 104 lowers Km, while acylation and deacylation still proceed slowly. Thus, a major role of Asp104Lys in SHV-1 is in overcoming the barrier to unfavorable side chain interactions and substrate binding.

Since the atomic structures of the Asp104Lys and the doubly substituted Asp104Lys Gly238Ser variants in SHV are not available, one must resort to a model to interpret these findings. The experimental foundation of our model is centered largely on the observation that (i) the Asp104Lys substitution significantly contributes to cefotaxime resistance and that (ii) Lys is a positively charged amino acid at pH 7.0, is flexible, and can assume multiple conformations in a protein. Based upon our data and a construction of the active site of the doubly substituted Asp104Lys Gly238Ser variant (Fig. 1), we propose that at a neutral pH, a H bond from the protonated, highly flexible Lys side chain directed towards the active site could interact with the oxime group of cefotaxime to stabilize the Michaelis-Menten complex and improve the ability of the enzyme to achieve saturation (lower Km). In this argument, the Ala237 backbone carbonyl and the side chains of Asn132 and Asp104Lys would form stabilizing H bonds with the oxyimino-cephalosporin's C-7 acrylamide linkage. Therefore, we maintain that Lys at position 104 in SHV may serve a direct substrate binding role, at least with regard to cefotaxime. This explanation agrees favorably with the analysis of TEM Glu104Lys by Sowek et al. (30). By extension, a similar argument would apply to Arg at this position.


Figure 1
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  		FIG. 1. A representation of the structure of the doubly substituted Asp104Lys Gly238Ser ß-lactamase using Insight II. Represented here is the protonated, highly flexible Asp104Lys side chain positioned towards the active site of the SHV-2 ß-lactamase. We propose that the protons of the NH3+ of Asp104Lys could directly interact with the oxime group of cefotaxime to increase affinity for cefotaxime.

 
How does one explain the other changes in ampicillin and cephalothin resistance? Petit et al. have advanced that the effect of mutations at position 104 directly or indirectly affect the SDN loop (Ser130-Asp131-Asn132) (23). This would also be consistent with our data and explains the decreased kcat/Km of ampicillin by Asp104Lys. However, more complex interactions than those just affecting the SDN loop are possible. Our data suggest that certain substitutions at position 104 may alter the pitch and shape (topology) of the loop spanning positions 101 to 111 (101-111 loop) and affect the position of Tyr105 as it stacks with the thiazolidine ring of penicillin or ß-lactamase inhibitors (8, 20). Thus, position 104 (like Tyr105 and Gly238) serves a structural role in SHV. This hypothesis is consistent with those of structural studies performed with TOHO-1 ß-lactamase (28). An inspection of the 101-111 loop of class A ß-lactamases (TOHO-1, TEM-52, TEM-64, PER-1, and the Asp104Lys SHV variant) reveals that subtle differences in conformation exist in this region of these five class A enzymes (Fig. 2). To our knowledge, the impact of individual amino acids affecting the overall shape and substrate specificity of this region has not been carefully scrutinized. Like the b3 ß-strand on the opposite side of the binding cavity, steric movement of the 101-111 loop may play a supportive role in the alteration of substrate specificity for penicillins and cephalosporins in the SHV ß-lactamase (13, 19, 28).


Figure 2
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  		FIG. 2. A representation of the structure of the TEM-52, TEM-64, PER-1, TOHO-1, and SHV Asp104Lys ß-lactamases using Insight II. Here, we show the relationship of the residue at Ambler position 104 and the position of residue 105 in the 101-111 loop. Glu166 and Ser70 are also shown.

 
This complex role of position 104 led us to wonder whether mutations at Asp104 and Gly238 are additive or synergistic. Efficient catalysis of penicillins and cephalosporins depends upon the binding of these substrates and their transition states to the ß-lactamase. Since ß-lactamases follow classical Michaelis-Menten kinetics, it is possible to deduce the energy changes in transition state binding from steady-state kinetics (5). This analysis has been applied to a variety of mutants of TEM (32, 33, 37) and SHV (10) as well as other enzymes of biological importance (subtilisin and tyrosyl-tRNA synthetase) (5, 36).

The contribution of each substitution to the overall catalytic efficiency of the ß-lactamase for ß-lactams was calculated. If substitutions of different amino acid residues are independent (introduce no structural change in the enzyme or enzyme substrate complex), the overall change in interaction energy ({Delta}GI) of the enzyme substrate transition state will be directly additive (the sum of {Delta}{Delta}G terms is near zero). Hence, the relationship is shown as follows: {Delta}{Delta}G1,2 = {Delta}{Delta}G1 + {Delta}{Delta}G2 +{Delta}G1. We examined these effects in the Asp104Lys Gly238Ser ß-lactamase for cephalothin (Fig. 3). Investigating cephalothin interaction in the Asp104Lys Gly238Ser cycle, we see that the change in interaction energy from the WT to the double mutant in the transition state is close to the algebraic sum of the free energy of the single mutants (Fig. 3b). Thus, the contribution of Asp104Lys is independent of Gly238Ser for the hydrolysis of cephalothin. Hence, we deduce that an additive spatial or electrostatic interaction occurs between ß-lactamase and the substrate when a basic positively charged residue (Lys) at Ambler position 104 is introduced in SHV-1 and SHV-2 ß-lactamases.


Figure 3
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  		FIG. 3. The changes in interaction energy of the enzyme and transition state are represented by the {Delta}{Delta}G terms (a). In (b), the calculations shown are from the kcat/Km ratio (units are µM–1 s–1) obtained for cephalothin.

 
In conclusion, our data highlight the importance of the Asp104 residue in SHV and reveal unpredicted findings. A novel appreciation of the significance of residues in the 101-111 loop in class A enzymes is also emerging. We show that single and double amino acid mutations affect the SHV ß-lactamase's gain and loss of function; each change may affect substrate binding and/or structure. Investigations are ongoing in our laboratories that explore the complex role of Asp104 in SHV, using transition state inhibitors that mimic these substrates. In light of the growing number of TEM and SHV variants being discovered, strategic drug design must take into account the interactions of multiple altered residues. We must anticipate that combinations of substitutions in the 101-111 loop will profoundly affect Km and turnover.


   ACKNOWLEDGMENTS
 
This work was supported by the Steris Foundation, a Merit Review award, and NIH grant R01AI063517-01 to R.A.B. J.M.T. and M.W.R. were supported in part by National Institutes of Health grant T32 GM07250 and the Case Medical Scientist Training Program. M.S.H. was supported by the Department of Veterans Affairs Advanced Career Development Award. M.P.-C. was supported by NIH grants GM 54072 and DK 53053.


   FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Diseases Section, Louis Stokes Cleveland Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4399. Fax: (216) 231-3482. E-mail: robert.bonomo{at}med.va.gov. Back

{triangledown} Published ahead of print on 18 September 2006. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, December 2006, p. 4124-4131, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00848-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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