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Cited by 6Enzyme Structure and FunctionAmino Acid Enzymes and MetabolismBiochemical and Molecular Research

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2-oxoglutarate triggers assembly of active dodecameric Methanosarcina mazei glutamine synthetase

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Herdering, Eva, Reif-Trauttmansdorff, Tristan, Kumar, Anuj, Habenicht, Tim, Hochberg, Georg, Bohn, Stefan, Schuller, Jan, Schmitz, Ruth Anne·2025·eLife
DOI 10.7554/elife.97484PMC PMC11957540

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Abstract

Glutamine synthetases (GS) are central enzymes essential for the 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) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK 1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA 1 ) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA 1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA 1 . The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK 1 . The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK 1 and crucially depends on the presence of 2-OG.

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Research works printed but not linked: 59

  1. Almassy, 1986, Novel subunit-subunit interactions in the structure of glutamine synthetase, Nature, 323, 304, 10.1038/323304a0 · DOI
  2. Bolay, 2018, The distinctive regulation of cyanobacterial glutamine synthetase, Life, 8, 10.3390/life8040052 · DOI
  3. Brown, 1994, Evolutionary relationships of bacterial and archaeal glutamine synthetase genes, Journal of Molecular Evolution, 38, 566, 10.1007/BF00175876 · DOI
  4. Cohen-Kupiec, 1999, Function and regulation of glnA in the methanogenic archaeon Methanococcus maripaludis, Journal of Bacteriology, 181, 256, 10.1128/JB.181.1.256-261.1999 · DOI
  5. Deuel, 1970, Bacillus subtilis Glutamine Synthetase, Journal of Biological Chemistry, 245, 5195, 10.1016/S0021-9258(18)62741-3 · DOI
  6. dos Santos Moreira, 2019, Glutamine synthetase structure‐catalysis relationship—Recent advances and applications, WIREs Computational Molecular Science, 9, 10.1002/wcms.1399 · DOI
  7. Ehlers, 2005, Unique mechanistic features of post-translational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability, Molecular Microbiology, 55, 1841, 10.1111/j.1365-2958.2005.04511.x · DOI
  8. Emsley, 2010, Features and development of Coot, Acta Crystallographica. Section D, Biological Crystallography, 66, 486, 10.1107/S0907444910007493 · DOI · Paper page
  9. Fedorova, 2013, Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis, FEBS Letters, 587, 1293, 10.1016/j.febslet.2013.03.015 · DOI
  10. Fisher, 1999, Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!, Molecular Microbiology, 32, 223, 10.1046/j.1365-2958.1999.01333.x · DOI · Paper page
  11. Fisher, 2008, Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes, PNAS, 105, 1014, 10.1073/pnas.0709949105 · DOI
  12. Forchhammer, 1999, The PII protein in synechococcus PCC 7942 senses and signals 2-oxoglutarate under ATP-replete conditions, 549, 10.1007/978-1-4615-4827-0_63 · DOI
  13. García-Domínguez, 1999, Glutamine synthetase inactivation by protein-protein interaction, PNAS, 96, 7161, 10.1073/pnas.96.13.7161 · DOI
  14. Gunka, 2012, Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation, Molecular Microbiology, 85, 213, 10.1111/j.1365-2958.2012.08105.x · DOI
  15. Gutt, 2021, High complexity of Glutamine synthetase regulation in Methanosarcina mazei: Small protein 26 interacts and enhances glutamine synthetase activity, The FEBS Journal, 288, 5350, 10.1111/febs.15799 · DOI
  16. Hanahan, 1983, Studies on transformation of Escherichia coli with plasmids, Journal of Molecular Biology, 166, 557, 10.1016/s0022-2836(83)80284-8 · DOI
  17. Hauf, 2016, The molecular basis of TnrA control by glutamine synthetase in Bacillus subtilis, The Journal of Biological Chemistry, 291, 3483, 10.1074/jbc.M115.680991 · DOI
  18. He, 2009, Crystal structure of Saccharomyces cerevisiae glutamine synthetase Gln1 suggests a nanotube-like supramolecular assembly, Proteins, 76, 249, 10.1002/prot.22403 · DOI
  19. Herrero, 2001, Nitrogen control in cyanobacteria, Journal of Bacteriology, 183, 411, 10.1128/JB.183.2.411-425.2001 · DOI
  20. Huang, 2022, Structural basis for the helical filament formation of Escherichia coli glutamine synthetase, Protein Science, 31, 10.1002/pro.4304 · DOI
  21. Inoue, 1990, High efficiency transformation of Escherichia coli with plasmids, Gene, 96, 23, 10.1016/0378-1119(90)90336-p · DOI
  22. Jäger, 2009, Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability, PNAS, 106, 21878, 10.1073/pnas.0909051106 · DOI
  23. Jiang, 1998, Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli, Biochemistry, 37, 12795, 10.1021/bi9802420 · DOI
  24. Klähn, 2015, The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7, PNAS, 112, E6243, 10.1073/pnas.1508412112 · DOI
  25. Klähn, 2018, A glutamine riboswitch is A key element for the regulation of glutamine synthetase in cyanobacteria, Nucleic Acids Research, 46, 10082, 10.1093/nar/gky709 · DOI
  26. Legrain, 1982, Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis, European Journal of Biochemistry, 123, 611, 10.1111/j.1432-1033.1982.tb06576.x · DOI
  27. Liaw, 1993, Feedback inhibition of fully unadenylylated glutamine synthetase from Salmonella typhimurium by glycine, alanine, and serine, PNAS, 90, 4996, 10.1073/pnas.90.11.4996 · DOI
  28. Liaw, 1994, Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes, Biochemistry, 33, 675, 10.1021/bi00169a007 · DOI
  29. Lie, 2007, Diverse homologues of the archaeal repressor NrpR function similarly in nitrogen regulation, FEMS Microbiology Letters, 271, 281, 10.1111/j.1574-6968.2007.00726.x · DOI
  30. Liebschner, 2019, Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix, Acta Crystallographica. Section D, Structural Biology, 75, 861, 10.1107/S2059798319011471 · DOI · Paper page
  31. Magasanik, 1993, The regulation of nitrogen utilization in enteric bacteria, Journal of Cellular Biochemistry, 51, 34, 10.1002/jcb.240510108 · DOI
  32. Masalkar, 2015, Glutamine synthetase isoforms in nitrogen-fixing soybean nodules: distinct oligomeric structures and thiol-based regulation, FEBS Letters, 589, 215, 10.1016/j.febslet.2014.11.048 · DOI
  33. Müller, 2024, Differences in regulation mechanisms of glutamine synthetases from methanogenic archaea unveiled by structural investigations, Communications Biology, 7, 10.1038/s42003-023-05726-w · DOI
  34. Murray, 2013, Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism, The Journal of Biological Chemistry, 288, 35801, 10.1074/jbc.M113.519496 · DOI
  35. Pedro-Roig, 2013, Regulation of ammonium assimilation in Haloferax mediterranei: interaction between glutamine synthetase and two GlnK proteins, Biochimica et Biophysica Acta, 1834, 16, 10.1016/j.bbapap.2012.10.006 · DOI
  36. Petrovska, 2014, Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation, eLife, 3, 10.7554/eLife.02409 · DOI
  37. Pettersen, 2021, UCSF ChimeraX: Structure visualization for researchers, educators, and developers, Protein Science, 30, 70, 10.1002/pro.3943 · DOI
  38. Prasse, 2018, Small RNAs involved in regulation of nitrogen metabolism, Microbiology Spectrum, 6, 10.1128/microbiolspec.rwr-0018-2018 · DOI
  39. Punjani, 2017, cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination, Nature Methods, 14, 290, 10.1038/nmeth.4169 · DOI · Paper page
  40. Reitzer, 2003, Nitrogen assimilation and global regulation in Escherichia coli, Annual Review of Microbiology, 57, 155, 10.1146/annurev.micro.57.030502.090820 · DOI
  41. Schumacher, 2023, M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation, Nature Communications, 14, 10.1038/s41467-023-43243-w · DOI
  42. Shapiro, 1970, [130] glutamine synthetase (Escherichia coli), 910, 10.1016/0076-6879(71)17305-3 · DOI
  43. Stadtman, 1990, [60] discovery of glutamine synthetase cascad, 793, 10.1016/0076-6879(90)82062-7 · DOI
  44. Stadtman, 2001, The story of glutamine synthetase regulation, The Journal of Biological Chemistry, 276, 44357, 10.1074/jbc.R100055200 · DOI
  45. Stadtman, 2004, Regulation of glutamine synthetase activity, EcoSal Plus, 1, 10.1128/ecosalplus.3.6.1.6 · DOI
  46. Travis, 2022, Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria, Nature Communications, 13, 10.1038/s41467-022-31573-0 · DOI
  47. Valentine, 1968, Regulation of glutamine synthetase XII: electron microscopy of the enzyme from Escherichia coli, Biochemistry, 7, 2143, 10.1021/bi00846a017 · DOI
  48. van Heeswijk, 2013, Nitrogen assimilation in Escherichia coli: putting molecular data into a systems perspective, Microbiology and Molecular Biology Reviews, 77, 628, 10.1128/MMBR.00025-13 · DOI
  49. van Rooyen, 2011, Crystal structure of Type III glutamine synthetase: surprising reversal of the inter-ring interface, Structure, 19, 471, 10.1016/j.str.2011.02.001 · DOI
  50. Veit, 2005, Effects of nitrogen and carbon sources on transcription of soluble methyltransferases in Methanosarcina mazei strain Go1, Journal of Bacteriology, 187, 6147, 10.1128/JB.187.17.6147-6154.2005 · DOI
  51. Weidenbach, 2008, Insights into the NrpR regulon in Methanosarcina mazei Gö1, Archives of Microbiology, 190, 319, 10.1007/s00203-008-0369-3 · DOI
  52. Weidenbach, 2010, NrpRII mediates contacts between NrpRI and general transcription factors in the archaeon Methanosarcina mazei Gö1, The FEBS Journal, 277, 4398, 10.1111/j.1742-4658.2010.07821.x · DOI
  53. Wisedchaisri, 2010, Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR, Structure, 18, 1512, 10.1016/j.str.2010.08.014 · DOI
  54. Woolfolk, 1967, Regulation of glutamine synthetase. 3. Cumulative feedback inhibition of glutamine synthetase from Escherichia coli, Archives of Biochemistry and Biophysics, 118, 736, 10.1016/0003-9861(67)90412-2 · DOI
  55. Yamashita, 1989, Refined atomic model of glutamine synthetase at 3.5 A resolution, The Journal of Biological Chemistry, 264, 17681, 10.2210/pdb2gls/pdb · DOI
  56. Kumar A, Schuller JM, Schmitz RA. 2025. Cryo-EM structure of the active dodecameric Methanosarcina mazei glutamine synthetase. RCSB Protein Data Bank. https://www.rcsb.org/structure/8S59. 8S59.
  57. Murray DS, Chinnam N, Tonthat NK, Whitfill T, Wray LV, Fisher SH, Schumacher MA. 2013. B. subtilis glutamine synthetase structures reveal large active site conformational changes and basis for isoenzyme specific regulation: structure of apo form of GS. Worldwide Protein Data Bank. · DOI
  58. Schumacher MA, Salinas R, Travis BA, Singh RR, Lent N. 2023. Cryo-EM structure of the Methanosarcina mazei glutamine synthetase (GS) with Met-Sox-P and ADP. Worldwide Protein Data Bank. · DOI
  59. Schumacher MA, Salinas R, Travis BA, Singh RR, Lent N. 2023. Cryo-EM structure of the Methanosarcina mazei apo glutamin synthetase structure: dodecameric form. Worldwide Protein Data Bank. · DOI

Metadata

Authors
Herdering, E., Reif-Trauttmansdorff, T., Kumar, A., Habenicht, T., Hochberg, G., Bohn, S., Schuller, J., & Schmitz, R. A.
Affiliations
  • Christian-Albrechts-Universität zu Kiel (DE)
  • Philipps University of Marburg (DE)
  • Helmholtz Munich (DE)
Corresponding author
  • Schuller, Jan
  • Schmitz, Ruth Anne
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2025-03-31
Volume
13
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.97484
PMC
PMC11957540
OpenAlex
W4399082889
Cited by
6
References
1
Citations by year
2026: 2 · 2025: 4
Type
posted content
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Deutsche Forschungsgemeinschaft (Schm1052/20-2, SCHU 3364/1-1)
  • European Research Council (10.3030/101075992)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Amino Acid Enzymes and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
Keywords
glutamine synthetase, Methanosarcina mazei, alpha-ketoglutarate, allosteric regulation, nitrogen metabolism, methanogenic archaea, enzyme oligomerization, ammonium assimilation
MeSH terms
Glutamate-Ammonia Ligase, Ketoglutaric Acids, Methanosarcina, Archaeal Proteins, Cryoelectron Microscopy, Protein Multimerization

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