Advertisement

Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

The gdhA1 point mutation in Escherichia coli K12 CLR207 alters a key lysine residue of glutamate dehydrogenase

Abstract

gdhA1 is a spontaneous mutant of Escherichia coli that causes complete loss of activity of the NADP-specific glutamate dehydrogenase (GDH) encoded by the gdhA gene. The gdhA1 mutational site has been identified by recombinational mapping, polymerase chain reaction (PCR) amplification and DNA sequencing, as an A to G transition at nucleotide 274 of the gdhA coding sequence, resulting in an amino acid change of lysine 92 to glutamic acid. The mutant enzyme forms hybrid hexamers with a wild-type GDH, providing a useful system for analysis of conformational integrity of mutational variants.

This is a preview of subscription content, log in to check access.

References

  1. Baker PJ, Britton KL, Engel PC, Farrants GW, Lilley KS, Rice DW, Stillman TJ (1992) Subunit assembly and active site location in the structure of glutamate dehydrogenase. Proteins 12:75–86

  2. Bankier AT, Weston KM, Barrell BG (1987) Random cloning and sequencing by the M13/dideoxynucleotide chain termination method. Methods Enzymol 155:51–93

  3. Berberich MA (1972) A glutamate-dependent phenotype in E. coli K12: the result of two mutations. Biochem Biophys Res Commun 47:1498–1503

  4. Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2998

  5. Katz L, Kingsbury DT, Helinski DR (1973) Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid — protein relaxation complex. J Bacteriol 114:577–591

  6. Lilley KS, Engel PC (1988) Identification of lysine residues involved in substrate binding at the active site of NAD+ glutamate dehydrogenase of Clostridium symbiosum. Biochem Soc Trans 16:875–876

  7. Mattaj LW, McPherson MJ, Wootton JC (1982) Localisation of a strongly conserved section of coding sequence in glutamate dehydrogenase genes. FEBS Lett 147:21–25

  8. McPherson MJ, Wootton JC (1983) Complete nucleotide sequence of the Escherichia coli gdhA gene. Nucleic Acids Res 11:5257–5266

  9. McPherson MJ, Baron AJ, Jones KM, Price GJ, Wootton JC (1988) Multiple interactions of lysine-128 of Escherichia coli glutamate dehydrogenase revealed by site-directed mutagenesis studies. Protein Engineering 2:147–152

  10. McPherson MJ, Oliver RP, Gurr SJ (1992) The polymerase chain reaction. In: Gurr SJ, McPherson MJ, Bowles DJ (eds) Molecular plant pathology: a practical approach. IRL Press, Oxford 123–145

  11. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78

  12. Mountain A, McPherson MJ, Baron AJ, Wootton JC (1985) The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli. Mol Gen Genet 199:141–145

  13. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  14. Sanchez-Pescador R, Sanvicente E, Valle F, Bolivar F (1982) Recombinant plasmids carrying the glutamate dehydrogenase structural gene from Escherichia coli K12. Gene 17:1–8

  15. Wootton JC, McPherson MJ (1984) Genes of nitrate and ammonium assimilation. Annu Proc Phytochem Soc Europe 23:89–114

Download references

Author information

Correspondence to Michael J. McPherson.

Additional information

Communicated by J. Lengeler

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jones, K.M., McPherson, M.J., Baron, A.J. et al. The gdhA1 point mutation in Escherichia coli K12 CLR207 alters a key lysine residue of glutamate dehydrogenase. Molec. Gen. Genet. 240, 286–289 (1993). https://doi.org/10.1007/BF00277068

Download citation

Key words

  • gdhA1 point mutation
  • Deletion analysis
  • Recombination frequency
  • PCR
  • DNA sequencing