Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1996 Sep;40(9):2152–2159. doi: 10.1128/aac.40.9.2152

Recovery of active beta-lactamases from Proteus vulgaris and RTEM-1 hybrid by random mutagenesis by using a dnaQ strain of Escherichia coli.

S M Hosseini-Mazinani 1, E Nakajima 1, Y Ihara 1, K Z Kameyama 1, K Sugimoto 1
PMCID: PMC163490  PMID: 8878598

Abstract

Proteus vulgaris and RTEM-1 beta-lactamases that belong to molecular class A with 37% amino acid similarity were examined to find the relationship between amino acid residues and activity of enzymes. MICs of ampicillin were > 2,000 micrograms/ml for Escherichia coli cells producing these enzymes. We have made 18 hybrid genes by substituting the coding region of the P. vulgaris beta-lactamase gene with the equivalent portions from the RTEM-1 gene. Most of these hybrids produced inactive proteins, but a few hybrid enzymes had partial or trace activity. From one of the hybrid genes (MIC of ampicillin, 100 micrograms/ml), we recovered three kinds of active mutants which provided ampicillin MICs of 1,000 micrograms/ml by the selection of spontaneous mutations in a dnaQ strain of E. coli. In these mutants, Leu-148, Met-182, and Tyr-274 were replaced with Val, Thr, and His, respectively. These amino acids have not been identified as residues with functional roles in substrate hydrolysis. Furthermore, from these hybrid mutants, we obtained a second set of mutants which conferred ampicillin MICs of 1,500 micrograms/ml. Interestingly, the second mutations were limited to these three amino acid substitutions. These amino acid residues which do not directly interact with substrates have an effect on enzyme activity. These mutant enzymes exhibited lower K(m) values for cephaloridine than both parental enzymes.

Full Text

The Full Text of this article is available as a PDF (358.6 KB).

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. Adachi H., Ohta T., Matsuzawa H. Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin. J Biol Chem. 1991 Feb 15;266(5):3186–3191. [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Asada K., Sugimoto K., Oka A., Takanami M., Hirota Y. Structure of replication origin of the Escherichia coli K-12 chromosome: the presence of spacer sequences in the ori region carrying information for autonomous replication. Nucleic Acids Res. 1982 Jun 25;10(12):3745–3754. doi: 10.1093/nar/10.12.3745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Chang Y. H., Labgold M. R., Richards J. H. Altering enzymatic activity: recruitment of carboxypeptidase activity into an RTEM beta-lactamase/penicillin-binding protein 5 chimera. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2823–2827. doi: 10.1073/pnas.87.7.2823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dalbadie-McFarland G., Neitzel J. J., Richards J. H. Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis. Biochemistry. 1986 Jan 28;25(2):332–338. doi: 10.1021/bi00350a008. [DOI] [PubMed] [Google Scholar]
  9. Datz M., Joris B., Azab E. A., Galleni M., Van Beeumen J., Frère J. M., Martin H. H. A common system controls the induction of very different genes. The class-A beta-lactamase of Proteus vulgaris and the enterobacterial class-C beta-lactamase. Eur J Biochem. 1994 Nov 15;226(1):149–157. doi: 10.1111/j.1432-1033.1994.tb20036.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dideberg O., Charlier P., Wéry J. P., Dehottay P., Dusart J., Erpicum T., Frère J. M., Ghuysen J. M. The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution. Biochem J. 1987 Aug 1;245(3):911–913. doi: 10.1042/bj2450911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dufresne J., Vézina G., Levesque R. C. Cloning and expression of the imipenem-hydrolyzing beta-lactamase operon from Pseudomonas maltophilia in Escherichia coli. Antimicrob Agents Chemother. 1988 Jun;32(6):819–826. doi: 10.1128/aac.32.6.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Isbell R. J., Fowler R. G. Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3'----5' exonuclease (proofreading) activity. Mutat Res. 1989 Aug;213(2):149–156. doi: 10.1016/0027-5107(89)90146-2. [DOI] [PubMed] [Google Scholar]
  17. Jaurin B., Grundström T. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of beta-lactamases of the penicillinase type. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4897–4901. doi: 10.1073/pnas.78.8.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jelsch C., Lenfant F., Masson J. M., Samama J. P. Beta-lactamase TEM1 of E. coli. Crystal structure determination at 2.5 A resolution. FEBS Lett. 1992 Mar 9;299(2):135–142. doi: 10.1016/0014-5793(92)80232-6. [DOI] [PubMed] [Google Scholar]
  19. Joris B., Ghuysen J. M., Dive G., Renard A., Dideberg O., Charlier P., Frère J. M., Kelly J. A., Boyington J. C., Moews P. C. The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family. Biochem J. 1988 Mar 1;250(2):313–324. doi: 10.1042/bj2500313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Joyce C. M., Grindley N. D. Method for determining whether a gene of Escherichia coli is essential: application to the polA gene. J Bacteriol. 1984 May;158(2):636–643. doi: 10.1128/jb.158.2.636-643.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Knox J. R., Moews P. C. Beta-lactamase of Bacillus licheniformis 749/C. Refinement at 2 A resolution and analysis of hydration. J Mol Biol. 1991 Jul 20;220(2):435–455. doi: 10.1016/0022-2836(91)90023-y. [DOI] [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. Lenfant F., Petit A., Labia R., Maveyraud L., Samama J. P., Masson J. M. Site-directed mutagenesis of beta-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes. Eur J Biochem. 1993 Nov 1;217(3):939–946. doi: 10.1111/j.1432-1033.1993.tb18324.x. [DOI] [PubMed] [Google Scholar]
  24. Martinez E., Bartolomé B., de la Cruz F. pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene. 1988 Aug 15;68(1):159–162. doi: 10.1016/0378-1119(88)90608-7. [DOI] [PubMed] [Google Scholar]
  25. Matagne A., Misselyn-Bauduin A. M., Joris B., Erpicum T., Granier B., Frère J. M. The diversity of the catalytic properties of class A beta-lactamases. Biochem J. 1990 Jan 1;265(1):131–146. doi: 10.1042/bj2650131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsubara N., Yotsuji A., Kumano K., Inoue M., Mitsuhashi S. Purification and some properties of a cephalosporinase from Proteus vulgaris. Antimicrob Agents Chemother. 1981 Jan;19(1):185–187. doi: 10.1128/aac.19.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moews P. C., Knox J. R., Dideberg O., Charlier P., Frère J. M. Beta-lactamase of Bacillus licheniformis 749/C at 2 A resolution. Proteins. 1990;7(2):156–171. doi: 10.1002/prot.340070205. [DOI] [PubMed] [Google Scholar]
  28. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  29. Nelson F. K., Friedman S. M., Smith G. P. Filamentous phage DNA cloning vectors: a noninfective mutant with a nonpolar deletion in gene III. Virology. 1981 Jan 30;108(2):338–350. doi: 10.1016/0042-6822(81)90442-6. [DOI] [PubMed] [Google Scholar]
  30. Péduzzi J., Reynaud A., Baron P., Barthélémy M., Labia R. Chromosomally encoded cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A. Biochim Biophys Acta. 1994 Jul 20;1207(1):31–39. doi: 10.1016/0167-4838(94)90048-5. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Schultz S. C., Richards J. H. Site-saturation studies of beta-lactamase: production and characterization of mutant beta-lactamases with all possible amino acid substitutions at residue 71. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1588–1592. doi: 10.1073/pnas.83.6.1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Strynadka N. C., Adachi H., Jensen S. E., Johns K., Sielecki A., Betzel C., Sutoh K., James M. N. Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 A resolution. Nature. 1992 Oct 22;359(6397):700–705. doi: 10.1038/359700a0. [DOI] [PubMed] [Google Scholar]
  34. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  35. Takano K., Nakabeppu Y., Maki H., Horiuchi T., Sekiguchi M. Structure and function of dnaQ and mutD mutators of Escherichia coli. Mol Gen Genet. 1986 Oct;205(1):9–13. doi: 10.1007/BF02428026. [DOI] [PubMed] [Google Scholar]
  36. Takeshita S., Sato M., Toba M., Masahashi W., Hashimoto-Gotoh T. High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. Gene. 1987;61(1):63–74. doi: 10.1016/0378-1119(87)90365-9. [DOI] [PubMed] [Google Scholar]
  37. Tamaki M., Nukaga M., Sawai T. Replacement of serine 237 in class A beta-lactamase of Proteus vulgaris modifies its unique substrate specificity. Biochemistry. 1994 Aug 23;33(33):10200–10206. doi: 10.1021/bi00199a049. [DOI] [PubMed] [Google Scholar]
  38. Thornewell S. J., Waley S. G. Site-directed mutagenesis and substrate-induced inactivation of beta-lactamase I. Biochem J. 1992 Dec 15;288(Pt 3):1045–1051. doi: 10.1042/bj2881045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Venkatachalam K. V., Huang W., LaRocco M., Palzkill T. Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime. J Biol Chem. 1994 Sep 23;269(38):23444–23450. [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES