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

Identification of an essential cysteinyl residue for the structure of glutamine synthetase α from Phaseolus vulgaris

  • 180 Accesses

  • 5 Citations


We have studied the possible role, in a plant glutamine synthetase (GS), of the different cysteinyl residues present in this enzyme. For this purpose we carried out the site-directed mutagenesis of the cDNA for α-GS polypeptide from Phaseolus vulgaris in the positions corresponding to Cys-92, Cys-159, and Cys-179, followed by heterologous expression in E. coli and enzymatic characterisation of WT and mutant proteins. The results show that neither Cys-92 nor Cys-179 residues were essential for enzyme activity, but the replacement of Cys-159 by alanine or serine strongly affects the quaternary structure and function of the GS enzyme molecule, resulting in a complete loss of enzymatic activity. Other studies using sulfhydryl specific reagents such as pHMB (p-hydroxymercuribenzoate) or DTNB (5,5′-dithiobis-2-nitrobenzoate) confirmed that the profound inhibition produced is associated with an important alteration of the quaternary structure of GS, and suggest that Cys-159 might be the residue responsible for the enzyme inhibition. All these results suggest that the Cys-159 residue is essential for the enzyme structure. The results are also consistent with previous reports based on classical biochemistry studies indicating the presence of essential cysteinyl residues for the enzyme activity of higher plant GS.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7



Glutamine synthetase






  1. Bernard SM, Habash DZ (2009) The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol 182:608–620

  2. Betti M, Márquez AJ, Yanes C, Maestre A (2002) ATP binding to purified homopolymeric plant glutamine synthetase studied by isothermal titration calorimetry. Thermochim Acta 394:63–71

  3. Betti M, Arcondeguy T, Márquez AJ (2006) Molecular analysis of two mutants from Lotus japonicus deficient in plant glutamine synthetase: functional properties of purified GLN2 enzymes. Planta 224:1068–1079

  4. Boksha IS, Schonfeld HJ, Langen H, Muller F, Tereshkina EB, Burbaeva GS (2002) Glutamine synthetase isolated from human brain: octameric structure and homology of partial primary structure with human liver glutamine synthetase. Biochemistry (Mosc) 67:1012–1020

  5. Choi YA, Kim SG, Kwon YM (1999) The plastidic glutamine synthetase activity is directly modulated by means of redox change at two unique cysteinyl residues. Plant Sci 149:175–182

  6. Clemente MT, Márquez AJ (1999a) Functional importance of Asp56 from the α-polypeptide of Phaseolus vulgaris glutamine synthetase. An essential residue for transferase but not for biosynthetic enzyme activity. Eur J Biochem 264:453–460

  7. Clemente MT, Márquez AJ (1999b) Site-directed mutagenesis of Glu-297 from the α-polypeptide of Phaseolus vulgaris glutamine synthetase alters kinetic and structural properties and confers resistance to l-methionine sulfoximine. Plant Mol Biol 40:835–845

  8. Clemente MT, Márquez AJ (2000) Site-directed mutagenesis of Cys-92 from the α-polypeptide of Phaseolus vulgaris glutamine synthetase reveals that this highly conserved residue is not essential for enzyme activity but it is involved in thermal stability. Plant Sci 154:189–197

  9. Eisenberg D, Gill HS, Pfluegl GMU, Rotstein SH (2000) Structure–function relationships of glutamine synthetases. Biochim Biophys Acta 1477:122–145

  10. Ericson MC, Brunn SA (1985) Cysteinyl residues at the active site of glutamine synthetase from spinach leaves. Biochem Biophys Res Commun 17:527–531

  11. Evstigneeva ZG, Pushkin AV, Akentyeva NP, Kretovich WL (1985) Active site of glutamine synthetase from chloroplast and cytosol of pea leaves. Physiol Vég 23:861–868

  12. Forde BG, Cullimore JV (1989) The molecular biology of glutamine synthetase in higher plants. Oxf Surv Plant Mol Cell Biol 6:247–296

  13. He Y, Gui L, Liu Y, Du Y, Zhou Y, Li P, Zhou C (2009) Crystal structure of Saccharomyces cerevisiae glutamine synthetase Gln1 suggests a nanotube-like supramolecular assembly. Proteins 76:249–254

  14. Hirel BG, Lea PJ (2002) The biochemistry, molecular biology, and genetic manipulation of primary ammonia assimilation. In: Foyer CH, Noctor G (eds) Photosynthetic nitrogen assimilation and associated carbon and respiratory metabolism. Kluwer, Dordrecht, pp 71–92

  15. Johnson RJ, Piskiewicz D (1985) Primary structure of peptides from bovine brain glutamine synthetase. Comparison with sequences of glutamine synthetases from other organisms. Biochim Biophys Acta 827:439–446

  16. Krajewski WW, Collins R, Holmberg-Schiavone L, Jones TA, Karlberg T, Mowbray SL (2008) Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design. J Mol Biol 375:217–228

  17. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685

  18. Lakowicz JR (1999) Principles of fluorescence spectroscopy. Plenum Publishers, New York, pp 185–210

  19. Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100:95–97

  20. Lea PJ (1991) The inhibition of ammonia assimilation: a mechanism of herbicide action. In: Baker NR, Percival MO (eds) Herbicides. Elsevier, Amsterdam, pp 267–298

  21. Lehrer SS (1971) Solute perturbation of protein fluorescence. The quenching of the tryptophanyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10:3254–3263

  22. Limami AR, Phillipson B, Ameziane R, Pernollet N, Jiang Q, Roy R, Deleens E, Chaumont-Bonnet M, Gresshoff PM, Hirel B (1999) Does root glutamine synthetase control plant biomass production in Lotus japonicus L.? Planta 209:495–502

  23. Limami AR, Rouillon C, Glevarec G, Gallais A, Hirel B (2002) Genetic and physiological analysis of germination efficiency in maize in relation to nitrogen metabolism reveals the importance of cytosolic glutamine synthetase. Plant Physiol 130:1860–1870

  24. Llorca O, Betti M, González JM, Valencia A, Márquez AJ, Valpuesta JM (2006) The three-dimensional structure of an eukaryotic glutamine synthetase: functional implications of its oligomeric structure. J Struct Biol 156:469–479

  25. Mäck G (1998) Glutamine synthetase isoenzymes, oligomers and subunits from hairy roots of Beta vulgaris L. var. lutea. Planta 205:113–120

  26. Márquez AJ, Betti M, García-Calderón M, Pal’ove-Balang P, Díaz P, Monza J (2005a) Nitrate assimilation in Lotus japonicus. J Exp Bot 56:1729–1739

  27. Márquez AJ, Betti M, García-Calderón M, Estivill G, Credali A, Pajuelo P, Orea A, Clemente MT, Pajuelo E, Galván F (2005b) Nitrate and ammonium assimilatory enzymes. In: Márquez AJ (ed) Lotus japonicus handbook. Springer, Dordrecht, pp 315–328

  28. Motohashi K, Kondoh A, Stumpp MT, Hisabori T (2001) Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci USA 98:11224–11229

  29. Pesole G, Bozzetti MP, Lanave C, Preparata G, Saccone C (1991) Glutamine synthetase gene evolution: a good molecular clock. Proc Natl Acad Sci USA 15:522–526

  30. Pushkin AV, Tsuprun VL, Dzhokharidze TZ, Evstingneeva ZG, Kretovich WL (1981) Glutamine synthetase from the pumpkin leaf cytosol. Biophys Acta 662:160–162

  31. Rao DR, Beyreuther K, Jaenicke L (1973) A comparative study of pig and sheep-brain glutamine synthetases: tryptic peptides and thiol groups. Eur J Biochem 15:582–592

  32. Rees T, Larson TR, Heldens J, Huning F (1995) In situ glutamine synthetase activity in a marine unicellular alga (development of a sensitive colorimetric assay and the effects of nitrogen status on enzyme activity). Plant Physiol 109:1405–1410

  33. Riddles PW, Blakeley RL, Zerner B (1983) Reassessment of Ellman’s reagent. Methods Enzymol 91:49–60

  34. Seabra AR, Carvalho H, Pereira PJ (2009) Crystallization and preliminary crystallographic characterization of glutamine synthetase from Medicago truncatula. Acta Crystallogr Sect F Struct Biol Cryst Commun 65:1309–1312

  35. Simonović AD, Gaddameedhi S, Anderson MD (2004) In-gel precipitation of enzymatically released phosphate. Anal Biochem 334:312–317

  36. Tavernier V, Cadiou S, Pageau K, Laugé R, Reisdorf-Cren M, Langin T, Masclaux-Daubresse C (2007) The plant nitrogen mobilization promoted by Colleotrichum lindemuthianum in Phaseolus leaves depends on fungus pathogenicity. J Exp Bot 58:3351–3360

  37. Tsuprun VL, Samsonidze TG, Radukina NA, Pushkin AV, Evstigneeva ZG, Kretovich WL (1980) Electron microscopy of glutamine synthetase from pea leaf chloroplasts. Biochim Biophys Acta 20:1–4

  38. Unno H, Uchida T, Sugawara H, Kurisu G, Sugiyama T, Yamaya T, Sakakibara H, Hase T, Kusunoki M (2006) Atomic structure of plant glutamine synthetase: a key enzyme for plant productivity. J Biol Chem 281:29287–29296

Download references


Authors thank funding of projects BFU2005-03120 and BIO2008-03213 from Spanish Ministry of Science and Junta de Andalucía (BIO-163; P-07-CVI-3026). G.E. was the recipient of a FPI fellowship from Junta de Andalucía. R.B. carried out his M.Sc. Thesis project by agreement with École Polytechnique Fédéral de Lausanne (Switzerland).

Author information

Correspondence to Antonio J. Márquez.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Estivill, G., Guardado, P., Buser, R. et al. Identification of an essential cysteinyl residue for the structure of glutamine synthetase α from Phaseolus vulgaris . Planta 231, 1101–1111 (2010). https://doi.org/10.1007/s00425-010-1115-z

Download citation


  • Ammonium assimilation
  • Cysteine
  • Glutamine synthetase
  • Quaternary structure