Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Dec;178(24):7248–7253. doi: 10.1128/jb.178.24.7248-7253.1996

Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216.

R K Voladri 1, M K Tummuru 1, D S Kernodle 1
PMCID: PMC178640  PMID: 8955409

Abstract

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus beta-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences located close to the active site, we hypothesized that these differences might explain the kinetic heterogeneity of the wild-type beta-lactamases. To test this hypothesis, genes encoding the type A, C, and D beta-lactamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of asparagine for serine at residue 216 of type A beta-lactamase resulted in a kinetic profile indistinguishable from that of type C beta-lactamase, whereas the substitution of serine for asparagine at the same site in the type C enzyme produced a kinetic type A mutant. Similar bidirectional substitutions identified the threonine-to-alanine difference at residue 128 as being responsible for the kinetic differences between the type A and D enzymes. Neither residue 216 nor 128 has previously been shown to be kinetically important among serine-active-site beta-lactamases.

Full Text

The Full Text of this article is available as a PDF (252.3 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. Ambler R. P. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci. 1980 May 16;289(1036):321–331. doi: 10.1098/rstb.1980.0049. [DOI] [PubMed] [Google Scholar]
  3. Bonfiglio G., Livermore D. M. beta-Lactamase types amongst Staphylococcus aureus isolates in relation to susceptibility to beta-lactamase inhibitor combinations. J Antimicrob Chemother. 1994 Mar;33(3):465–481. doi: 10.1093/jac/33.3.465. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chan P. T. Nucleotide sequence of the Staphylococcus aureus PC1 beta-lactamase gene. Nucleic Acids Res. 1986 Jul 25;14(14):5940–5940. doi: 10.1093/nar/14.14.5940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  7. Delaire M., Labia R., Samama J. P., Masson J. M. Site-directed mutagenesis at the active site of Escherichia coli TEM-1 beta-lactamase. Suicide inhibitor-resistant mutants reveal the role of arginine 244 and methionine 69 in catalysis. J Biol Chem. 1992 Oct 15;267(29):20600–20606. [PubMed] [Google Scholar]
  8. East A. K., Curnock S. P., Dyke K. G. Change of a single amino acid in the leader peptide of a staphylococcal beta-lactamase prevents the appearance of the enzyme in the medium. FEMS Microbiol Lett. 1990 Jun 1;57(3):249–254. doi: 10.1016/0378-1097(90)90075-2. [DOI] [PubMed] [Google Scholar]
  9. East A. K., Dyke K. G. Cloning and sequence determination of six Staphylococcus aureus beta-lactamases and their expression in Escherichia coli and Staphylococcus aureus. J Gen Microbiol. 1989 Apr;135(4):1001–1015. doi: 10.1099/00221287-135-4-1001. [DOI] [PubMed] [Google Scholar]
  10. Galetto D. W., Johnston J. L., Archer G. L. Molecular epidemiology of trimethoprim resistance among coagulase-negative staphylococci. Antimicrob Agents Chemother. 1987 Nov;31(11):1683–1688. doi: 10.1128/aac.31.11.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Herzberg O., Moult J. Bacterial resistance to beta-lactam antibiotics: crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.5 A resolution. Science. 1987 May 8;236(4802):694–701. doi: 10.1126/science.3107125. [DOI] [PubMed] [Google Scholar]
  12. Herzberg O. Refined crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.0 A resolution. J Mol Biol. 1991 Feb 20;217(4):701–719. doi: 10.1016/0022-2836(91)90527-d. [DOI] [PubMed] [Google Scholar]
  13. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J Bacteriol. 1982 May;150(2):804–814. doi: 10.1128/jb.150.2.804-814.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Huletsky A., Knox J. R., Levesque R. C. Role of Ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling. J Biol Chem. 1993 Feb 15;268(5):3690–3697. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Johnston L. H., Dyke K. G. Stability of penicillinase plasmids in Staphylococcus aureus. J Bacteriol. 1971 Jul;107(1):68–73. doi: 10.1128/jb.107.1.68-73.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kernodle D. S., McGraw P. A., Stratton C. W., Kaiser A. B. Use of extracts versus whole-cell bacterial suspensions in the identification of Staphylococcus aureus beta-lactamase variants. Antimicrob Agents Chemother. 1990 Mar;34(3):420–425. doi: 10.1128/aac.34.3.420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kernodle D. S., Stratton C. W., McMurray L. W., Chipley J. R., McGraw P. A. Differentiation of beta-lactamase variants of Staphylococcus aureus by substrate hydrolysis profiles. J Infect Dis. 1989 Jan;159(1):103–108. doi: 10.1093/infdis/159.1.103. [DOI] [PubMed] [Google Scholar]
  20. Kernodle D. S., Zygmunt D. J., McGraw P. A., Chipley J. R. Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography. Antimicrob Agents Chemother. 1990 Nov;34(11):2177–2183. doi: 10.1128/aac.34.11.2177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Lacey R. W., Grinsted J. Linkage of fusidic acid resistance to the penicillinase plasmid in Staphylococcus aureus. J Gen Microbiol. 1972 Dec;73(3):501–508. doi: 10.1099/00221287-73-3-501. [DOI] [PubMed] [Google Scholar]
  24. Medeiros A. A. Beta-lactamases. Br Med Bull. 1984 Jan;40(1):18–27. doi: 10.1093/oxfordjournals.bmb.a071942. [DOI] [PubMed] [Google Scholar]
  25. Miller S., Janin J., Lesk A. M., Chothia C. Interior and surface of monomeric proteins. J Mol Biol. 1987 Aug 5;196(3):641–656. doi: 10.1016/0022-2836(87)90038-6. [DOI] [PubMed] [Google Scholar]
  26. NOVICK R. P. ANALYSIS BY TRANSDUCTION OF MUTATIONS AFFECTING PENICILLINASE FORMATION IN STAPHYLOCOCCUS AUREUS. J Gen Microbiol. 1963 Oct;33:121–136. doi: 10.1099/00221287-33-1-121. [DOI] [PubMed] [Google Scholar]
  27. Osuna J., Viadiu H., Fink A. L., Soberón X. Substitution of Asp for Asn at position 132 in the active site of TEM beta-lactamase. Activity toward different substrates and effects of neighboring residues. J Biol Chem. 1995 Jan 13;270(2):775–780. [PubMed] [Google Scholar]
  28. Paul G. C., Gerbaud G., Bure A., Philippon A. M., Pangon B., Courvalin P. TEM-4, a new plasmid-mediated beta-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Escherichia coli. Antimicrob Agents Chemother. 1989 Nov;33(11):1958–1963. doi: 10.1128/aac.33.11.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Petit A., Sirot D. L., Chanal C. M., Sirot J. L., Labia R., Gerbaud G., Cluzel R. A. Novel plasmid-mediated beta-lactamase in clinical isolates of Klebsiella pneumoniae more resistant to ceftazidime than to other broad-spectrum cephalosporins. Antimicrob Agents Chemother. 1988 May;32(5):626–630. doi: 10.1128/aac.32.5.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. RICHMOND M. H. PURIFICATION AND PROPERTIES OF THE EXOPENICILLINASE FROM STAPHYLOCOCCUS AUREUS. Biochem J. 1963 Sep;88:452–459. doi: 10.1042/bj0880452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rosdahl V. T. Naturally occurring constitutive -lactamase of novel serotype in Staphylococcus aureus. J Gen Microbiol. 1973 Jul;77(1):229–231. doi: 10.1099/00221287-77-1-229. [DOI] [PubMed] [Google Scholar]
  32. Rosdahl V. T. Penicillinase production in Staphylococcus aureus strains of clinical importance. Dan Med Bull. 1986 Aug;33(4):175–184. [PubMed] [Google Scholar]
  33. Ross G. W., Chanter K. V., Harris A. M., Kirby S. M., Marshall M. J., O'Callaghan C. H. Comparison of assay techniques for beta-lactamase activity. Anal Biochem. 1973 Jul;54(1):9–16. doi: 10.1016/0003-2697(73)90241-8. [DOI] [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Waley S. G. A spectrophotometric assay of beta-lactamase action on penicillins. Biochem J. 1974 Jun;139(3):789–790. doi: 10.1042/bj1390789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang P. Z., Novick R. P. Nucleotide sequence and expression of the beta-lactamase gene from Staphylococcus aureus plasmid pI258 in Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. J Bacteriol. 1987 Apr;169(4):1763–1766. doi: 10.1128/jb.169.4.1763-1766.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  38. Zygmunt D. J., Stratton C. W., Kernodle D. S. Characterization of four beta-lactamases produced by Staphylococcus aureus. Antimicrob Agents Chemother. 1992 Feb;36(2):440–445. doi: 10.1128/aac.36.2.440. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES