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• W2041442194 abstract "The conserved Class A β-lactamase active site residue Tyr-105 was substituted by saturation mutagenesis in TEM-1 β-lactamase from Escherichia coli in order to clarify its role in enzyme activity and in substrate stabilization and discrimination. Minimum inhibitory concentrations were calculated for E. coli cells harboring each Y105X mutant in the presence of various penicillin and cephalosporin antibiotics. We found that only aromatic residues as well as asparagine replacements conferred high in vivo survival rates for all substrates tested. At position 105, the small residues alanine and glycine provide weak substrate discrimination as evidenced by the difference in benzylpenicillin hydrolysis relative to cephalothin, two typical penicillin and cephalosporin antibiotics. Kinetic analyses of mutants of interest revealed that the Y105X replacements have a greater effect on Km than kcat, highlighting the importance of Tyr-105 in substrate recognition. Finally, by performing a short molecular dynamics study on a restricted set of Y105X mutants of TEM-1, we found that the strong aromatic bias observed at position 105 in Class A β-lactamases is primarily defined by a structural requirement, selecting planar residues that form a stabilizing wall to the active site. The adopted conformation of residue 105 prevents detrimental steric interactions with the substrate molecule in the active site cavity and provides a rationalization for the strong aromatic bias found in nature at this position among Class A β-lactamases. The conserved Class A β-lactamase active site residue Tyr-105 was substituted by saturation mutagenesis in TEM-1 β-lactamase from Escherichia coli in order to clarify its role in enzyme activity and in substrate stabilization and discrimination. Minimum inhibitory concentrations were calculated for E. coli cells harboring each Y105X mutant in the presence of various penicillin and cephalosporin antibiotics. We found that only aromatic residues as well as asparagine replacements conferred high in vivo survival rates for all substrates tested. At position 105, the small residues alanine and glycine provide weak substrate discrimination as evidenced by the difference in benzylpenicillin hydrolysis relative to cephalothin, two typical penicillin and cephalosporin antibiotics. Kinetic analyses of mutants of interest revealed that the Y105X replacements have a greater effect on Km than kcat, highlighting the importance of Tyr-105 in substrate recognition. Finally, by performing a short molecular dynamics study on a restricted set of Y105X mutants of TEM-1, we found that the strong aromatic bias observed at position 105 in Class A β-lactamases is primarily defined by a structural requirement, selecting planar residues that form a stabilizing wall to the active site. The adopted conformation of residue 105 prevents detrimental steric interactions with the substrate molecule in the active site cavity and provides a rationalization for the strong aromatic bias found in nature at this position among Class A β-lactamases. During the past decades, β-lactamase production (EC 3.5.2.6) has become a significant problem in bacterial strain resistance to widely used clinical antibiotics. Among these enzymes, the prevalent type has always been the Class A active site serine hydrolase β-lactamases, which have become model enzymes extensively studied by protein engineering with respect to site-directed or combinatorial mutagenesis (1Orencia M.C. Yoon J.S. Ness J.E. Stemmer W.P. Stevens R.C. Nat. Struct. Biol. 2001; 8: 238-242Crossref PubMed Scopus (194) Google Scholar, 2Matagne A. Fre're J.M. Biochim. Biophys. Acta. 1995; 1246: 109-127Crossref PubMed Scopus (104) Google Scholar, 3Matagne A. Lamotte-Brasseur J. Fre're J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar, 4Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar, 5Yang Y. Rasmussen B.A. Shlaes D.M. Pharmacol. Ther. 1999; 83: 141-151Crossref PubMed Scopus (91) Google Scholar), structure determination (6Minasov G. Wang X. Shoichet B.K. J. Am. Chem. Soc. 2002; 124: 5333-5340Crossref PubMed Scopus (185) Google Scholar, 7Chen C.C. Herzberg O. Biochemistry. 2001; 40: 2351-2358Crossref PubMed Scopus (36) Google Scholar, 8Strynadka N.C.J. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N.G. Nature. 1992; 359: 700-705Crossref PubMed Scopus (533) Google Scholar, 9Tranier S. Bouthors A.T. Maveyraud L. Guillet V. Sougakoff W. Samama J.P. J. Biol. Chem. 2000; 275: 28075-28082Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Shimamura T. Ibuka A. Fushinobu S. Wakagi T. Ishiguro M. Ishii Y. Matsuzawa H. J. Biol. Chem. 2002; 277: 46601-46608Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and molecular simulations (11Meroueh S.O. Roblin P. Golemi D. Maveyraud L. Vakulenko S.B. Zhang Y. Samama J.P. Mobashery S. J. Am. Chem. Soc. 2002; 124: 9422-9430Crossref PubMed Scopus (47) Google Scholar, 12Díaz N. Sordo T.L. Merz Jr., K.M. Suárez D. J. Am. Chem. Soc. 2003; 125: 672-684Crossref PubMed Scopus (48) Google Scholar, 13Castillo R. Silla E. Tuñón I. J. Am. Chem. Soc. 2002; 124: 1809-1816Crossref PubMed Scopus (31) Google Scholar). Over the years Escherichia coli TEM-1 β-lactamase has become an impressive example of the rapid evolution rate of proteins occurring within natural bacterial isolates subjected to selective pressure. Ever since the clinical introduction of β-lactam compounds and the discovery of TEM-1 β-lactamase, both in the 1940s (14Abraham E.P. Chain E. Nature. 1940; 146: 837Crossref Scopus (558) Google Scholar), natural mutations have generated a large number of single and multiple mutants of this enzyme (for an extensive list see www.lahey.org/Studies/temtable.asp). The high rate of occurrence of mutated enzymes capable of hydrolyzing higher generation cephalosporins has stimulated research of β-lactamase adaptation to these new substrates in order to understand the molecular basis of this evolutionary chain of events. Consequently, a number of studies have successfully predicted the in vitro appearance of new mutations conferring resistance before their appearance in natural isolates (for an overall view, see Ref 1Orencia M.C. Yoon J.S. Ness J.E. Stemmer W.P. Stevens R.C. Nat. Struct. Biol. 2001; 8: 238-242Crossref PubMed Scopus (194) Google Scholar). To provide more information regarding these mutations in enzyme catalysis and/or substrate stabilization, multiple mutagenesis replacements have been undertaken in Class A β-lactamases to residues in close proximity to the active site cavity that are most likely to be in direct contact with the substrate, allowing for modified catalytic parameters (2Matagne A. Fre're J.M. Biochim. Biophys. Acta. 1995; 1246: 109-127Crossref PubMed Scopus (104) Google Scholar, 3Matagne A. Lamotte-Brasseur J. Fre're J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar, 4Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar). In conjunction with the SDN loop (3Matagne A. Lamotte-Brasseur J. Fre're J.M. Biochem. J. 1998; 330: 581-598Crossref PubMed Scopus (321) Google Scholar), the Class A conserved residue Tyr-105 delineates one of the edges of the active site cavity of TEM-1 as a result of the position of its side chain near the thiazolidine ring of penicillin substrates and the dihydrothiazine ring of cephalosporin substrates. To date, two independent mutagenesis studies were performed to modify Tyr-105 to Phe and Cys on the Class A β-lactamases of Bacillus licheniformis and Bacillus cereus, respectively (15Escobar W.A. Miller J. Fink A.L. Biochem. J. 1994; 303: 555-558Crossref PubMed Scopus (14) Google Scholar, 16Di Gleria K. Halliwell C.M. Jacob C. Hill H.A. FEBS Lett. 1997; 400: 155-157Crossref PubMed Scopus (28) Google Scholar). The B. licheniformis Y105F mutant displayed a 52% catalytic efficiency toward benzylpenicillin compared with its corresponding wild-type enzyme. Based on these results, the Y105F mutation rules out a critical role of the hydroxyl group of the wild-type Tyr residue toward enzyme activity or stability. Moreover, the B. cereus Y105C mutant displayed native-like catalytic activity under standard nitrocefin assay conditions, demonstrating that the aromatic moiety at this position is not essential for enzyme activity or stability. On the other hand, the location and the strong conservation of aromatic character of this residue among Class A β-lactamases suggest that residue 105 may play a role in substrate recognition and/or stabilization. This hypothesis is based on crystallographic data of many Class A enzymes showing that the Tyr-105 side chain is significantly displaced upon binding of substrates or mechanism-based inhibitors as a result of a flipping motion (7Chen C.C. Herzberg O. Biochemistry. 2001; 40: 2351-2358Crossref PubMed Scopus (36) Google Scholar, 17Chen C.C. Rahil J. Pratt R.F. Herzberg O. J. Mol. Biol. 1993; 234: 165-178Crossref PubMed Scopus (92) Google Scholar, 18Guo F. Huynh J. Dmitrienko G.I. Viswanatha T. Clarke A.J. Biochim. Biophys. Acta. 1999; 1431: 132-147Crossref PubMed Scopus (7) Google Scholar, 19Wang X. Minasov G. Blázquez J. Caselli E. Prati F. Shoichet B.K. Biochemistry. 2003; 42: 8434-8444Crossref PubMed Scopus (45) Google Scholar, 20Maveyraud L. Mourey L. Kotra L.P. Pedelacq J.D. Guillet V. Mobashery S. Samama J.P. J. Am. Chem. Soc. 1998; 120: 9748-9752Crossref Scopus (129) Google Scholar, 21Strynadka N.C.J. Martin R. Jensen S.E. Gold M. Jones J.B. Nat. Struct. Biol. 1996; 3: 688-695Crossref PubMed Scopus (99) Google Scholar). In fact, it has been suggested that the Tyr-105 side chain may “stack” with the thiazolidine ring of penicillins (21Strynadka N.C.J. Martin R. Jensen S.E. Gold M. Jones J.B. Nat. Struct. Biol. 1996; 3: 688-695Crossref PubMed Scopus (99) Google Scholar, 22Strynadka N.C.J. Jensen S.E. Alzari P.M. James M.N.G. Nat. Struct. Biol. 1996; 3: 290-297Crossref PubMed Scopus (127) Google Scholar) as well as form van der Waals and hydrophobic interactions with the benzyl side chains of substrates (7Chen C.C. Herzberg O. Biochemistry. 2001; 40: 2351-2358Crossref PubMed Scopus (36) Google Scholar, 12Díaz N. Sordo T.L. Merz Jr., K.M. Suárez D. J. Am. Chem. Soc. 2003; 125: 672-684Crossref PubMed Scopus (48) Google Scholar) or inhibitors (18Guo F. Huynh J. Dmitrienko G.I. Viswanatha T. Clarke A.J. Biochim. Biophys. Acta. 1999; 1431: 132-147Crossref PubMed Scopus (7) Google Scholar, 19Wang X. Minasov G. Blázquez J. Caselli E. Prati F. Shoichet B.K. Biochemistry. 2003; 42: 8434-8444Crossref PubMed Scopus (45) Google Scholar), suggestive of its active participation in substrate and inhibitor positioning at the active site. As a result of this characteristic, residue 105 has been considered a determinant of susceptibility to mechanism-based inhibitors (23Page M.G.F. Drug Resist. Updat. 2000; 3: 109-125Crossref PubMed Scopus (70) Google Scholar). To our knowledge, apart from the Y105F and Y105C mutations (15Escobar W.A. Miller J. Fink A.L. Biochem. J. 1994; 303: 555-558Crossref PubMed Scopus (14) Google Scholar, 16Di Gleria K. Halliwell C.M. Jacob C. Hill H.A. FEBS Lett. 1997; 400: 155-157Crossref PubMed Scopus (28) Google Scholar), no further site-directed mutagenesis studies have been undertaken specifically at this critical active site position. Huang et al. (24Huang W.Z. Petrosino J. Hirsch M. Shenkin P.S. Palzkill T. J. Mol. Biol. 1996; 258: 688-703Crossref PubMed Scopus (158) Google Scholar) previously reported a three-codon-based combinatorial mutagenesis for the entire gene of TEM-1 but conducted no detailed analysis of residue 105 with respect to substrate recognition. Nevertheless, their results suggest that only the Y105H mutant is able to confer wild-type activity for ampicillin upon selecting for survival in the presence of 1 mg/ml antibiotic. However, these Y105X mutations were performed only with other simultaneous mutations at positions 103 and 104. Although the high activities observed with the Y105F and Y105C mutants suggest that hydrophobicity may be a determining factor at this active-site position, the effects of variables such as side-chain volume, polarity, and flexibility at this position have not been specifically addressed. Thus, the importance of sequence conservation at position 105 is unclear, and the role of residue 105 has yet to be elucidated in detail with respect to enzyme stability and substrate stabilization and discrimination for Class A β-lactamases. Consequently, to clarify the importance of residue 105 in TEM-1 β-lactamase as well as its potential role in other Class A β-lactamases, we performed saturation mutagenesis at position 105 on TEM-1. In vivo antibiotic susceptibility tests and in vitro kinetic studies were carried out using penicillin as well as first- and third-generation cephalosporin substrates to assess the impact of the mutations on substrate recognition and enzyme catalysis. Finally, molecular modeling studies of mutants of interest were undertaken to evaluate the structural importance of residue 105 in substrate stabilization. Our results identify residue 105 as a weak substrate determinant of TEM-1 β-lactamase. Reagents—Restriction and DNA-modifying enzymes were purchased from MBI Fermentas (Burlington, ON) and New England Biolabs, Ltd. (Mississauga, ON). Ampicillin was obtained from BioShop Canada, Inc. (Burlington, ON), and benzylpenicillin, cephalothin, cefazolin, cefotaxime, and Fast-Flow DEAE-Sepharose were purchased from Sigma-Aldrich. Nitrocefin was purchased from Calbiochem. Bacterial Strains and Plasmids—E. coli XL1-Blue (supE44, hsdR17, recA1, endA1, gyrA46, thi, relA1, lac F′ [proAB+, lacIq, lacZΔM15, Tn10(tetr)]) was used for the propagation and expression of all plasmids. Plasmid pQE32Chl in which the blaTEM-1 gene of pQE32 (Qiagen, Mississauga, ON) was replaced by a chloramphenicol acetyltransferase gene was a generous gift from François-Xavier Campbell-Valois and Stephen W. Michnick (Département de Biochimie, Université de Montréal, QC) and was used for protein expression. It was maintained using 12.5 μg/ml chloramphenicol (Chl). 1The abbreviations used are: Chl, chloramphenicol; AMP, ampicillin; BZ, benzylpenicillin; CF, cephalothin; CZ, cefazolin; MIC, minimum inhibitory concentration. Plasmid pBR322, which contains the wild-type blaTEM-1 gene (without the V84I and A184V mutations) was kindly provided by Luis A. Rokeach (Département de Biochimie, Université de Montréal, QC). Oligonucleotides and Saturation Mutagenesis—Oligonucleotide primers used for mutagenesis were synthesized by Alpha DNA (Montréal, QC) and Integrated DNA Technologies (Coralville, IA). Oligonucleotide primers used for DNA sequencing were synthesized by Li-Cor Biotechnology (Lincoln, NB). The Y105X mutants of TEM-1 were constructed using the site overlap extension mutagenesis method (25Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). The 861-bp blaTEM-1 gene was PCR-amplified from plasmid pBR322 using the terminal oligonucleotides BamHITEMF (5′-CACACAGGATCCACATGAGTATTCAACATTTCCGT-3′) and TEMHindIIIR (5′-ACACACAAGCTTTTACCAATGCTTAATCAGTGA-3′) containing the BamHI and HindIII restriction sites (underlined), respectively. The 19-amino acid possibilities at codon 105 were introduced by a set of three complementary pairs of degenerate oligonucleotides (only the coding strands are shown): TEM105NTSF (5′-ATGACTTGGTTGAGNTSTCACCAGTCACAG-3′), TEM105NGSF (5′-ATGACTTGGTTGAGNGSTCACCAGTCACAG-3′), and VMS105F (5′-ATGACTTGGTTGAGVMSTCACCAGTCACAG-3′). The use of three separate degenerate oligonucleotides encoding the possibilities NTS, NGS, and VMS instead of a single NNS codon was justified by the elimination of the wild-type tyrosine residue as well as the three stop codons. The recombinant TEM genes were digested with BamHI/HindIII and cloned into BamHI/HindIII-digested and calf intestinal alkaline phosphatase-treated pQE32Chl before electroporation into E. coli XL1-Blue cells. Colonies were individually picked after selection on a Luria-Bertani (LB) medium containing 12.5 μg/ml Chl, and the sequence of each mutant was confirmed by the dideoxy chain termination method with the Thermo Sequenase Cycle Sequencing kit (Upstate Biotechnology Corp., Cleveland, OH) using a dye-labeled primer and a Li-Cor automated sequencer (Lincoln, NB). Expression and Purification of Mutant β-Lactamases—An overnight culture of each XL1-Blue/pQE32Chl-TEM(Y105X) clone was used to inoculate 50 ml of LB that was grown with agitation at 37 °C until A600 nm = 0.6. After the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside, the cultures were propagated for an additional 3 h. After induction, the cells were pelleted by centrifugation (30 min, 3000 × g, 4 °C), resuspended in 10 ml of 10 mm Tris-Cl buffer, pH 7.0, and separated in 1-ml aliquots. A gentle lysis of the outer membrane of E. coli was performed by 3–4 1.5-min freeze-thaw cycles using a dryice/ethanol and a 37 °C water bath followed by a centrifugation (15 min, 20,000 × g, 4 °C) to collect the supernatant. Purification of TEM(Y105X) mutants of interest was performed according to the following single-step anion-exchange chromatography procedure. All steps were undertaken at 4 °C with a flow rate of 1 ml/min on a System Gold high performance liquid chromatography apparatus (Beckman Coulter Canada, Inc., Mississauga, ON). Supernatant (10 ml) was applied to a DEAE-Sepharose column (2 × 25 cm) followed by a wash of 2.5 column volumes with 10 mm Tris-Cl, pH 7.0, buffer. Mutant enzymes were eluted with a linear gradient of 10–150 mm Tris-Cl, pH 7.0, buffer (1.5 column volume), and a subsequent wash with the same buffer was finally performed (2.5 column volumes). Fractions containing β-lactamase activity were identified by a qualitative nitrocefin hydrolysis test and pooled for subsequent analysis. Aliquots of each clone were analyzed by SDS-PAGE gel, and the purity was estimated in all cases to be between 80 and 90% using the public domain image analysis software Scion Image (National Institutes of Health, rsb.info.nih.gov/nih-image). The column was regenerated by applying 5 volumes of 8 m urea followed by 5 volumes of 10 mm Tris-Cl, pH 7.0, buffer between each purification. No significant β-lactamase activity carryover was detected by nitrocefin assay upon running a mock purification (cells expressing no TEM-1 β-lactamase) following a purification of the native TEM-1. Antibiotic Susceptibility—Minimum inhibitory concentrations (MICs) were determined by broth microdilutions according to Cantu et al. (26Cantu III, C. Huang W. Palzkill T. J. Biol. Chem. 1996; 271: 22538-22545Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The ranges of antibiotic concentrations tested were as follows (by stepwise 2-fold increases): 125–4000 μg/ml for benzylpenicillin and ampicillin, 4–500 μg/ml for cephalothin, 1–125 μg/ml for cefazolin, and 4–500 ng/ml for cefotaxime. Each MIC determination was performed at least in triplicate in independent experiments. Enzyme Kinetics—The kinetic constants Km and kcat for benzylpenicillin and cephalothin were determined at room temperature in 50 mm sodium phosphate buffer, pH 7.0, for the mutants presented in Table III. The following extinction coefficients (27Bouthors A.T. Dagoneau-Blanchard N. Naas T. Nordmann P. Jarlier V. Sougakoff W. Biochem. J. 1998; 330: 1443-1449Crossref PubMed Scopus (33) Google Scholar) and concentration ranges were used: Δϵ232 nm = 1100 m-1 cm-1 for benzylpenicillin (50–200 μm) and Δϵ262 nm = 7960 m-1 cm-1 for cephalothin (30–300 μm). Substrate hydrolysis was monitored according to initial steady-state velocities for a minimum of six substrate concentrations generally flanking the Km values (when the molar extinction coefficients and the Km value allowed it) using a Cary 100 Bio UV-visible spectrophotometer (Varian Canada, Inc., Montréal, QC). For each assay the concentration of enzyme was kept at least 500 times lower than substrate for benzylpenicillin (BZ) and was generally 50 times lower for cephalothin (CF). The kinetic parameters were determined from the rates of hydrolysis calculated from the initial linear portion of the curve and fitted to a Lineweaver-Burk (1/[S] versus 1/[V]) and a Hanes ([S] versus [S]/V) plot. In most cases initial rates were also analyzed with the software Graphpad Prism (Graphpad Software, San Diego, CA) by a non-linear regression curve corresponding to the Michaelis-Menten equation. The kcat parameter was determined using the equation kcat = Vmax/[E], where the concentration of enzyme was determined by a Bio-Rad protein assay kit (Bio-Rad) taking into account its purity, estimated as described above.Table IIIKinetic parameters for wild-type TEM-1 β-lactamase and Y105X mutant derivativesSubstrateTEM-1 variantkcatkcat relative to wild typeKmKm relative to wild typekcat/Kmkcat/Km relative to wild types-1μmm-1 s-1BZWild type1240 ± 1251.0043 ± 91.002.9 × 1071.00Y105D255 ± 200.21369 ± 238.586.9 × 1050.02Y105G1203 ± 3500.97152 ± 613.537.9 × 1060.27Y105N1616 ± 3391.30276 ± 846.425.9 × 1060.20Y105R525 ± 700.42156 ± 383.623.4 × 1060.12Y105W900 ± 780.7323 ± 90.533.9 × 1071.34CFWild type105 ± 111.00177 ± 271.005.9 × 1051.00Y105D51 ± 110.492860 ± 54016.21.8 × 1040.03Y105G45 ± 180.431630 ± 7509.212.8 × 1040.05Y105N61 ± 170.58303 ± 971.712.0 × 1050.34Y105R90 ± 280.86546 ± 343.081.6 × 1050.27Y105W65 ± 80.6274 ± 220.428.8 × 1051.49 Open table in a new tab Computer Modeling—All computations were performed with the InsightII package, version 2000 (Accelrys, San Diego, CA). The BIOPOLYMER module was used to modify molecular structures, and all energy minimizations and molecular dynamics calculations were performed with the DISCOVER module using the consistent valence force field. The dynamic trajectories were analyzed using the DECIPHER module. We performed the simulations with the coordinates of an acyl-enzyme intermediate of TEM-1 complexed with benzylpenicillin (BZ), and we created an acyl-enzyme model of TEM-1 complexed with CF to compare the behavior of this substrate with that observed for BZ over the course of a 200-ps dynamics. We also conducted molecular dynamics studies using the coordinates of an apoenzyme structure of TEM-1 with residue 105 mutated, to assess conformation before substrate binding. Comparison of these systems (BZ acyl-enzyme, CF acylenzyme, and apoenzyme) allowed for the evaluation of the conformation of residue 105 before and after substrate recognition. The 1.8-Å crystallographic structure of the E. coli TEM-1 β-lactamase (Protein Data Bank, Brookhaven National Laboratory, code 1BTL) (28Jelsch C. Mourey L. Masson J.M. Samama J.P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar) was used for the starting coordinates for apoenzyme calculations, and the 1.7-Å crystallographic structure of a E166N deacylation-defective mutant of the same enzyme (PDB code 1FQG) (8Strynadka N.C.J. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N.G. Nature. 1992; 359: 700-705Crossref PubMed Scopus (533) Google Scholar) was used for the starting coordinates for calculations involving benzylpenicillin-bound enzyme. The crystallographic water molecules of both enzymes were conserved. The active site SO4 molecule was deleted from 1BTL, and the E166N mutation of 1FQG was reverted to wild type. Hydrogen atoms were added at the normal ionization state of the amino acids at pH 7.0. For 1FQG, the atomic potentials of the BZ substrate were fixed according to the consistent valence force field atom types recommended by the manufacturer. These coordinates served as the starting points for all the subsequent calculations in presence (1FQG) or absence (1BTL) of BZ. The Y105G, Y105L, Y105N, Y105Q, Y105R, and Y105W mutations were introduced. Before minimization, the tryptophan side chain was repositioned according to the crystal structure of the structurally homologous Class A PER-1 β-lactamase (PDB code 1E25) (9Tranier S. Bouthors A.T. Maveyraud L. Guillet V. Sougakoff W. Samama J.P. J. Biol. Chem. 2000; 275: 28075-28082Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Residues contained in a simulation area within 15 Å from any atom of BZ (1FQG) or residue 105 (1BTL) were allowed to move as well as the loop encompassing residues 96–116, with the remainder of the protein being fixed. A layer of 5 Å of explicit water was added to the surface of this assembly, and a nonbonded cutoff of 20 Å was fixed to reduce the time of calculation. Each structure was energy-minimized by applying 100 steps of steepest descents followed by a conjugate gradient minimization until convergence of 0.001 kcal mol-1 Å-1. A short molecular dynamics simulation was performed starting from the energy-minimized structures. Accordingly, the molecular system was allowed to equilibrate at 310 K for 100 fs followed by the actual simulation to explore conformational space for 200 ps at the same temperature (time step = 1 fs). Snapshots were taken each picosecond, generating 201 different conformers. At this point the trajectories obtained for each mutant were analyzed. To create the acyl-enzyme intermediate models of TEM-1 with cephalothin (TEM-CF and Y105G-CF), the atomic coordinates of CF were taken from the crystal structure of an acyl-enzyme intermediate of TOHO-1 β-lactamase (PDB code 1IYP) (10Shimamura T. Ibuka A. Fushinobu S. Wakagi T. Ishiguro M. Ishii Y. Matsuzawa H. J. Biol. Chem. 2002; 277: 46601-46608Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and fitted to the 1.8-Å crystallographic structure of the E. coli TEM-1 β-lactamase (PDB code 1BTL). The active site SO4 molecule was deleted from 1BTL, and the CF substrate was positioned according to BZ in 1FQG by applying torsions to the Cβ-Oγ bond of Ser-70. Hydrogen atoms were adjusted to pH 7, and the atomic potentials of CF were adjusted as described herein. An energy minimization was performed on this structure by applying 100 steps of steepest descents followed by a conjugate gradient minimization until convergence of 0.001 kcal mol-1Å-1. This structure served as the starting coordinate for all subsequent steps of minimization and dynamics studies, which were performed as described above. Antibiotic Susceptibility—We mutated position 105 (Tyr) of TEM-1 β-lactamase to the 19 other possibilities and confirmed through DNA sequencing of the entire gene that no secondary mutations had occurred. To assess the effect of the Y105X replacements on the capacity of TEM-1 to hydrolyze penicillin-type and cephalosporin antibiotics, MICs were calculated for E. coli XL1-Blue cells alone or expressing TEM-1 mutants. The antibiotics used are presented in Fig. 1. Table I presents MIC values determined for all mutants toward two classical penicillin-type substrates, BZ and ampicillin (AMP) as well as two typical first-generation cephalosporins, cephalothin (CF) and cefazolin (CZ). Thus, in vivo cell survival at higher antibiotic concentrations reflects a higher rate of hydrolysis by the enzyme. Because they are performed in vivo, MIC values serve as points of comparison rather than precise values and do not directly correlate with the Km or kcat parameters of each mutant enzyme. They nevertheless offer a rapid, qualitative assessment of the efficiency of mutants and allow for the identification of mutants deemed to be the most interesting with respect to detailed kinetic analysis.Table IMICs of E. coli XL1-Blue cells expressing TEM-1 β-lactamase with Tyr-105 replacements Open table in a new tab As seen in Table I, the range of resistance is higher for the penicillin substrates (500–4000 μg/ml for BZ and 500–7500 μg/ml for AMP) than for the cephalosporin substrates (16–125 μg/ml for CF and 4–250 μg/ml for CZ). This result was expected since Class A β-lactamases hydrolyze penicillins much more efficiently than cephalosporins, the latter having been historically developed to counteract the appearance of natural resistance to penicillins (29Bush K. Mobashery S. Adv. Exp. Med. Biol. 1998; 456: 71-98Crossref PubMed Scopus (109) Google Scholar, 30Page M.I. Adv. Phys. Org. Chem. 1987; 23: 165-270Google Scholar). Nonetheless, the 20 mutants allow for a comparable breadth of resistance with respect to these four substrates. Thus, the ratio between the least and the most active mutants for BZ hydrolysis is approximately one order of magnitude (500 μg/ml relative to 4000 μg/ml), similar to that observed for CF hydrolysis (16 μg/ml relative to 125 μg/ml). AMP and CZ allow for slightly broader ranges but remain in the same range of resistance as BZ and CF, respectively. This breadth of resistance illustrates that position 105 replacements can alter enzyme efficiency up to a factor of 10-fold. Nonetheless, this relatively weak effect confirms previous observations suggesting that position 105 cannot be considered essential for enzyme catalysis in Class A β-lactamases (15Escobar W.A. Miller J. Fink A.L. Biochem. J. 1994; 303: 555-558Crossref PubMed Scopus (14) Google Scholar, 31Wolozin B.L. Myerowitz R. Pratt R.F. Biochim. Biophys. Acta. 1982; 701: 153-163Crossref PubMed Scopus (5) Google Scholar). To understand the source of the effect, we examined the following hypotheses; (a) disparity in enzyme solubility conferred by the Y105X replacements, (b) changes in periplasmic localization efficiency of some Y105X mutants, (c) an active site cavity disruption modifying substrate recognition or catalytic turnover. We verified that the mutations at position 105 have little effe" @default.
• W2041442194 created "2016-06-24" @default.
• W2041442194 creator A5024277895 @default.
• W2041442194 creator A5027368726 @default.
• W2041442194 creator A5077525668 @default.
• W2041442194 date "2004-10-01" @default.
• W2041442194 modified "2023-10-03" @default.
• W2041442194 title "Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase" @default.
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