Current Microbiology

, Volume 30, Issue 6, pp 367–372 | Cite as

Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe

  • Angelos Perysinakis
  • James R. Kinghorn
  • Constantin Drainas
Article

Abstract

Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamineoxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as Km values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.

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Literature Cited

  1. 1.
    Barel I, Bignell G, Simpson A, MacDonald D (1988) Isolation of a DNA fragment which complements glutamine synthetase deficient strains of S. pombe. Curr Genet 13:487–494Google Scholar
  2. 2.
    Beaucamp K, Bergmeyer HU, Beutler OH (1974) Coenzymes, metabolites and other biochemical reagents. In: HU Bergmeyer (ed) Methods of enzymatic analysis, vol. 1. New York: Academic Press, pp 523–556Google Scholar
  3. 3.
    Bergmeyer HU, Bernt E, Grassl M, Michal G (1974) Evaluation of experimental results. In: HU Bergmeyer (ed) Methods of enzymatic analysis, vol. 1. New York: Academic Press, pp 308–317Google Scholar
  4. 4.
    Bogonez E, Satrustegui J, Machado A (1985) Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae. J Gen Microbiol 131:1425–1432Google Scholar
  5. 5.
    Brenchley JE, Magasanik B (1974) Mutants of Klebsiella aerogenes lacking glutamate dehydrogenase. J Bacteriol 117: 544–550Google Scholar
  6. 6.
    Brown CM, Burn VL, Johnson B (1973) Presence of glutamate synthase in fission yeasts and its possible role in ammonia assimilation. Nature New Biol 246:115–116Google Scholar
  7. 7.
    Bueno R, Pahel G, Magasanik B (1985) Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli. J Bacteriol 164:816–822Google Scholar
  8. 8.
    Doherty D (1970) L-Glutamate dehydrogenases (yeast). Methods Enzymol 17A:850–856Google Scholar
  9. 9.
    Feng J, Atkinson MR, McCleary W, Stock JB, Wanner BL, Ninfa AJ (1992) Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J Bacteriol 174:6061–6070Google Scholar
  10. 10.
    Green J, Large PJ (1984) Regulation of the key enzymes of methylated amine metabolism in Candida boidinii. J Gen Microbiol 130:1947–1959Google Scholar
  11. 11.
    Gutz H, Heslot H, Leupold U, Loprieno N (1977) Schizosaccharomyces pombe. In: RC King (ed) Handbook of genetics, vol. I. New York: Plenum Press, pp 395–446Google Scholar
  12. 12.
    Hernandez G, Mora J (1986) Glutamine synthesis regulates sucrose catabolism in Neurospora crassa. J Gen Microbiol 132:3315–3323Google Scholar
  13. 13.
    Holmes AR, Collings A, Farnden KJF, Shepherd MG (1989) Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase. J Gen Microbiol 135:1423–1430Google Scholar
  14. 14.
    Johnson B, Brown CM (1974) The enzymes of ammonia assimilation in Schizosaccharomyces spp. J Gen Microbiol 85:169–172Google Scholar
  15. 15.
    Kohli J (1987) Genetic nomenclature and gene list of the fission yeast Schizosaccharomyces pombe. Curr Genet 11:575–589Google Scholar
  16. 16.
    Kohli J, Hottinger H, Munz P, Strauss A, Thuriaux P (1977) Genetic marring in Schizosaccharomyces pombe by mitotic and meiotic analysis and induced haploidization. Genetics 87:471–489Google Scholar
  17. 17.
    Kusnan MB, Berger MG, Fock HP (1987) The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans. J Gen Microbiol 133:1235–1242Google Scholar
  18. 18.
    Lomnitz A, Calderon J, Hernandez G, Mora J (1987) Functional analysis of ammonium assimilation enzymes in Neurospora crassa. J Gen Microbiol 133:2333–2340Google Scholar
  19. 19.
    Lowry OH, Rosebrough NJ, Farr AL, Randal RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  20. 20.
    Mandal AK, Ghosh S (1993) Isolation of a glutamate synthase (GOGAT)-negative, pleiotropically N utilization-defective mutant of Azospirillum brasilense: cloning and partial characterization of GOGAT structural gene. J Bacteriol 175:8024–8029Google Scholar
  21. 21.
    Meeks JC, Wolk CP, Lockau W, Schilling N, Shaffer PW, Chien WS (1978) Pathways of assimilation of [13N] N2 and 13NH4 + by cyanobacteria with and without heterocysts. J Bacteriol 134:125–130Google Scholar
  22. 22.
    Meers JL, Tempest DW, Brown CM (1970) “Glutamine(amide): 2-oxoglutarate amino transferase oxido-reductase (NADP),” an enzyme involved in the synthesis of glutamate by some bacteria. J Gen Microbiol 64:187–194Google Scholar
  23. 23.
    Ninfa AJ, Bennett RL (1991) Identification of the site of autophosphorylation of the bacterial protein kinase/phosphatase 372-1. J Biol Chem 266:6888–6893Google Scholar
  24. 24.
    Perysinakis A, Kinghorn JR, Drainas C (1994) Biochemical and genetical studies of NADP-specific glutamate dehydrogenase in the fission yeast Schizosaccharomyces pombe. Curr Genet 26:315–320Google Scholar
  25. 25.
    Sanchez F, Calva E, Campomanes M, Blanco L, Guzman J, Saborio JL, Palacios R (1980) Heterogeneity of glutamine synthetase polypeptides in Neurospora crassa. J Biol Chem 255:2231–2234Google Scholar
  26. 26.
    Van Andel JG, Brown CM (1977) Ammonia assimilation in the fission yeast Schizosaccharomyces pombe 972. Arch Microbiol 111:265–270Google Scholar
  27. 27.
    Weiss V, Magasanik B (1988) Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli. Proc Natl Acad Sci USA 85:8919–8923Google Scholar
  28. 28.
    Westby CA, Enderlin CS, Steinberg NA, Joseph CM, Meeks JC (1987) Assimilation of 13NH4 + by Azospirillum brasilense grown under nitrogen limitation and excess. J Bacteriol 169: 4211–4214Google Scholar
  29. 29.
    Woolfolk CA, Shapiro B, Stadtman ER (1966) Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch Biochem Biophys 116:177–192Google Scholar
  30. 30.
    Zwart KB, Harder W (1983) Regulation of the metabolism of some alkylated amines in the yeasts Candida utilis and Hansenula polymorpha. J Gen Microbiol 129:3157–3169Google Scholar

Copyright information

© Springer-Verlag New York Inc 1995

Authors and Affiliations

  • Angelos Perysinakis
    • 1
  • James R. Kinghorn
    • 2
  • Constantin Drainas
    • 1
  1. 1.Sector of Organic Chemistry and Biochemistry, Department of ChemistryUniversity of IoanninaIoanninaGreece
  2. 2.Division of Plant Ecology and Evolution, School of Biological and Medical SciencesUniversity of St. AndrewsSt. AndrewsScotland, UK

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