Structure–function analysis of {alpha}-helix H4 using PSE-4 as a model enzyme representative of class A ß-lactamases

Annie Savoie1, François Sanschagrin1, Timothy Palzkill2, Normand Voyer3 and Roger C. Levesque1,4

1 Microbiologie Moléculaire et Génie des Protéines, Pavillon Charles-Eugène Marchand, Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4, 2 Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA and 3 Département de Chimie, Pavillon Vachon, Faculté des Sciences et de Génie, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We extracted maximum information for structure–function analysis of the PSE-4 class A ß-lactamase by random replacement mutagenesis of three contiguous codons in the H4 {alpha}-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129. These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model. Structure–function studies on positions 125–129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 {alpha}-helix structure together, thus maintaining the important Ser130–Asp131–Asn132 motif positioned towards the active site. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4. The Tyr 125 of the mutant YAM formed an edge to face {pi}{pi} interaction with Phe 124 which also interacts with the Trp 210 with the same interactions. Antibiotic susceptibilities showed that amino acid changes in the the H4 {alpha}-helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors. However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 {alpha}-helix region than clavulanic acid, an inhibitor of the oxypenam class. The analysis of H4 {alpha}-helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.

Keywords: antibiotic resistance/ß-lactamase/{pi}-{pi} interactions/protein structure


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The control of bacterial pathogens causing infectious diseases using ß-lactam antibiotics resulted in the development of several bacterial resistance mechanisms to these drugs. In Gram-negative bacteria, the cleavage of the amide bond of the ß-lactam ring by bacterial ß-lactamases is the major cause of bacterial resistance to these compounds (Bush et al., 1995Go). ß-Lactamases are grouped into four classes based on the primary sequence homology (Ambler, 1980Go; Joris et al., 1988Go). A Ser70 amino acid residue forms the active site for classes A, C and D, whereas a zinc atom is found in class B carbapenem metallo-ß-lactamases (Ambler et al., 1991Go; Joris et al., 1988Go; Carfi et al., 1995Go). Since their discovery, extensive work has been done to elucidate their biophysical and biochemical properties to understand steps in enzyme catalysis and inhibition. In recent years data from both kinetic and high-resolution crystallographic studies have been used to understand the structural–functional roles of amino acids at or near the active site.

The Ser130–Asp131–Asn132 motif, conserved in all class A enzymes including carbenicillin-hydrolyzing enzymes such as PSE-4, and its importance in catalysis have been the subject of several studies. Ser130 has a structural role in maintaining the active site by a hydrogen bond with Lys234 (Jacob et al., 1990Go; Moews et al., 1990Go). It also interacts both with penicillin and with cephalosporin during hydrolysis in having a critical role for substrate specificity and in substrate binding (Juteau et al., 1992Go). Ser130 has a catalytic role in proton shuttle towards the nitrogen atom of the ß-lactam ring (Moews et al., 1990Go) and contributes to initial binding with inhibitors, as demonstrated with clavulanate (Imtiaz et al., 1993Go) and with sulbactam (Imtiaz et al., 1994Go). Asp131 was considered as a key residue in protein structural stability (Jacob et al., 1990Go) and Asn132 contributed in positioning the substrate into the active site by forming a hydrogen bond with the carbonyl group of the antibiotic (Herzberg and Moult, 1987Go; Moews et al., 1990Go). However, no studies were done on positions near the Ser130–Asp131–Asn132 motif and their effects on the structure or the function of the enzyme. In TEM-type enzymes, replacement of one or three similar amino acids not located in the vicinity of the active site but sites close to it caused changes in kinetic profiles against broad-spectrum ß-lactam antibiotics (Petit et al., 1989Go). In the H4 and H5 {alpha}-helices of TEM, it has been found that Ala125 did not tolerate any substitution and that Ala134 was invariant and conserved in all class A enzymes (Huang et al., 1996Go). Another study showed that position 128 (Thr128 Ala mutant) was responsible for kinetic differences among two wild-type Staphylococcus aureus enzymes (Voladri et al., 1996Go).

The model enzyme used in this study was PSE-4, a carbenicillin-hydrolyzing ß-lactamase found predominantly in Pseudomonas aeruginosa (Williams et al., 1984Go; Wiedemann et al, 1989Go). Molecular modeling of a three-dimensional structure of PSE-4 permitted the identification of possible amino-acid residues in the H4 {alpha}-helix to be critical for the structure and/or for the function of the protein. In this approach, a particular focus was to identify possible atomic interactions of the PSE-4 enzyme with suicide inhibitors. These five amino acids are found at the end of the fourth {alpha}-helix (H4 in PSE-4) of the all {alpha} domain (Herzberg et al., 1987) and flanks a conserved box of amino acids, the Ser130–Asp131–Asn132 motif invariant in all class A ß-lactamases (Joris et al., 1991Go) and as depicted in Figure 1Go. To extract maximum information for these positions selected in H4 (amino acids 125–129 in PSE-4), two libraries were created by random replacement mutagenesis at these conserved amino acid positions. We present data in an attempt to define the possible functions of these amino acids at positions 125–129 with results supported by determination of minimal inhibitory concentrations for several ß-lactam antibiotics, by kinetic values and by information obtained from the secondary structures using circular dichroism and by molecular modeling of a three-dimensional structure of PSE-4.



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Fig. 1. Cartoon representation of secondary structures of PSE-4 built by homology using data from the atomic coordinates of S.aureus PC1 ß-lactamase, B.licheniformis 749/C and TEM-1.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

All antibiotics were purchased from Sigma (Oakville, Ont.), except carbenicillin, which was purchased from Gibco BRL, nitrocefin from Oxoid (Nepean, Canada), and ticarcillin disodium, which was kindly provided by SmithKline Beecham Pharmaceuticals (Oakville, Ont.). The mechanism-based inhibitors sulbactam sodium, lithium clavulanate and tazobactam were kindly supplied by Pfizer (Groton, CT), SmithKline Beecham Pharmaceuticals and SynPhar Laboratories (Edmonton, Alb.), respectively. Polynucleotide kinase and the restriction endonucleases BamHI and HindIII were purchased from New England Biolabs (Mississauga, Ont). The Muta-gene phagemid in vitro mutagenesis kit was obtained from Bio-Rad (Mississauga, Ont.). Media and broth were purchased from Difco (Detroit, MI).

Bacterial strains and phagemid

Four strains of Escherichia coli were used. All protein expressions were performed into E.coli BL21({lambda}DE3) [F-ompT [lon] hsds (r-m-) an E.coli B strain with a {lambda} prophage carrying the T7 RNA polymerase gene]. E.coli CJ236 [ung, dut, thi, rel A; pCJ105 (Cmr)] was used to prepare uracilated single stranded DNA. E.coli DH5{alpha} F' (LT1) [F'/endA1 hsdR17 (r-m+) supE44 thi-1 gyrA (Nalr) relA1 {triangleup}(lacIZYA-argF)U169 deoR (f80dlac{triangleup}(lacZ)M15)] was used for antibiotic susceptibility testing and for protein characterization in immunoblots. E.coli JM101 [F' traD36lacIq{triangleup}(lacZ)M15 proA+B/supE thi{triangleup}(lac-proAB)] was used in the second mutagenesis step for the construction of both libraries of mutants. Phagemid pMON711 contains a 1.1 kb EcoRI + HindIII DNA fragment coding for blaPSE-4 gene (Boissinot and Levesque, 1990Go) in the cloning vector pBGS19+ (Spratt et al., 1986Go). After transformation and purification of colonies on solid media, a single bacterial clone was selected and plasmid size confirmed. The plasmid pMON711 containing blaPSE-4 was verified by sequencing the entire structural gene using a Pharmacia T7 sequencing kit (Baie d'Urfé, Qc.) using five 17-mer internal oligonucleotide primers. The sequenced gene had 100% nucleotide identity with the sequence published previously (Boissinot and Levesque, 1990Go).

DNA sequencing

DNA sequencing reactions were carried out with Taq- sequencing dye-terminator cycle sequencing ready reactions from ABI loaded on an ABI 373 (Perken-Elmer Applied Biosystems, Foster City, CA). The nucleotide sequences were done for both DNA strands using plasmid DNA (Qiagen, Santa Clarita, CA). The complete blaPSE-4 genes were sequenced for all mutants. Chromatograms of electrophoretic signals were analyzed by Factura (ABI, version 1.2.0) and Sequence Navigator (ABI, version 1.0.1) and alignment of primary structures was performed using the software Wisconsin Package Version 10.0, Genetics Computer Group (GCG) (Madison, WI).

Random replacement mutagenesis and oligonucleotides used

Preparation of single-stranded DNA from phagemid pMON711 was done according to standard methods (Sambrook et al., 1989Go). In vitro DNA synthesis was done with single-stranded uracilated DNA with the Bio-Rad Muta-Gene kit. The mutagenesis scheme was performed using Kunkel's method (Kunkel et al., 1987Go) with the strategy of Petrosino and Palzkill (1996). After ligation, DNA was inserted in E.coli CJ236 by electroporation. Selection of clones was done on TSA (Difco) plates containing 50 µg/ml of kanamycin and 30 µg/ml of chloramphenicol. After phenotypic selection, clones were purified as single colonies and the nucleotide sequence determined at each mutagenesis step.

Four oligonucleotide primers were synthesized with 394 DNA/RNA synthesizer from Perken-Elmer ABI Applied Biosystems. The first primer 5'-GATGCGTGCTTCGCAAGGATCCGCTATGACTACAAGTGAT-3' and the second primer 5'-TGCTTCGCAACTATGAGGATCCGCTACAAGTGATAATACT-3' were used in the first mutagenesis step for making libraries 125–127 and 127–129. The underlined portion indicates the BamHI inserted in the three codons to be randomized, a G nucleotide was added to change the ORF, thus permitting the phenotypic selection of ampicillin susceptibility. The two oligonucleotides: 5'-ATGATGCGTGCTTCNNC/GNNC/GNNC/GACTACAAGTGATAA-3' and 5'-CGT-GCTTCGCAACTNNC/GNNC/GNNC/GGAGTGATAATACT-GC-3' served in the second step randomizing the targeted positions for libraries 125–127 and 127–129. These two 37-mers contains two 14 nucleotide arms flanking 9 nucleotide randomized codons (NNC/GNNC/GNNC/G).

Preliminary characterization of mutants

E.coli clones that were kanamycin-resistant were tested for susceptibility to ampicillin (1500 and 3000 µg/ml), piperacillin (150 and 500 µg/ml), cephaloridine (15 µg/ml), cephalexin (10 µg/ml) and clavulanate (8 µg/ml) in combination with ampicillin (4.8 µg/ml). From 30 to 40 clones selected from each group were chosen for further characterizations. These characterizations included ampicillin (3000 and 1500 µg/ml), piperacillin (150, 300 and 400 µg/ml), cephaloridine (15 µg/ml), cephalexin (10 µg/ml) and with inhibitor clavulanate at 8 µg/ml with ticarcillin (50 µg/ml), amoxycillin (140 µg/ml) and ampicillin (2.5 and 5 µg/ml), where 20 mutants were kept for antibiotic susceptibility analysis.

Antibiotic susceptibility testing

Minimal inhibitory concentrations (MICs) were determined by using the microdilution method in Mueller–Hinton broth in 96-well microtiter plates. Selected ß-lactamase-producing E.coli DH5{alpha} cells were grown to 109 CFU/ml and diluted to 105 CFU/ml, inoculated with 100 µl of broth and serial twofold dilutions for each antibiotic tested. Plates were examined after 20 h at 37°C and the lowest concentration of antibiotic, which inhibited visual growth, was estimated as the MIC value. E.coli ATCC 25922 served as a define control where MIC values were determined from the US National Committee of Clinical Laboratory Standards.

Immunoblots of ß-lactamases

Expression of ß-lactamases was verified by Western transfer using anti-PSE-4 polyclonal antibodies and enzyme extracts prepared by osmotic shock and by sonication. Cultures of 5 ml of E.coli DH5{alpha} were grown overnight in tryptic soy broth supplemented with 50 µg/ml of kanamycin and ampicillin, and cells were pelleted and resuspended in 0.3 volumes (ml) per mg of pellet in 50 mM potassium phosphate buffer (pH 7.0), sonicated with a micro-probe for 10–20 s at level 5 intensity using a VirSonic 475 apparatus (VirTis, Gardiner, NY). The sonicated cells were microcentrifuged for 15 min at 4°C and supernatants were loaded on 12% sodium dodecyl sulfate polyacrylamide gel for SDS–PAGE and separated by electrophoresis for 1.5 h at 17 V/cm in a minigel apparatus (Mini-PROTEAN II, Bio-Rad, Mississauga, Ont., Canada). Proteins were electrotransferred (Towbin et al., 1979Go) on to PVDF membranes, labeled with anti-PSE-4 polyclonal antibodies (diluted to 1:20 000) and detected with a BM chemiluminescence Western blotting kit (mouse/rabbit) (Boehringer Mannheim, Laval, Qc.).

ß-Lactamase purifications

Bacterial cells were grown at 37°C, 250 r.p.m., in Terrific Broth (Difco) containing 50 µg/ml of kanamycin and ampicillin; after 3 h 1 mM isopropyl-1-thio-ß-D-galactopyranoside was added and incubated overnight. The periplasmic proteins (including ß-lactamases) were isolated by osmotic shock (Neu and Heppel, 1965Go). The crude extract obtained was filtered on PD-10 Sephadex 25 columns (Pharmacia) and directly loaded on an Econo-Pac Q anion-exchange chromatography cartridge (Bio-Rad). The wild-type PSE-4 enzyme eluted with ~30 mM NaCl and mutant enzymes eluted with a continuous salt gradient between 22 and 98 mM NaCl concentrations. Fractions containing ß-lactamase proteins were identified with nitrocefin and by SDS–PAGE. Proteins from selected fractions were concentrated and buffers exchanged by ultrafiltration with Centricon 10 and by Centriprep 10 (Amicon Canada, Oakville, Ont.). The concentrated fractions were loaded on a sieve chromatography apparatus using HiPrep 26/60 Sephacryl S-100 (Pharmacia) with a filtration step of 50 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, at a flow rate of 1.3 ml/min. Chromatographic columns were run on a ConSep LC100 apparatus (Millipore, Mississauga, Ont.). Fractions were selected as above and enzyme purity was estimated from SDS–PAGE gels stained with SYPRO Orange protein gel stain (Bio-Rad, Mississauga, Ont.) and protein density calculated by using the computer software NIH Image version 1.60 (NIH, USA). Enzymes were kept in aliquots at –20°C in 50% glycerol and 300 µg/ml ultrapure bovine serum albumin (NEB, Mississauga, Ont.).

Enzyme kinetics

Kinetics were measured at 30°C in 50 mM sodium phosphate buffer, pH 7.0, in a 1 ml cuvette reaction volume with a Cary 1 spectrophotometer (Varian, Mississauga, Ont.). Hydrolysis for ampicillin ({Delta}{varepsilon} = 912 M–1 cm–1) and carbenicillin ({Delta}{varepsilon} = 1190 M–1 cm–1) was monitored at 232 and 280 nm, respectively. Kinetic parameters Vmax and Km were determined by rates of hydrolysis calculated from the initial velocity in the linear portion, with the same cuvette and a least-squares calculation. Enzyme concentrations used for the wild-type PSE-4 purified protein were 3.2 and 22.5 nM for the mutant PSE-3 YAM ß-lactamase. Concentrations of the substrates tested varied from 10 to 800 µM. The inhibition parameters Ki for the three mechanism-based inhibitors, clavulanate, tazobactam and sulbactam, were determined by using carbenicillin as a reporter substrate. This dissociation constant was determined by direct non-linear regressions using the hyperbolic Henri–Michaelis equation, where the concentration of inhibitor was constant in the reaction and the concentration of the reporter substrate in excess varied. These experiments were carried out for the wild-type PSE-4 with 25 and 50 µM for clavulanate, 1.25 and 2.5 µM for tazobactam and 10 and 20 µM for sulbactam. The third plot represented the 0 µM concentration of inhibitor in the reactions. For the YAM mutant concentrations varied from 7 and 10 µM for clavulanate, 0.75 and 1.25 µM for tazobactam and 7 and 10 µM for sulbactam. All experiments were carried out in triplicate. Analysis of enzyme kinetic data was done with the Leonora software for robust regression analysis of enzyme data and a bi-weighting regression system (Cornish-Bowden, 1995Go).

The molar inhibition–enzyme ratio (% I/E) was determined after 18 h by using the technique of progressive inhibition described previously (Bush et al., 1993Go). The reporter substrate carbenicillin was in excess at 700 µM in a 1 ml cuvette reaction volume. Reaction rates were monitored for 20 and 45 min with clavulanate and sulbactam. Protein concentrations were 3 µM for PSE-4 and 2.5 µM for YAM.

Circular dichroism

Circular dichroic (CD) spectra were recorded on a Jasco Model J-710 spectropolarimeter (Jasco, Easton, MD). The protein concentrations were 131.4 µM for PSE-4 wild-type and 31.6 µM for the mutant YAM, in a 50 mM sodium phosphate buffer, pH 7.0. Measurements were obtained from 5 to 85°C using increments of 10°C. For each increment, 15 scans were collected from 190 to 250 nm in a 0.2 mm pathlength cuvette. The ellipticity value was calculated using Equation 1 (Walton, 1981Go). The instrument sensitivity factor (f) was 5 mdeg, the residue mean molecular weight (M) was 109.4 g/mol and the path length (l) was 0.02 cm.

Computer-assisted modeling

A three-dimensional structure of PSE-4 was constructed using atomic coordinates from Staphylococcus aureus PC1, Bacillus licheniformis 749/C and TEM-1 ß-lactamases which shared 34.7, 28 and 40.4% identity with PSE-4 (Herzberg, 1991Go; Knox and Moews, 1991Go; Jelsch et al., 1993Go). The homology software in the InsightII package (version 95.0, MSI) was used for molecular modeling on a Silicon Graphics Elan R4000 workstation.


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 Materials and methods
 Results
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Random replacement mutagenesis and preliminary characterization of mutants

The construction of two randomized libraries at positions Ala125–Thr126–Met127 and Met127–Thr128–Thr129 by replacement with all codon substitutions was used to extract maximum information. A total of 32 768 randomized sequences were obtained for each library. Phenotypic analysis of E.coli expressing PSE-4 mutants from the first antibiotic screening on plates permitted 20 mutants to be chosen from each library, that were kept for further analysis of MICs. The nucleotide sequences were determined for the complete bla PSE-4 genes and codon substitutions were compared with antibiotic phenotypes. From 20 sequences, six amino acid substitutions were found at amino acid positions 125–127 (Table IGo). From the 127–129 library, we obtained 12 mutants with substitutions (of 20 sequenced) (Table IIGo). In both cases, we noted that Met127 was conserved in all PSE-4 mutants selected as depicted in Tables I and IIGoGo.


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Table I. Minimum inhibitory concentrations for mutants from library 125–127 (µg/ml)
 

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Table II. Minimum inhibitory concentrations for mutants from library 127–129 (µg/ml)
 
Antibiotic susceptibility

Antibiotic susceptibility testing for 18 mutants are shown in Tables I and IIGoGo. When a penam ß-lactam antibiotic was used alone, no significant changes were apparent in MIC values when comparing the MIC of E.coli producing wild-type PSE-4 with the MICs from the 18 E.coli producing mutant PSE-4 ß-lactamases. However, significant decreases in MICs were observed when a mechanism-based inhibitor was used in combination with a ß-lactam antibiotic. The clavulanate combination with either ampicillin or with carbenicillin gave decreases in MIC values between 4- and 18-fold for several mutants. As a general rule, tazobactam combined with carbenicillin was less effective in inhibition than the combination with piperacillin. In combination with carbenicillin, tazobactam gave the most significant decreases in MICs, where a 21-fold decrease was obtained for the Thr126Met substitution (mutant AMM in Table IGo). Interestingly, all mutants had significant decreases in susceptibility when piperacillin was in combination with tazobactam; the decreases ranged from 8- to 30-fold. The sulbactam combination with either carbenicillin or with ampicillin gave the most significant decreases in susceptibility of all combinations used, up to 126-fold being observed with Lys125–Thr126–Met127 and Tyr125–Ala126–Met127 substitutions (see LTM and YAM mutants, Table IGo). With sulbactam, the most significant decreases were observed when the mutations where at position 125 and/or 126; the same observations were true as in the carbenicillin–tazobactam combination. The mutants AMM, LTM and YAM gave a 32- to 64-fold decrease when carbenicillin was used (MICs from 39 to 78 µg/ml) and a 78- to 126-fold decrease (MICs from 1 to 2 µg/ml) when ampicillin was used (Table IGo).

ß-Lactamase expression

Figure 2Go shows the expression levels of the ß-lactamase PSE-4 mutants estimated by immunoblotting. For mutants having substitutions at 125 and/or 126, the only mutant that gave levels of expression similar to the wild-type protein were PSE-4 mutants VTM and YAM from the library 125–127. In the 127–129 library, only MVY and MVL PSE-4 mutants had similar expression levels to the wild-type PSE-4. However, low but detectable levels of protein expression were also observed for PSE-4 mutants MVW and MTY. A total of 12 PSE-4 mutant proteins from both libraries were not detectable by immunoblots. Since enzyme degradation could occur during the lengthy process preceding the blotting experiments, we measured enzyme activity just after the sonication step as shown in Figure 2Go. There were no correlations between the MICs (Tables I and IIGoGo) and the quantities of enzyme detected by immunoblotting.



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Fig. 2. Immunoblots of PSE-4 wild-type and mutant ß-lactamases from the two libraries constructed. (A) Library 125–127. Wild-type PSE-4 having the Ala125–Tyr–126–Met127 motif. (B), (C) Library 127–129. Wild-type PSE-4, positive control, i.e. represented by the sequence 127MTT.

 
Enzyme kinetics

To understand the effects of substitutions in the H4 {alpha}-helix region of the PSE-4 enzyme, the mutant YAM from library 125–127 was chosen for further analysis by enzyme kinetics. Purified proteins from the mutant and the wild-type PSE-4 enzymes were used to determine Km, kcat and kcat/Km, inhibition parameters Ki and molar ratios of inhibition (% I/E). As shown in Table IIIGo, the turnover number kcat for the YAM substitution mutant was compared with PSE-4 ATM wild-type and had a significant decrease of 16-fold for carbenicillin. A 4-fold decrease was obtained for kcat when ampicillin was used as substrate. However, the overall catalytic efficiency (kcat/Km) of the mutant protein having YAM compared with ATM was lower with ampicillin than with carbenicillin owing to a lower affinity for ampicillin (higher Km value). The double mutations in the H4 region, YAM versus ATM, had slightly increased affinity for carbenicillin, but the corresponding value of kcat/Km was not significantly changed when compared with the wild-type value (3.8x106 s–1 µM–1 for YAM versus 17x106 s–1 µM–1 for wild-type ATM).


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Table III. Substrate profile and kinetic parameters for wild-type PSE-4 and YAM mutant
 
Interactions with inhibitors

We determined the apparent affinity of the PSE-4 enzyme having the ATM to YAM substitutions for three mechanism-based inhibitors because MIC values for this mutant were decreased dramatically (from a ratio of 8 with clavulanate combinations to 15 with sulbactam combinations and to 70–156 using tazobactam combinations, Table IVGo). The Ki value showed a significant decrease of 22-fold for clavulanate with a Ki of 1 µM for YAM compared with 22 µM for PSE-4 (Table IVGo); while MIC values decreased ~10-fold (from 313 to 39 µg/ml) with this inhibitor (Table IIGo). Decreases of Ki values were 4-fold with sulbactam (Table IVGo), while changes in MICs values between the wild-type and YAM mutant were between 90- and 100-fold with sulbactam. Curiously, there was no significant change in Ki value with tazobactam compared with the 10–15-fold changes in MIC. The molar ratio of inhibition was determined with clavulanate and sulbactam due to their effects on the apparent inhibition affinity (Ki) and MIC values. By calculating the % I/E relative values (Table IVGo), a significant difference was noted between the molar ratio of inhibition with clavulanate (relative ratio of 6.3), indicating a more efficient turnover between the mutant YAM and wild-type ATM; sulbactam gave a 4-fold difference (ratio of 0.25) while tazobactam gave no significant difference (ratio of 0.82).


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Table IV. Ki values (µM) and molar ratio of inhibition (%I/E) with selected inhibitors for wild-type PSE-4 and YAM mutant
 
Circular dichroism

We studied the effects of amino acid changes in the H4 {alpha}-helix secondary structure by circular dichroism. The CD plots obtained at 25°C for the wild-type PSE-4 protein and the protein having the 125ATM->125YAM substitutions are shown in Figure 3aGo. Figure 3bGo shows the thermal denaturation profile of helicity characterized by the negative band at 222 nm for both mutant YAM and wild-type PSE-4. The structural helicity for PSE-4 is stable until 55°C, where increasing the temperature decreases ellipticity, as shown by smaller negative values at 222 nm in Figure 3bGo. This conformational change suggests a loss of helical content leaving the core ß-sheet structure, which is more stable. On comparing the thermal denaturation profile of the mutant protein YAM with the wild-type PSE-4 (or ATM), a different structural behavior is noted regarding the temperature at which the ellipticity is augmented (Figure 3bGo). The ellipticity starts to increase slightly around 75°C for YAM compared with 55°C for PSE-4, but the helicity for YAM is not entirely lost at 85°C and it is still possible to observe at this temperature the typical 208 and 222 nm negative bands.



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Fig. 3. (A) CD spectra of mutant ß-lactamase YAM ({triangleup}) and wild-type PSE-4 ({circ}) at 25°C. (B) Thermal denaturation profile as measured by the change in ellipticity at 222 nm for YAM and PSE-4 wild-type.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Elucidating how amino acid substitutions alter enzyme ß-lactamase specificity or activity for given ß-lactam antibiotics or mechanism-based inhibitors is a crucial step in understanding the molecular interactions necessary for rational drug design. We present data in an attempt to define the roles of amino acids found at positions 125–129 in the H4 {alpha}-helix of PSE-4 used as a model enzyme for carbenicillin-hydrolysing enzyme and representative of class A ß-lactamase. Random replacement mutagenesis was used to construct two random libraries, 125–127 and 127–129. A total of 18 mutants were compared with the wild-type PSE-4 for the amino acid sequences and their antibiotic susceptibilities. One mutant, YAM, was kept for further analysis by using kinetics, CD and computer-assisted molecular modeling of a PSE-4 three-dimensional structure.

The study identified 43 amino acids that did not tolerate substitutions in the mature form of the TEM-1 enzyme in which the 263 amino acid residues were randomized; among them 23 had previously been identified as being invariant in class A ß-lactamases. Ala125 of the H4 {alpha}-helix region was one of 43 amino acids identified as being invariant in TEM-1 (Huang et al., 1996Go). Ala125 is also conserved in PSE-4 (Boissinot and Levesque, 1990Go). In the six PSE-4 mutants characterized from the library 125–127, four had substitutions for Ala125. Three amino acids were in the same electrochemical family as Ala (Trp, Val, Leu) and the fourth mutant had the hydrophilic Tyr substitution. All four mutant proteins expressed in E.coli conserved an activity towards ß-lactam antibiotics shown by MIC data, demonstrating that PSE-4 can tolerate substitutions at Ala125. In contrast, no mutants had substitutions for Met127 from the 18 selected. The Met127 could be attributed to maintaining the H4 {alpha}-helix structure and positioning the Ser130–Asn131–Asp132 motif towards the active site. Indeed, Ser130 has been shown to be essential in maintaining the active site but also is critical for substrate specificity and for binding (Juteau et al., 1992Go).

On examining a multiple alignment for positions 125–127 of class A enzymes, only Ala and Val are found at position 125 and Ile, Val, Leu and Met are found at position 127. Knowing that Ala125 is conserved and that an Ile 127 can be substituted by Leu and Met in TEM-1 ß-lactamase suggests differences in PSE-4 distinct from TEM-1. Single amino acid substitutions at positions near the active site can be responsible for different substrate profiles (Petit et al., 1989Go; Voladri et al., 1996Go) where this was demonstrated with a substitution in the H4 {alpha}-helix between wild-type variants of S.aureus ß-lactamases belonging to class A enzymes. Kinetic heterogeneity in type A enzyme was exhibited with a Thr125 Ala change in the S.aureus enzyme. In PSE-4 mutants, different kinetic profiles from the wild-type were also obtained with mechanism-based inhibitors, suggesting a possible role of atomic interaction with inhibitors with the {alpha}-helix H4 region including the conserved Met127.

As shown in Tables I and IIGoGo with inhibitors, significant changes from susceptibility towards resistance were noticed when each amino acid position from 125 to 129 excluding Met127 were substituted; these findings were especially true when the Ala125 or Thr126 of the wild-type PSE-4 enzyme contained either a single or double substitution (lower MICs). Analysis of the possible changes in the secondary structures as shown between PSE-4 and the YAM mutant by CD could indicate a more stable structure. Although they conferred similar susceptibility patterns to the wild-type PSE-4 when ß-lactams were used alone in MICs, several mutant enzymes obtained from both libraries could not be detected by immunoblotting (Figure 2Go). It has been shown that the decreased steady-state expression of TEM mutant ß-lactamases not detected by immunoblotting could be due to the increased turnover of the mutant enzyme (Palzkill et al., 1994Go). Although some mutant enzymes were previously less stable, the half-life was long enough to confer similar resistance to ß-lactams to the wild-type PSE-4, indicating that the mutations at positions 125–129 were responsible for the higher susceptibilities obtained in MICs towards the suicide inhibitors.

The kinetic data obtained with carbenicillin and ampicillin shown in Table IIIGo indicated that mutations at Ala125 and Thr126 did not affect the overall catalytic efficiency of the enzyme, but caused a lower catalytic turnover towards the antibiotic; especially in the case of carbenicillin. On looking at both antibiotic structures, carbenicillin has a COOH group compared with an NH2 group for ampicillin in the R1 lateral side-chains. This difference between the two antibiotics is not apparent in either the kcat or the Km values for PSE-4. The kcat value is influenced by both first-order rate constants that characterize the acylation step (k2) and the hydrolysis step (k3). The lower kcat value obtained with carbenicillin for the mutant YAM having the Ala125 Thr126 change for Tyr Ala indicated that the double mutations has affected one or both of these steps. The global change of {alpha}-helices in PSE-4 secondary structure detected by CD observed a local change in H4 region in the three-dimensional structure that could influence the lower regeneration of the enzyme towards carbenicillin.

The MICs indicated that the YAM mutant having the Ala125 Thr126 change for Tyr Ala was more sensitive towards the suicide inhibitors than wild-type PSE-4. The two different inhibition values, Ki and %I/E ratio obtained with clavulanate may be due to the differences in reaction time from which the velocity value was measured. Ki values were obtained from the initial velocities in the linear portion of the hydrolysis plot which corresponded to the first 10 s of the reaction, while the molar ratios of inhibition values were calculated using the maximum velocity value in the carbenicillin hydrolysis obtained near the 30–45 min reaction period.

Analysis of total {alpha}-helix and ß-sheet content of the wild-type PSE-4 compared with the YAM mutant showed that PSE-4 started to lose helicity at 55°C compared with 75°C for YAM. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of YAM and PSE 4. The Tyr125 of the mutant YAM formed possibly an edge-to-face {pi}{pi} interaction with Phe124 which also interacts similarly with Trp210. {pi}{pi} interactions either face-to-face or edge-to-face have been shown to stabilize protein structure and to be implicated in many biological phenomena (Burley and Petsko, 1986Go, 1988Go). In Figure 4Go, the phenyl ring of Phe124 is perfectly nested to form favorable `sandwich' aromatic–aromatic interactions that should enhance the structural stability of both {alpha}-helices H-4 and H-9. These interactions could explain the increased stability to thermal denaturation of PSE-4 YAM mutant as compared with PSE-4 wild-type enzyme seen in the CD studies. Although the CD data are interesting, it will be important to determine the stability of the enzymes on the basis of their activities. Note that the apparent Tm that can be deduced from Figure 3Go for the wild-type PSE-4 is about 60°C and is significantly higher than the TEM value determined by fluorescence, 51°C (Vanhove et al., 1995Go). Electrostatic attractions between amino acids found on H4 and H9 {alpha}-helices created a slight movement of the Ser130–Asp131–Asn132 motif within the active site. The movement of the Ser130–Asp131–Asn132 motif could alter clavulanate binding where the affinity was higher (lower Ki) and the catalytic turnover of the mutant YAM enzyme towards carbenicillin. These results obtained by modeling of the PSE-4 structure must be interpreted with caution; the PSE-4 crystal structure will forthcoming soon (N.Strynadka and R.C.Levesque, unpublished work).



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Fig. 4. Overlap of two minimized structures, mutant YAM (pink) and wild-type PSE-4 (yellow), where an edge-to-face is seen in the {alpha}-helices H4 and H9 region between Phe124, Tyr125 and Trp210 in YAM. By electrostatic attractions, this edge-to-face causes a movement in the H4 region where a spatial shift is seen for the Ser130–Asp131–Asn132 motif between YAM and PSE-4.

 
PSE-4 and other CARB enzymes have the property of being particularly active against carbenicillin, but no studies have pinpointed to this subtle substrate specificity. The differences in substrate profiles between two ß-lactamases has been shown to be caused by a single or a few amino acid changes as with third-generation cephalosporins. The data reported here indicate that PSE-4 has subtle differences from TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. We showed that the H4 {alpha}-helix possibly interacts with mechanism based-inhibitors. However, kinetic analysis showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 {alpha}-helix region than clavulanic acid, an inhibitor of the oxypenam class.


    Notes
 
4 To whom correspondence should be addressed E-mail: rclevesq{at}rsvs.ulaval.ca Back


    Acknowledgments
 
R.C.L. is a Research Scholar of Exceptional Merit from the Fonds de la Recherche en Santé du Québec. Work carried out in R.C.L.'s laboratory is funded funded by the Medical Research Council of Canada, FCAR and the Canadian Centers of Excellence via CBDN. We express our gratitude to L.Eltis, Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, for using the LEONORA software and suggestions in enzymology, and to R.Micetich and colleagues at Synphar Laboratories Inc., Edmonton, Alberta, for gifts of tazobactam.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received July 15, 1999; revised November 10, 1999; accepted January 5, 2000.