Differences in the regulation mechanisms of the glutamine synthetase from methanogenic archaea unveiled by structural investigations

Abstract

Glutamine synthetases catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be tightly regulated to fit cellular needs. While their catalytic mechanisms and regulation are well-characterized in bacteria and eukaryotes, only limited knowledge exists about the archaeal representatives. Here, we natively purified the glutamine synthetases fromMethanothermococcus thermolithotrophicusandMethermicoccus shengliensis, two thermophilic methanogens belonging to different orders. Biochemical investigations combined with X-ray crystallography unveiled the first structures of archaeal glutamine synthetases and highlighted differences in their regulation. The enzyme fromM. thermolithotrophicusis inactive in its resting state and employs 2-oxoglutarate as an on-switch. The 2-oxoglutarate acts as a sensor of cellular nitrogen deficiency, and its reported cellular concentration remarkably overlays with that required for the enzyme activation. Its binding to an allosteric pocket leads to the reconfiguration of the active site and promotes a catalytically competent state. The homolog fromM. shengliensisdoes not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited by glutamine, as shown for bacterial homologs. The glutamine inhibition depends on a key arginine residue from the Asp50ʹ-loop. The arginine is substituted by a glycine inM. thermolithotrophicus, abolishing the inhibitory effect. While the effectors are surprisingly different, the molecular switch controlling the glutamine synthetase activity is fundamentally the same and depends on the correct positioning of the Asp50ʹ-loop and a catalytic arginine. Residue conservation suggests that both regulation mechanisms are widespread and not mutually exclusive across archaea.

2-oxoglutarate triggers assembly of active dodecamericMethanosarcina mazeiglutamine synthetase

“…For bacteria it is known, that GS can be feedback inhibited. Very recently, the first feedback inhibition of an archaeal GS by glutamine has been reported for Methermicoccus shengliensis GS (Müller et al, 2023). The specific arginine residue identified to be relevant for the feedback inhibition is R66.…”