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

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Abstract

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.

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Abbreviations

GS:

Glutamine synthetase

pHMB:

p-Hydroxymercuribenzoate

DTNB:

5,5′-Dithiobis-2-nitrobenzoate

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Acknowledgments

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).

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Correspondence to Antonio J. Márquez.

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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

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Keywords

  • Ammonium assimilation
  • Cysteine
  • Glutamine synthetase
  • Quaternary structure