Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Mar 15;330(Pt 3):1443–1449. doi: 10.1042/bj3301443

Role of residues 104, 164, 166, 238 and 240 in the substrate profile of PER-1 beta-lactamase hydrolysing third-generation cephalosporins.

A T Bouthors 1, N Dagoneau-Blanchard 1, T Naas 1, P Nordmann 1, V Jarlier 1, W Sougakoff 1
PMCID: PMC1219294  PMID: 9494118

Abstract

The class A beta-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum beta-lactamases (ESBLs), catalyses the hydrolysis of oxyimino-beta-lactams such as cefotaxime (CTX), ceftazidime (CAZ) and aztreonam (AZT). Molecular modelling was used to identify in PER-1 the amino acid residues corresponding to those found at positions 104, 164, 238 and 240 in the TEM-type ESBLs, which are critical for hydrolysis of oxyimino-beta-lactams. The function of these residues in PER-1 was assessed by site-directed mutagenesis. In this enzyme, residue 104 could be either a glutamine, an asparagine or a threonine. The Gln-->Gly mutation did not significantly affect the catalytic efficiency, while Asn-->Gly and Thr-->Glu resulted in a marked decrease in catalytic activity, probably due to the alteration of a hydrogen bond network connecting the putative Asn-104 residue to Asn-132 and Glu-166. Replacement of Ala-164 by Arg in PER-1 resulted in a mutant with no detectable activity, thus suggesting that Ala-164 is important for catalysis and stability of PER-1. Conversely, Ser-238-->Gly and Gly-240-->Glu had little effect on kcat and Km values. Finally, the replacement of the catalytic residue Glu-166 by an alanine resulted in a complete loss of activity for CTX and a marked decrease of kcat for CAZ and AZT. These results suggest that Glu-166 is an important residue in PER-1. However, residues other than Glu-166 could contribute in maintaining residual activity towards oxyimino-beta-lactams in the Ala-166 mutant.

Full Text

The Full Text of this article is available as a PDF (435.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ambler R. P., Coulson A. F., Frère J. M., Ghuysen J. M., Joris B., Forsman M., Levesque R. C., Tiraby G., Waley S. G. A standard numbering scheme for the class A beta-lactamases. Biochem J. 1991 May 15;276(Pt 1):269–270. doi: 10.1042/bj2760269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bush K., Jacoby G. A., Medeiros A. A. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother. 1995 Jun;39(6):1211–1233. doi: 10.1128/aac.39.6.1211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Escobar W. A., Tan A. K., Lewis E. R., Fink A. L. Site-directed mutagenesis of glutamate-166 in beta-lactamase leads to a branched path mechanism. Biochemistry. 1994 Jun 21;33(24):7619–7626. doi: 10.1021/bi00190a015. [DOI] [PubMed] [Google Scholar]
  4. Escobar W. A., Tan A. K., Lewis E. R., Fink A. L. Site-directed mutagenesis of glutamate-166 in beta-lactamase leads to a branched path mechanism. Biochemistry. 1994 Jun 21;33(24):7619–7626. doi: 10.1021/bi00190a015. [DOI] [PubMed] [Google Scholar]
  5. Guillaume G., Vanhove M., Lamotte-Brasseur J., Ledent P., Jamin M., Joris B., Frère J. M. Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies. J Biol Chem. 1997 Feb 28;272(9):5438–5444. doi: 10.1074/jbc.272.9.5438. [DOI] [PubMed] [Google Scholar]
  6. Jacob F., Joris B., Lepage S., Dusart J., Frère J. M. Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis. Biochem J. 1990 Oct 15;271(2):399–406. doi: 10.1042/bj2710399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jacoby G. A., Medeiros A. A. More extended-spectrum beta-lactamases. Antimicrob Agents Chemother. 1991 Sep;35(9):1697–1704. doi: 10.1128/aac.35.9.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  9. Joris B., Ledent P., Dideberg O., Fonzé E., Lamotte-Brasseur J., Kelly J. A., Ghuysen J. M., Frère J. M. Comparison of the sequences of class A beta-lactamases and of the secondary structure elements of penicillin-recognizing proteins. Antimicrob Agents Chemother. 1991 Nov;35(11):2294–2301. doi: 10.1128/aac.35.11.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Knox J. R., Moews P. C., Escobar W. A., Fink A. L. A catalytically-impaired class A beta-lactamase: 2 A crystal structure and kinetics of the Bacillus licheniformis E166A mutant. Protein Eng. 1993 Jan;6(1):11–18. doi: 10.1093/protein/6.1.11. [DOI] [PubMed] [Google Scholar]
  11. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Matagne A., Frère J. M. Contribution of mutant analysis to the understanding of enzyme catalysis: the case of class A beta-lactamases. Biochim Biophys Acta. 1995 Jan 19;1246(2):109–127. doi: 10.1016/0167-4838(94)00177-i. [DOI] [PubMed] [Google Scholar]
  14. Maveyraud L., Saves I., Burlet-Schiltz O., Swarén P., Masson J. M., Delaire M., Mourey L., Promé J. C., Samama J. P. Structural basis of extended spectrum TEM beta-lactamases. Crystallographic, kinetic, and mass spectrometric investigations of enzyme mutants. J Biol Chem. 1996 May 3;271(18):10482–10489. doi: 10.1074/jbc.271.18.10482. [DOI] [PubMed] [Google Scholar]
  15. McClary J. A., Witney F., Geisselsoder J. Efficient site-directed in vitro mutagenesis using phagemid vectors. Biotechniques. 1989 Mar;7(3):282–289. [PubMed] [Google Scholar]
  16. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  17. Neugebauer K., Sprengel R., Schaller H. Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium. Nucleic Acids Res. 1981 Jun 11;9(11):2577–2588. doi: 10.1093/nar/9.11.2577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nordmann P., Naas T. Sequence analysis of PER-1 extended-spectrum beta-lactamase from Pseudomonas aeruginosa and comparison with class A beta-lactamases. Antimicrob Agents Chemother. 1994 Jan;38(1):104–114. doi: 10.1128/aac.38.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nordmann P., Ronco E., Naas T., Duport C., Michel-Briand Y., Labia R. Characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1993 May;37(5):962–969. doi: 10.1128/aac.37.5.962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. O'Callaghan C. H., Morris A., Kirby S. M., Shingler A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother. 1972 Apr;1(4):283–288. doi: 10.1128/aac.1.4.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Palzkill T., Le Q. Q., Venkatachalam K. V., LaRocco M., Ocera H. Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase. Mol Microbiol. 1994 Apr;12(2):217–229. doi: 10.1111/j.1365-2958.1994.tb01011.x. [DOI] [PubMed] [Google Scholar]
  22. Peitsch M. C. ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem Soc Trans. 1996 Feb;24(1):274–279. doi: 10.1042/bst0240274. [DOI] [PubMed] [Google Scholar]
  23. Petit A., Maveyraud L., Lenfant F., Samama J. P., Labia R., Masson J. M. Multiple substitutions at position 104 of beta-lactamase TEM-1: assessing the role of this residue in substrate specificity. Biochem J. 1995 Jan 1;305(Pt 1):33–40. doi: 10.1042/bj3050033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pridmore R. D. New and versatile cloning vectors with kanamycin-resistance marker. Gene. 1987;56(2-3):309–312. doi: 10.1016/0378-1119(87)90149-1. [DOI] [PubMed] [Google Scholar]
  25. Raquet X., Lamotte-Brasseur J., Fonzé E., Goussard S., Courvalin P., Frère J. M. TEM beta-lactamase mutants hydrolysing third-generation cephalosporins. A kinetic and molecular modelling analysis. J Mol Biol. 1994 Dec 16;244(5):625–639. doi: 10.1006/jmbi.1994.1756. [DOI] [PubMed] [Google Scholar]
  26. Saqi M. A., Sayle R. PdbMotif--a tool for the automatic identification and display of motifs in protein structures. Comput Appl Biosci. 1994 Sep;10(5):545–546. doi: 10.1093/bioinformatics/10.5.545. [DOI] [PubMed] [Google Scholar]
  27. Saves I., Burlet-Schiltz O., Maveyraud L., Samama J. P., Promé J. C., Masson J. M. Mass spectral kinetic study of acylation and deacylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant. Biochemistry. 1995 Sep 19;34(37):11660–11667. doi: 10.1021/bi00037a003. [DOI] [PubMed] [Google Scholar]
  28. Sougakoff W., Petit A., Goussard S., Sirot D., Bure A., Courvalin P. Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae. Gene. 1989 May 30;78(2):339–348. doi: 10.1016/0378-1119(89)90236-9. [DOI] [PubMed] [Google Scholar]
  29. Sutcliffe J. G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. doi: 10.1073/pnas.75.8.3737. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES