Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Sep;177(17):4921–4926. doi: 10.1128/jb.177.17.4921-4926.1995

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

H Mori 1, A Iida 1, S Teshiba 1, T Fujio 1
PMCID: PMC177266  PMID: 7665468

Abstract

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).

Full Text

The Full Text of this article is available as a PDF (300.5 KB).

Selected References

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

  1. BROCKMAN R. W. BIOCHEMICAL ASPECTS OF MERCAPTOPURINE INHBITION AND RESISTANCE. Cancer Res. 1963 Sep;23:1191–1201. [PubMed] [Google Scholar]
  2. Brunngraber E. F., Chargaff E. A nucleotide phosphotransferase from Escherichia coli. Purification and properties. Biochemistry. 1973 Jul 31;12(16):3005–3012. doi: 10.1021/bi00740a009. [DOI] [PubMed] [Google Scholar]
  3. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  4. Hammer-Jespersen K., Buxton R. S., Hansen T. D. A second purine nucleoside phosphorylase in Escherichia coli K-12. II. Properties of xanthosine phosphorylase and its induction by xanthosine. Mol Gen Genet. 1980;179(2):341–348. doi: 10.1007/BF00425462. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Harlow K. W., Nygaard P., Hove-Jensen B. Cloning and characterization of the gsk gene encoding guanosine kinase of Escherichia coli. J Bacteriol. 1995 Apr;177(8):2236–2240. doi: 10.1128/jb.177.8.2236-2240.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  8. Hirano M., Shigesada K., Imai M. Construction and characterization of plasmid and lambda phage vector systems for study of transcriptional control in Escherichia coli. Gene. 1987;57(1):89–99. doi: 10.1016/0378-1119(87)90180-6. [DOI] [PubMed] [Google Scholar]
  9. Hoffmeyer J., Neuhard J. Metabolism of exogenous purine bases and nucleosides by Salmonella typhimurium. J Bacteriol. 1971 Apr;106(1):14–24. doi: 10.1128/jb.106.1.14-24.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hove-Jensen B., Nygaard P. Role of guanosine kinase in the utilization of guanosine for nucleotide synthesis in Escherichia coli. J Gen Microbiol. 1989 May;135(5):1263–1273. doi: 10.1099/00221287-135-5-1263. [DOI] [PubMed] [Google Scholar]
  11. Jensen K. F. Purine-nucleoside phosphorylase from Salmonella typhimurium and Escherichia coli. Initial velocity kinetics, ligand banding, and reaction mechanism. Eur J Biochem. 1976 Jan 15;61(2):377–386. doi: 10.1111/j.1432-1033.1976.tb10031.x. [DOI] [PubMed] [Google Scholar]
  12. Jochimsen B., Nygaard P., Vestergaard T. Location on the chromosome of Escherichia coli of genes governing purine metabolism. Adenosine deaminase (add), guanosine kinase (gsk) and hypoxanthine phosphoribosyltransferase (hpt). Mol Gen Genet. 1975 Dec 30;143(1):85–91. doi: 10.1007/BF00269424. [DOI] [PubMed] [Google Scholar]
  13. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  14. Koszalka G. W., Vanhooke J., Short S. A., Hall W. W. Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12. J Bacteriol. 1988 Aug;170(8):3493–3498. doi: 10.1128/jb.170.8.3493-3498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Miyamoto K., Nakahigashi K., Nishimura K., Inokuchi H. Isolation and characterization of visible light-sensitive mutants of Escherichia coli K12. J Mol Biol. 1991 Jun 5;219(3):393–398. doi: 10.1016/0022-2836(91)90180-e. [DOI] [PubMed] [Google Scholar]
  17. Pierre K. J., LePage G. A. Formation of inosine-5'-monophosphate by a kinase in cell-free extracts of Ehrlich ascites cells in vitro. Proc Soc Exp Biol Med. 1968 Feb;127(2):432–440. doi: 10.3181/00379727-127-32709. [DOI] [PubMed] [Google Scholar]
  18. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  19. Pratt D., Subramani S. Nucleotide sequence of the Escherichia coli xanthine-guanine phosphoribosyl transferase gene. Nucleic Acids Res. 1983 Dec 20;11(24):8817–8823. doi: 10.1093/nar/11.24.8817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Richardson K. K., Fostel J., Skopek T. R. Nucleotide sequence of the xanthine guanine phosphoribosyl transferase gene of E. coli. Nucleic Acids Res. 1983 Dec 20;11(24):8809–8816. doi: 10.1093/nar/11.24.8809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]

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

RESOURCES