Mutational analysis of the inactivating factors, IF7 and IF17 from Synechocystis sp. PCC 6803: critical role of arginine amino acid residues for glutamine synthetase inactivation

The Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by a process that involves protein-protein interaction with two inactivating factors (IF7 and IF17). IF7 is a natively unfolded, 65-residue-long protein, homologous to the carboxy-terminal region of IF17. Both proteins have abundance of positively charged amino acid residues and a high isoelectric point. In this study, we analyse the IF amino acid residues involved in GS inactivation by a mutational approach, both in vitro and in vivo. The results clearly indicate that the GS-IF complex formation must be determined mainly by electrostatic interactions. We have identified three conserved arginine residues of IF7 and IF17 that are essential for the interaction of these proteins with GS. All these residues map in the homologous region of IFs. Furthermore, in vitro analysis of a truncated IF17 protein without the 82-residue-long amino-terminal part, together with the analysis of a Synechocystis strain expressing a chimeric protein, containing this amino-terminal part of IF17 fused to IF7, demonstrates that amino-terminal region of IF17 mostly confers a higher stability to this protein.     

Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.     

A core of three amino acids at the carboxyl‐terminal region of glutamine synthetase defines its regulation in cyanobacteria

Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein–protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site‐directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C‐terminus of Synechocystis GS. A protein–protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction.