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. 1996 Apr;178(7):1777–1781. doi: 10.1128/jb.178.7.1777-1781.1996

Two forms of the nucleoside diphosphate kinase of Pseudomonas aeruginosa 8830: altered specificity of nucleoside triphosphate synthesis by the cell membrane-associated form of the truncated enzyme.

S Shankar 1, S Kamath 1, A M Chakrabarty 1
PMCID: PMC177868  PMID: 8606147

Abstract

Nucleoside diphosphate kinase (EC 2.7.4.6) (Ndk) is a ubiquitous enzyme functioning in the intracellular distribution of terminal phosphate bond energy among the various nucleotides used in synthetic and regulatory functions in cells. We have previously reported that in Pseudomonas aeruginosa, this important enzyme is transcriptionally regulated by the gene algR2 and posttranslationally regulated by a phosphoprotein phosphatase for the phosphorylated form of Ndk. We report here that an intracellular protease cleaves the 16-kDa form of Ndk to a 12-kDa form that undergoes autophosphorylation with an efficiency almost identical to that of the 16-kDa form. The 12-kDa form was found to be predominantly associated with the P. aeruginosa cell membrane fraction, whereas the 16-kDa form was predominantly cytoplasmic. In the membrane-associated state, the 12-kDa form of Ndk was found to synthesize GTP in preference to other nucleoside triphosphates. The specificity toward GTP synthesis could be abolished by the addition of Tween 20 or Triton X-100. The activity itself could be abolished by the addition of anti-Ndk antibody to the assay mixture. The formation of the 12-kDa form of Ndk and its association with the cell membrane were found to be related to the growth stage of P. aeruginosa, with less than 1% of the 12-kDa Ndk detectable in the membrane fraction at early log phase in comparison with the levels present at late stationary phase.

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Selected References

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  1. Ahnn J., March P. E., Takiff H. E., Inouye M. A GTP-binding protein of Escherichia coli has homology to yeast RAS proteins. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8849–8853. doi: 10.1073/pnas.83.23.8849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Almaula N., Lu Q., Delgado J., Belkin S., Inouye M. Nucleoside diphosphate kinase from Escherichia coli. J Bacteriol. 1995 May;177(9):2524–2529. doi: 10.1128/jb.177.9.2524-2529.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  4. Bominaar A. A., Tepper A. D., Véron M. Autophosphorylation of nucleoside diphosphate kinase on non-histidine residues. FEBS Lett. 1994 Oct 10;353(1):5–8. doi: 10.1016/0014-5793(94)00997-x. [DOI] [PubMed] [Google Scholar]
  5. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  6. Hoch J. A. Genetics of bacterial sporulation. Adv Genet. 1976;18:69–98. doi: 10.1016/s0065-2660(08)60437-x. [DOI] [PubMed] [Google Scholar]
  7. Kavanaugh-Black A., Connolly D. M., Chugani S. A., Chakrabarty A. M. Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5883–5887. doi: 10.1073/pnas.91.13.5883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kimura N., Shimada N. Membrane-associated nucleoside diphosphate kinase from rat liver. Purification, characterization, and comparison with cytosolic enzyme. J Biol Chem. 1988 Apr 5;263(10):4647–4653. [PubMed] [Google Scholar]
  9. Lacombe M. L., Wallet V., Troll H., Véron M. Functional cloning of a nucleoside diphosphate kinase from Dictyostelium discoideum. J Biol Chem. 1990 Jun 15;265(17):10012–10018. [PubMed] [Google Scholar]
  10. Leone A., Flatow U., King C. R., Sandeen M. A., Margulies I. M., Liotta L. A., Steeg P. S. Reduced tumor incidence, metastatic potential, and cytokine responsiveness of nm23-transfected melanoma cells. Cell. 1991 Apr 5;65(1):25–35. doi: 10.1016/0092-8674(91)90404-m. [DOI] [PubMed] [Google Scholar]
  11. Lerner C. G., Inouye M. Pleiotropic changes resulting from depletion of Era, an essential GTP-binding protein in Escherichia coli. Mol Microbiol. 1991 Apr;5(4):951–957. doi: 10.1111/j.1365-2958.1991.tb00770.x. [DOI] [PubMed] [Google Scholar]
  12. Mathews C. K. The cell-bag of enzymes or network of channels? J Bacteriol. 1993 Oct;175(20):6377–6381. doi: 10.1128/jb.175.20.6377-6381.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. May T. B., Shinabarger D., Boyd A., Chakrabarty A. M. Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa. J Biol Chem. 1994 Feb 18;269(7):4872–4877. [PubMed] [Google Scholar]
  14. Muñoz-Dorado J., Almaula N., Inouye S., Inouye M. Autophosphorylation of nucleoside diphosphate kinase from Myxococcus xanthus. J Bacteriol. 1993 Feb;175(4):1176–1181. doi: 10.1128/jb.175.4.1176-1181.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nickerson J. A., Wells W. W. The microtubule-associated nucleoside diphosphate kinase. J Biol Chem. 1984 Sep 25;259(18):11297–11304. [PubMed] [Google Scholar]
  16. Pall M. L. GTP: a central regulator of cellular anabolism. Curr Top Cell Regul. 1985;25:1–20. doi: 10.1016/b978-0-12-152825-6.50005-9. [DOI] [PubMed] [Google Scholar]
  17. Pall M. L., Robertson C. K. Growth regulation by GTP. Regulation of nucleotide pools in Neurospora by nitrogen and sulfur control systems. J Biol Chem. 1988 Aug 15;263(23):11168–11174. [PubMed] [Google Scholar]
  18. Penningroth S. M., Kirschner M. W. Nucleotide binding and phosphorylation in microtubule assembly in vitro. J Mol Biol. 1977 Oct 5;115(4):643–673. doi: 10.1016/0022-2836(77)90108-5. [DOI] [PubMed] [Google Scholar]
  19. Postel E. H., Berberich S. J., Flint S. J., Ferrone C. A. Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis. Science. 1993 Jul 23;261(5120):478–480. doi: 10.1126/science.8392752. [DOI] [PubMed] [Google Scholar]
  20. Ray N. B., Mathews C. K. Nucleoside diphosphokinase: a functional link between intermediary metabolism and nucleic acid synthesis. Curr Top Cell Regul. 1992;33:343–357. doi: 10.1016/b978-0-12-152833-1.50025-3. [DOI] [PubMed] [Google Scholar]
  21. Saeki T., Hori M., Umezawa H. Pyruvate kinase of Escherichia coli. Its role in supplying nucleoside triphosphates in cells under anaerobic conditions. J Biochem. 1974 Sep;76(3):631–637. doi: 10.1093/oxfordjournals.jbchem.a130607. [DOI] [PubMed] [Google Scholar]
  22. Schlictman D., Kavanaugh-Black A., Shankar S., Chakrabarty A. M. Energy metabolism and alginate biosynthesis in Pseudomonas aeruginosa: role of the tricarboxylic acid cycle. J Bacteriol. 1994 Oct;176(19):6023–6029. doi: 10.1128/jb.176.19.6023-6029.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schlictman D., Kubo M., Shankar S., Chakrabarty A. M. Regulation of nucleoside diphosphate kinase and secretable virulence factors in Pseudomonas aeruginosa: roles of algR2 and algH. J Bacteriol. 1995 May;177(9):2469–2474. doi: 10.1128/jb.177.9.2469-2474.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shankar S., Kavanaugh-Black A., Kamath S., Chakrabarty A. M. Characterization of a phosphoprotein phosphatase for the phosphorylated form of nucleoside-diphosphate kinase from Pseudomonas aeruginosa. J Biol Chem. 1995 Nov 24;270(47):28246–28250. doi: 10.1074/jbc.270.47.28246. [DOI] [PubMed] [Google Scholar]
  25. Steeg P. S., Bevilacqua G., Kopper L., Thorgeirsson U. P., Talmadge J. E., Liotta L. A., Sobel M. E. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst. 1988 Apr 6;80(3):200–204. doi: 10.1093/jnci/80.3.200. [DOI] [PubMed] [Google Scholar]
  26. Walton G. M., Gill G. N. Nucleotide regulation of a eukaryotic protein synthesis initiation complex;. Biochim Biophys Acta. 1975 May 1;390(2):231–245. doi: 10.1016/0005-2787(75)90344-5. [DOI] [PubMed] [Google Scholar]

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