Insertion Mutagenesis as a Tool in the Modification of Protein Function

The TEM-1 β-lactamase enzyme efficiently hydrolyzes β-lactam antibiotics such as ampicillin but cleaves third generation cephalosporin antibiotics poorly. Variant β-lactamases that conferred elevated levels of resistance to the cephalosporin ceftazidime were identified in a set of β-lactamase derivatives previously generated by pentapeptide scanning mutagenesis in which a variable 5-amino acid cassette was introduced randomly in the target protein. This mutagenesis procedure was also modified to allow the direct selection of variant β-lactamases with pentapeptide insertions that conferred extended substrate specificities. All insertions associated with enhanced resistance to ceftazidime were targetted to the 19-amino acid Ω-loop region, which forms part of the catalytic pocket of the β-lactamase enzyme. However, pentapeptide insertions in the C- and N-terminal halves of this region had different effects on the ability of the enzyme to hydrolyze ampicillin in vivo. Larger insertions that increased the length of the Ω-loop by up to 2-fold also retained catalytic activity toward ampicillin and/or ceftazidime in vivo. In accord with previous substitution mutation studies, these results emphasize the extreme flexibility of the Ω-loop with regards the primary structure requirements for ceftazidime hydrolysis by β-lactamase. The potential of pentapeptide scanning mutagenesis in mimicking evolution events that result from the insertion and excision of transposons in nature is discussed. The TEM-1 β-lactamase enzyme efficiently hydrolyzes β-lactam antibiotics such as ampicillin but cleaves third generation cephalosporin antibiotics poorly. Variant β-lactamases that conferred elevated levels of resistance to the cephalosporin ceftazidime were identified in a set of β-lactamase derivatives previously generated by pentapeptide scanning mutagenesis in which a variable 5-amino acid cassette was introduced randomly in the target protein. This mutagenesis procedure was also modified to allow the direct selection of variant β-lactamases with pentapeptide insertions that conferred extended substrate specificities. All insertions associated with enhanced resistance to ceftazidime were targetted to the 19-amino acid Ω-loop region, which forms part of the catalytic pocket of the β-lactamase enzyme. However, pentapeptide insertions in the C- and N-terminal halves of this region had different effects on the ability of the enzyme to hydrolyze ampicillin in vivo. Larger insertions that increased the length of the Ω-loop by up to 2-fold also retained catalytic activity toward ampicillin and/or ceftazidime in vivo. In accord with previous substitution mutation studies, these results emphasize the extreme flexibility of the Ω-loop with regards the primary structure requirements for ceftazidime hydrolysis by β-lactamase. The potential of pentapeptide scanning mutagenesis in mimicking evolution events that result from the insertion and excision of transposons in nature is discussed. Even prior to the clinical introduction of the first β-lactam antibiotic, penicillin, infectious bacteria resistant to the activity of this antibiotic were identified (1Frère J.-M. Mol. Microbiol. 1995; 16: 385-395Crossref PubMed Scopus (268) Google Scholar). This resistance subsequently proved to be due to the expression in these bacteria of one of four classes (A, B, C, and D) of β-lactamase enzyme that efficiently hydrolyzes the antibiotic substrate (2Ambler R.P. Phil. Trans. R. Soc. Lond. Ser. B. 1980; 289: 321-331Crossref PubMed Scopus (1430) Google Scholar). Furthermore, the introduction of later generation β-lactam antibiotics was followed inexorably by the appearance of resistant strains expressing mutated β-lactamase enzymes with correspondingly altered substrate specificities (1Frère J.-M. Mol. Microbiol. 1995; 16: 385-395Crossref PubMed Scopus (268) Google Scholar, 3Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar). To date over 50 naturally occurring class A TEM β-lactamase variants have been isolated. 1G. Jacoby and K. Bush, World Wide Web site,http://www.lahey.hitchcock.org/pages/lhc/studies/webt.htm. Among the variant β-lactamases that have emerged are derivatives that cleave third generation cephalosporin antibiotics, e.g.ceftazidime, which the wild-type protein recognizes poorly (3Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar). Natural mutations that give rise to these variants are confined to amino acids at positions 104, 164, and 237–240, which are close to or which form part of the catalytic pocket of TEM-1 β-lactamase (5Jacoby G.A. Medeiros A.A. Antimicrob. Agents Chemother. 1991; 35: 1697-1704Crossref PubMed Scopus (652) Google Scholar, 6Jelsch C. Mourey L. Masson J.-M. Samama J.-P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar, 7Strynadka 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). However, artificial substitution mutations at many positions in the Ω-loop (residues 161–179) also alter the specificity of the enzyme (8Delaire M. Lenfant F. Labia R. Masson J.-M. Protein Eng. 1991; 4: 805-810Crossref PubMed Scopus (47) Google Scholar, 9Guillaume G. Vanhove M. Lamotte-Brasseur J. Ledent P. Jamin M. Joris B. Frère J.-M. J. Biol. Chem. 1997; 272: 5438-5444Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 10Palzkill T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google Scholar, 11Petrosino J.F. Palzkill T. J. Bacteriol. 1996; 178: 1821-1828Crossref PubMed Google Scholar). Ω-Loop substitutions appear to increase the conformational flexibility of the catalytic region, thereby allowing access of the bulkier side chain of third generation cephalosporin substrates (9Guillaume G. Vanhove M. Lamotte-Brasseur J. Ledent P. Jamin M. Joris B. Frère J.-M. J. Biol. Chem. 1997; 272: 5438-5444Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 10Palzkill T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google Scholar, 11Petrosino J.F. Palzkill T. J. Bacteriol. 1996; 178: 1821-1828Crossref PubMed Google Scholar, 12Maveyraud L. Saves I. Burlet-Schiltz O. Swarén P. Masson J.-M. Delaire M. Mourey L. Promé J.-C. Samama J.-P. J. Biol. Chem. 1996; 271: 10482-10489Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 13Raquet X. Lamotte-Brasseur J. Fonze E. Goussard S. Courvalin P. Frère J.-M. J. Mol. Biol. 1994; 244: 625-639Crossref PubMed Scopus (124) Google Scholar). Pentapeptide scanning mutagenesis is a method by which a variable five amino acid cassette is introduced at random into a target protein (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar,15Cao Y. Hallet B. Sherratt D.J. Hayes F. J. Mol. Biol. 1997; 274: 39-53Crossref PubMed Scopus (26) Google Scholar). In this study, pentapeptide insertions were constructed in the active pocket Ω-loop of the TEM β-lactamase, which extended the activity spectrum of the enzyme to include the third generation cephalosporin, ceftazidime. The extreme tolerance of the Ω-loop to alteration was emphasized further by the construction of β-lactamase derivatives in which the length of this region was increased up to 2-fold but which retained catalytic activity toward ceftazidime and/or ampicillin in vivo. Escherichia colicultures were grown in L medium at 37 °C unless otherwise stated. The following antibiotics were added to L medium when required (Sigma): 10–5500 μg/ml ampicillin, 0.075–1.5 μg/ml ceftazidime, 50 μg/ml kanamycin, 50 μg/ml streptomycin, and 5 μg/ml tetracycline. Enzymes were obtained from New England Biolabs or Life Technologies, Inc.. Transposon Tn4430Ω5 is a derivative of Tn4430 that encodes kanamycin resistance (16Mahillon J. Lereclus D. EMBO J. 1988; 7: 1515-1526Crossref PubMed Scopus (83) Google Scholar). Plasmid pHT385 (16Mahillon J. Lereclus D. EMBO J. 1988; 7: 1515-1526Crossref PubMed Scopus (83) Google Scholar) was used as a source of Tn4430Ω5. The bla gene on plasmid pBR322 (17Bolivar F. Rodriguez R.L. Greene P.J. Betlach M.C. Heynecker H.C. Boyer H.W. Crosa J.H. Falkow S. Gene (Amst.). 1977; 2: 95-113Crossref PubMed Scopus (3527) Google Scholar) was used as the target for Tn4430Ω5 mutagenesis. E. coli FH1046A, which was obtained by cotransformation of pHT385 and pBR322 into strain XL1-Blue (18Bullock W.O. Fernandez J.M. Short J.M. BioTechniques. 1987; 5: 376-378Google Scholar), and the streptomycin-resistant strain DS941 (19Summers D.K. Sherratt D.J. EMBO J. 1988; 7: 851-858Crossref PubMed Scopus (106) Google Scholar) were used as donor and recipient, respectively, in conjugation trials. Strain DH5α (20Woodcock D.M. Crowther P.J. Doherty J. Jefferson S. Decruz E. Noyer-Weidner M. Smith S.S. Michael M.Z. Graham M.W. Nucleic Acids Res. 1989; 17: 3469-3478Crossref PubMed Scopus (640) Google Scholar) was used for plasmid propagation and in cloning experiments. Plasmid DNA was prepared and manipulated essentially as described by Sambrook et al. (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The maximum antibiotic concentrations allowing growth were determined by making 104 dilutions of overnight cultures grown in L broth containing tetracycline at 30 °C and spotting 2-μl volumes of these dilutions on L plates containing the appropriate concentration of ampicillin or ceftazidime. Results were read after incubation at 30 °C for 48 h. Nucleotide sequencing of insertion sites in pBR322::15-bp 2The abbreviation used is: bp, base pair(s).constructs was performed with a set of custom-madebla-specific primers using dye terminator chemistry after which samples were run on an ABI PRISM 377 DNA sequencer (Applied Biosystems, Warrington, Cheshire, UK). Pentapeptide scanning mutagenesis is a technique whereby 5-amino acid insertions are introduced at random in a target protein (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar). Briefly, a donor strain containing the target plasmid and pHT385, a conjugative Tn4430Ω5 delivery vector, is mated with a plasmid-free recipient strain. By plating the mating mix simultaneously on antibiotics selecting for the recipient, the target plasmid, and Tn4430Ω5, transconjugants containing pHT385::target plasmid cointegrates are isolated. This cointegrate resolves rapidly in vivo, regenerating pHT385 and the target plasmid into which a copy of Tn4430Ω5 has been inserted. Tn4430Ω5 contains KpnI sites located 5 bp from both ends of the transposon and duplicates 5 bp of target site sequence during transposition. By digesting the target with KpnI and the the of the transposon is to a target plasmid derivative containing a the insertion is in a this result in a 5-amino acid insertion in the target protein. conjugation of donor and recipient grown on were in of L and volumes of these were on an L incubation for at 37 the conjugation mix was in of L and volumes were on L plates containing streptomycin, and kanamycin to for transconjugants that following overnight incubation at 37 The levels of variant β-lactamase in cultures were by of grown at 30 °C with tetracycline selection were prepared as described by Palzkill et al. T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google Scholar). in the were by on a at for at in the were to by and β-lactamase protein was by with β-lactamase a to and were with a and was using Pentapeptide insertions in the β-lactamase protein are by the amino acid to the N-terminal side of the insertion followed by the sequence of the insertion acid to that R.P. Frère J.-M. J.-M. Joris B. M. G. J. 1991; PubMed Scopus Google Scholar). mutations of the resistance spectrum of the TEM-1 β-lactamase enzyme to include third generation which wild-type β-lactamase hydrolyzes poorly T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google Scholar, 11Petrosino J.F. Palzkill T. J. Bacteriol. 1996; 178: 1821-1828Crossref PubMed Google Scholar, 13Raquet X. Lamotte-Brasseur J. Fonze E. Goussard S. Courvalin P. Frère J.-M. J. Mol. Biol. 1994; 244: 625-639Crossref PubMed Scopus (124) Google Scholar, S. K. 1991; PubMed Scopus Google Scholar). the pentapeptide scanning mutagenesis a set of pentapeptide insertions was constructed previously in the β-lactamase protein by the bla gene of pBR322 (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar). By the maximum concentrations that growth at 30 °C on the cephalosporin antibiotic ceftazidime, the of these insertions on β-lactamase substrate specificity was The wild-type β-lactamase protein by pBR322 conferred resistance to μg/ml of ceftazidime the used in this The and variants had elevated ceftazidime resistance levels of and levels by these mutated β-lactamase were at expressing β-lactamases with pentapeptide insertions at were as to ceftazidime at as was a strain containing the wild-type protein. variants conferred different levels of ampicillin resistance (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar). the insertions that in increased resistance to ceftazidime ampicillin resistance B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google and and are located one amino acid in the active site Ω-loop This result in to substitution insertion mutations in the Ω-loop alter the resistance spectrum of the β-lactamase levels in of TEM-1 β-lactamase derivatives containing pentapeptide insertions in the and concentration allowing growth at 30 to to containing or 19-amino acid insertions at this had antibiotic as variants with pentapeptide containing or 19-amino acid insertions at this had antibiotic as variants with pentapeptide containing or 19-amino acid insertions at this had antibiotic as variants with pentapeptide containing or 19-amino acid insertions at this had antibiotic as variants with pentapeptide in a The of pentapeptide scanning mutagenesis in β-lactamase derivatives with altered substrate was further by the mutagenesis procedure to the direct selection of variant enzymes that conferred elevated levels of resistance to ceftazidime. the of positions at which mutations β-lactamase substrate specificity be (3Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar), a of containing a of insertions in bla was generated as Plasmid pBR322 was into a strain containing the Tn4430Ω5 donor plasmid, from this were into of L and dilutions of this were used as donor in mating with the streptomycin-resistant recipient, in which a cointegrate was to DS941 were by plating the mating mix on streptomycin, kanamycin, and tetracycline. containing were and plasmid DNA was from a of these of the cointegrate is in B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar, Y. Hallet B. Sherratt D.J. Hayes F. J. Mol. Biol. 1997; 274: 39-53Crossref PubMed Scopus (26) Google Scholar, J. Lereclus D. EMBO J. 1988; 7: 1515-1526Crossref PubMed Scopus (83) Google Scholar), this plasmid of pBR322 derivatives containing Tn4430Ω5 has been these Tn4430Ω5 insertions are randomly in the target plasmid (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar, Y. Hallet B. Sherratt D.J. Hayes F. J. Mol. Biol. 1997; 274: 39-53Crossref PubMed Scopus (26) Google Scholar). The plasmid was with KpnI and to a of pBR322 derivatives in which the of Tn4430Ω5 was but in which insertions in bla in elevated levels of resistance to ceftazidime were from this by the into strain DH5α and selecting at 30 °C on tetracycline and also on ceftazidime at a concentration of This of ceftazidime is the of resistance conferred by the wild-type β-lactamase protein this procedure plasmid DNA generated from a of were in of 30 experiments. from of the was further All of the pentapeptide insertions in these derivatives were targetted to the active site Ω-loop of the β-lactamase protein. insertions the Ω-loop were identified the variant β-lactamase at different positions the in β-lactamase derivatives with different pentapeptide insertions at both and The of mutations and described in the that were from the of pentapeptide insertions previously constructed in the β-lactamase protein (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar) in the direct selection trials. The resistance levels conferred by the variant β-lactamase enzymes were determined on both ampicillin and ceftazidime resistance levels the variants from to the of resistance associated with the wild-type protein. resistance levels are with conferred by substitution mutations in the Ω-loop T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google Scholar, 11Petrosino J.F. Palzkill T. J. Bacteriol. 1996; 178: 1821-1828Crossref PubMed Google Scholar). in the N-terminal of the Ω-loop activity toward In four insertions in the of this region retained different but levels of ampicillin results that insertions at positions in the Ω-loop the activity of the β-lactamase protein to include a cephalosporin In the results also insertions in the Ω-loop the ability to hydrolyze ampicillin, insertions in the of the have a insertions in the N-terminal All insertions generated by pentapeptide scanning mutagenesis include a KpnI site (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar). The on the β-lactamase substrate of Ω-loop insertions five amino acids was by a into site in the bla the and mutated that have and activity toward ampicillin, This in variant containing a of amino acids introduced at positions and The a of sites for that By digesting plasmid DNA containing the insertions with appropriate of these enzymes and the of derivatives β-lactamase with a of or amino acids at positions or were of the acid insertions at positions and altered the levels of resistance toward ceftazidime and ampicillin in with the derivatives containing pentapeptide insertions at these results that β-lactamase derivatives in which the length of the Ω-loop has been increased by up to 2-fold catalytic activity toward ampicillin and/or ceftazidime. The levels of variant β-lactamases during growth were by with pentapeptide insertions in the N-terminal of the Ω-loop that ampicillin were in the wild-type protein. In variants with pentapeptide insertions in the of this region that conferred ampicillin resistance were at levels from that of the wild-type protein results with previous that have a wild-type expression levels and the ability to hydrolyze ampicillin in β-lactamases with Ω-loop substitution mutations T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google J.F. Palzkill T. J. Bacteriol. 1996; 178: 1821-1828Crossref PubMed Google Scholar). The of the of the Ω-loop insertion on β-lactamase levels was also containing or 19-amino acid insertions at were at of the wild-type A acid insertion at this protein levels to of the wild-type In insertions of amino acids at β-lactamase concentrations to these conferred ceftazidime resistance insertions at of the β-lactamase enzyme insertions at This to the of ceftazidime resistance conferred by the the of derivatives with insertions of amino acids be altered such that the of the protein by the is The Ω-loop of TEM-1 β-lactamase contains required both for enzyme and for the of the active site C. Mourey L. Masson J.-M. Samama J.-P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar, 7Strynadka 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, 12Maveyraud L. Saves I. Burlet-Schiltz O. Swarén P. Masson J.-M. Delaire M. Mourey L. Promé J.-C. Samama J.-P. J. Biol. Chem. 1996; 271: 10482-10489Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). or substitution mutations in this region the resistance spectrum of β-lactamase to include third generation cephalosporin antibiotics that the wild-type enzyme hydrolyzes poorly (5Jacoby G.A. Medeiros A.A. Antimicrob. Agents Chemother. 1991; 35: 1697-1704Crossref PubMed Scopus (652) Google Scholar, M. Lenfant F. Labia R. Masson J.-M. Protein Eng. 1991; 4: 805-810Crossref PubMed Scopus (47) Google Scholar, 9Guillaume G. Vanhove M. Lamotte-Brasseur J. Ledent P. Jamin M. Joris B. Frère J.-M. J. Biol. Chem. 1997; 272: 5438-5444Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 10Palzkill T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google Scholar, 11Petrosino J.F. Palzkill T. J. Bacteriol. 1996; 178: 1821-1828Crossref PubMed Google Scholar, 12Maveyraud L. Saves I. Burlet-Schiltz O. Swarén P. Masson J.-M. Delaire M. Mourey L. Promé J.-C. Samama J.-P. J. Biol. Chem. 1996; 271: 10482-10489Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, S. K. 1991; PubMed Scopus Google Scholar). The that the Ω-loop is to mutations that β-lactamase substrate specificity previously pentapeptide insertions at a of different positions in this region increased by as as the of ceftazidime resistance conferred by the protein with the resistance levels are with in mutagenesis of the Ω-loop T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google Scholar, 11Petrosino J.F. Palzkill T. J. Bacteriol. 1996; 178: 1821-1828Crossref PubMed Google Scholar). insertions that increased the length of the Ω-loop as as 2-fold also retained catalytic activity in vivo. results emphasize further the flexibility of the Ω-loop with to the primary sequence requirements for ceftazidime as has been previously in substitution mutation T. Le Q.-Q. Venkatachalam K.V. LaRocco M. Ocera H. Mol. Microbiol. 1994; 12: 217-229Crossref PubMed Scopus (76) Google Scholar, 11Petrosino J.F. Palzkill T. J. Bacteriol. 1996; 178: 1821-1828Crossref PubMed Google Scholar). a in the catalytic region of a class β-lactamase variant has been also to substrate specificity to include M. S. K. K. K. M. T. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). and of that and/or mutations have into the by which Ω-loop mutations the resistance spectrum of β-lactamase (9Guillaume G. Vanhove M. Lamotte-Brasseur J. Ledent P. Jamin M. Joris B. Frère J.-M. J. Biol. Chem. 1997; 272: 5438-5444Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Maveyraud L. Saves I. Burlet-Schiltz O. Swarén P. Masson J.-M. Delaire M. Mourey L. Promé J.-C. Samama J.-P. J. Biol. Chem. 1996; 271: 10482-10489Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 13Raquet X. Lamotte-Brasseur J. Fonze E. Goussard S. Courvalin P. Frère J.-M. J. Mol. Biol. 1994; 244: 625-639Crossref PubMed Scopus (124) Google Scholar). of of an in which the substrate is to the active site of the enzyme J. J. S. 1980; PubMed Scopus Google Scholar). is required both for of the (9Guillaume G. Vanhove M. Lamotte-Brasseur J. Ledent P. Jamin M. Joris B. Frère J.-M. J. Biol. Chem. 1997; 272: 5438-5444Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and for of the 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, H. T. H. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, 1991; PubMed Scopus Google Scholar). The increased activity spectrum of enzymes with mutations of be due to a of effects of a catalytic from the active access by the side chain of to the active and of a of the that contains catalytic (9Guillaume G. Vanhove M. Lamotte-Brasseur J. Ledent P. Jamin M. Joris B. Frère J.-M. J. Biol. Chem. 1997; 272: 5438-5444Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Maveyraud L. Saves I. Burlet-Schiltz O. Swarén P. Masson J.-M. Delaire M. Mourey L. Promé J.-C. Samama J.-P. J. Biol. Chem. 1996; 271: 10482-10489Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). forms with and C. Mourey L. Masson J.-M. Samama J.-P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar, 7Strynadka 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). of that these alter the and of the Ω-loop such that both in of the and in the catalytic are thereby allowing access of substrates (3Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar, 12Maveyraud L. Saves I. Burlet-Schiltz O. Swarén P. Masson J.-M. Delaire M. Mourey L. Promé J.-C. Samama J.-P. J. Biol. Chem. 1996; 271: 10482-10489Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The altered resistance associated with the insertion mutations described in this be due to of the active site to by and substitution insertions as as amino acids have an on substrate specificity to that of substitution the of the amino acids from the of the the of the Ω-loop be as as in substitution pentapeptide insertions in both the and of the Ω-loop conferred ceftazidime enzymes containing insertions in the region levels of activity toward ampicillin, but variants with insertions in the region This the of N-terminal insertions to the and and of derivatives with Ω-loop insertions these DNA by are as an by for gene and gene The excision of transposons in pentapeptide scanning mutagenesis is in by the KpnI of be for amino acid insertions when protein are Scholar, 1993; PubMed Scopus Google Scholar). The excision of has also been as a by which mutations G. EMBO J. 1994; PubMed Scopus Google Scholar, J. EMBO J. 1994; PubMed Scopus Google Scholar). Pentapeptide scanning mutagenesis an for a naturally occurring of protein and for with catalytic In this a result is that of the β-lactamase pentapeptide insertions generated in a (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar) were to alter enzyme substrate specificity by increased levels of resistance to ceftazidime selection for this was at of the mutagenesis procedure The of pentapeptide scanning mutagenesis was emphasized further by the procedure to allow the direct selection of β-lactamase derivatives with substrate specificities. derivatives insertions at a of positions a 19-amino acid the (14Hallet B. Sherratt D.J. Hayes F. Nucleic Acids Res. 1997; 25: 1866-1867Crossref PubMed Scopus (77) Google Scholar) and levels of the mutated β-lactamases also that pentapeptide insertions protein and is to that by insertions enzyme substrate by the conformational flexibility of the in the catalytic mutations to the protein in Pentapeptide scanning mutagenesis has also been used to of the in activity in vivo, in the substrate target and in with the Y. Hallet B. Sherratt D.J. Hayes F. J. Mol. Biol. 1997; 274: 39-53Crossref PubMed Scopus (26) Google Scholar). The of pentapeptide scanning mutagenesis as a of enzyme activity is further Frère for β-lactamase and and for on the