Glutamine synthetases (GS) are central enzymes essential for nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG), which is the central signal for nitrogen starvation, is directly inducing the GS activity (Ehlers et al., 2005). Moreover, modulation of GlnA1 activity by protein-protein interactions with the two small proteins GlnK1 and small protein 26 (sP26) has been described (Ehlers et al., 2005; Gutt et al., 2021). Thereby, GlnK1 was found to interact with GlnA1 under nitrogen upshift by pull-down approaches, pointing towards an inhibitory function of GlnK1 under shifting conditions from nitrogen limitation to sufficiency (Ehlers et al., 2005). Additionally, the 2.5 kDa sP26 was described to interact with GlnA1, stabilizing the complex and enhancing GlnA1 activity (Gutt et al., 2021). Here, we aim to clarify the previously reported contradictory effects of GlnK1 on M. mazei GlnA1 by mass photometry (MP) analysis. The underlying molecular mechanism of the strong 2-OG activation of M. mazei GlnA1 was elucidated using MP. Moreover, we determined the cryo-electron microscopy single-particle structure of the purified GlnA1 dodecamer arguing for a unique regulation of M. mazei GS.

Ehlers, C., Weidenbach, K., Veit, K., Forchhammer, K., Schmitz, R.A, 2005. Unique mechanistic features of post-translational regulation of glutamine synthetase activity in M. mazei strain Gö1 in response to nitrogen availability: Regulation of glutamine synthetase activity in M. mazei. Mol. Microbiol. 55 https://doi.org/10.1111/j.1365-2958.2005.04511.x

Gutt, M., Jordan, B., Weidenbach, K., Gudzuhn, M., Kiessling, C., Cassidy, L., Helbig, A., Tholey, A., Pyper, D.J., Kubatova, N., Schwalbe, H., Schmitz, R.A., 2021. High complexity of Glutamine synthetase regulation in M. mazei: Small protein 26 interacts and enhances [...] FEBS J. https://doi.org/10.1111/febs.15799