Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Mar;83(6):1588–1592. doi: 10.1073/pnas.83.6.1588

Site-saturation studies of beta-lactamase: production and characterization of mutant beta-lactamases with all possible amino acid substitutions at residue 71.

S C Schultz, J H Richards
PMCID: PMC323128  PMID: 3513181

Abstract

A mutagenic technique that "saturates" a particular site in a protein with all possible amino acid substitutions was used to study the role of residue 71 in beta-lactamase (EC 3.5.2.6). Threonine is conserved at residue 71 in all class A beta-lactamases and is adjacent to the active site Ser-70. All 19 mutants of the enzyme were characterized by the penam and cephem antibiotic resistance they provided to Escherichia coli LS1 cells. Surprisingly, cells producing any of 14 of the mutant beta-lactamases displayed appreciable resistance to ampicillin; only cells with mutants having Tyr, Trp, Asp, Lys, or Arg at residue 71 had no observable resistance to ampicillin. However, the mutants are less stable to cellular proteases than wild-type enzyme is. These results suggest that Thr-71 is not essential for binding or catalysis but is important for stability of the beta-lactamase protein. An apparent change in specificity indicates that residue 71 influences the region of the protein that accommodates the side chain attached to the beta-lactam ring of the substrate.

Full text

PDF
1588

Images in this article

1591Image
on p.1591
1591Image
on p.1591
1591Image
on p.1591
1591Image
on p.1591

Selected References

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

  1. Ackers G. K., Smith F. R. Effects of site-specific amino acid modification on protein interactions and biological function. Annu Rev Biochem. 1985;54:597–629. doi: 10.1146/annurev.bi.54.070185.003121. [DOI] [PubMed] [Google Scholar]
  2. Barany F. Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4202–4206. doi: 10.1073/pnas.82.12.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  5. Craik C. S., Largman C., Fletcher T., Roczniak S., Barr P. J., Fletterick R., Rutter W. J. Redesigning trypsin: alteration of substrate specificity. Science. 1985 Apr 19;228(4697):291–297. doi: 10.1126/science.3838593. [DOI] [PubMed] [Google Scholar]
  6. Dalbadie-McFarland G., Cohen L. W., Riggs A. D., Morin C., Itakura K., Richards J. H. Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6409–6413. doi: 10.1073/pnas.79.21.6409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fisher J., Belasco J. G., Khosla S., Knowles J. R. beta-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin. Biochemistry. 1980 Jun 24;19(13):2895–2901. doi: 10.1021/bi00554a012. [DOI] [PubMed] [Google Scholar]
  8. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  9. Hecht M. H., Sturtevant J. M., Sauer R. T. Effect of single amino acid replacements on the thermal stability of the NH2-terminal domain of phage lambda repressor. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5685–5689. doi: 10.1073/pnas.81.18.5685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kadonaga J. T., Gautier A. E., Straus D. R., Charles A. D., Edge M. D., Knowles J. R. The role of the beta-lactamase signal sequence in the secretion of proteins by Escherichia coli. J Biol Chem. 1984 Feb 25;259(4):2149–2154. [PubMed] [Google Scholar]
  12. Knott-Hunziker V., Waley S. G., Orlek B. S., Sammes P. G. Penicillinase active sites: labelling of serine-44 in beta-lactamase I by 6beta-bromopenicillanic acid. FEBS Lett. 1979 Mar 1;99(1):59–61. doi: 10.1016/0014-5793(79)80248-3. [DOI] [PubMed] [Google Scholar]
  13. Matthew M., Hedges R. W. Analytical isoelectric focusing of R factor-determined beta-lactamases: correlation with plasmid compatibility. J Bacteriol. 1976 Feb;125(2):713–718. doi: 10.1128/jb.125.2.713-718.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  15. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [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. Norris K., Norris F., Christiansen L., Fiil N. Efficient site-directed mutagenesis by simultaneous use of two primers. Nucleic Acids Res. 1983 Aug 11;11(15):5103–5112. doi: 10.1093/nar/11.15.5103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Perry L. J., Wetzel R. Disulfide bond engineered into T4 lysozyme: stabilization of the protein toward thermal inactivation. Science. 1984 Nov 2;226(4674):555–557. doi: 10.1126/science.6387910. [DOI] [PubMed] [Google Scholar]
  19. Petsko G. A., Davenport R. C., Jr, Frankel D., RaiBhandary U. L. Probing the catalytic mechanism of yeast triose phosphate isomerase by site-specific mutagenesis. Biochem Soc Trans. 1984 Apr;12(2):229–232. doi: 10.1042/bst0120229. [DOI] [PubMed] [Google Scholar]
  20. Robey E. A., Schachman H. K. Site-specific mutagenesis of aspartate transcarbamoylase. Replacement of tyrosine 165 in the catalytic chain by serine reduces enzymatic activity. J Biol Chem. 1984 Sep 25;259(18):11180–11183. [PubMed] [Google Scholar]
  21. Seeburg P. H., Colby W. W., Capon D. J., Goeddel D. V., Levinson A. D. Biological properties of human c-Ha-ras1 genes mutated at codon 12. Nature. 1984 Nov 1;312(5989):71–75. doi: 10.1038/312071a0. [DOI] [PubMed] [Google Scholar]
  22. So M., Gill R., Falkow S. The generation of a ColE1-Apr cloning vehicle which allows detection of inserted DNA. Mol Gen Genet. 1975 Dec 30;142(3):239–249. doi: 10.1007/BF00425649. [DOI] [PubMed] [Google Scholar]
  23. Straus D., Raines R., Kawashima E., Knowles J. R., Gilbert W. Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2272–2276. doi: 10.1073/pnas.82.8.2272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Villafranca J. E., Howell E. E., Voet D. H., Strobel M. S., Ogden R. C., Abelson J. N., Kraut J. Directed mutagenesis of dihydrofolate reductase. Science. 1983 Nov 18;222(4625):782–788. doi: 10.1126/science.6356360. [DOI] [PubMed] [Google Scholar]
  25. Wells J. A., Vasser M., Powers D. B. Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites. Gene. 1985;34(2-3):315–323. doi: 10.1016/0378-1119(85)90140-4. [DOI] [PubMed] [Google Scholar]
  26. Wilkinson A. J., Fersht A. R., Blow D. M., Carter P., Winter G. A large increase in enzyme-substrate affinity by protein engineering. Nature. 1984 Jan 12;307(5947):187–188. doi: 10.1038/307187a0. [DOI] [PubMed] [Google Scholar]
  27. Winter G., Fersht A. R., Wilkinson A. J., Zoller M., Smith M. Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding. Nature. 1982 Oct 21;299(5885):756–758. doi: 10.1038/299756a0. [DOI] [PubMed] [Google Scholar]
  28. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES