Modulation of Glutamine Synthetase adenylylation by nitrogen availability enables one-step purification in distinct post-translational states.

The glutamine synthetase (GS) enzyme pathway promotes ammonium assimilation in bacteria and is a metabolic hub for glutamine and glutamate homeostasis. Bacterial GS can be reversibly inhibited through adenylylation as a response to nitrogen availability, carried out by the GlnE enzyme. The adenylylation changes GS catalytic and regulatory properties, such as the sensitivity to negative feedback by allosteric modulators and the preferred cofactor usage. In this way, the purification of GS in different modification states can be useful during the investigation of its regulatory properties. Here we show that just by changing nitrogen availability during cell growth it is possible to obtain adenylylated or unmodified GS enzymes after heterologous expression followed by a one-step purification. As a model, we expressed GS enzymes from the diazotrophic bacteria Herbaspirillum seropedicae and Azospirillum brasilense in the M9 media supplemented with ammonium or glutamine. The enzymes were purified by Ni-affinity chromatography. The data showed that just by varying the nitrogen source during protein expression it was possible to obtain GS in different adenylation status. The different adenylated isoforms of GS obtained were confirmed by electrophoretic mobility shifts and showed unique responses to Mg and Mn ions and feedback inhibition by amino acids. Finally, we show the unmodified GS can only bind the glutamate substrate when ATP is present. The method to purify GS on different adenylylation states in a single step described here will facilitate the characterization of this key metabolic enzyme in the future.