Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM
2021; Elsevier BV; Volume: 297; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2021.101317
ISSN1083-351X
AutoresM. Pathania, T. Tosi, Charlotte Millership, Fumiya Hoshiga, Rhodri M. L. Morgan, Paul S. Freemont, Angelika Gründling,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoCyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes. Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes. Nucleotide signaling molecules play important roles in helping bacteria to rapidly adapt to changing environmental conditions (1Hengge R. Gründling A. Jenal U. 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Cell. 2008; 30: 167-178Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 21Rosenberg J. Dickmanns A. Neumann P. Gunka K. Arens J. Kaever V. Stülke J. Ficner R. Commichau F.M. Structural and biochemical analysis of the essential diadenylate cyclase CdaA from Listeria monocytogenes.J. Biol. Chem. 2015; 290: 6596-6606Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 22Zheng C. Ma Y. Wang X. Xie Y. Ali M.K. He J. Functional analysis of the sporulation-specific diadenylate cyclase CdaS in Bacillus thuringiensis.Front. Microbiol. 2015; 6: 908Crossref PubMed Scopus (24) Google Scholar, 23Müller M. Deimling T. Hopfner K.P. Witte G. Structural analysis of the diadenylate cyclase reaction of DNA-integrity scanning protein A (DisA) and its inhibition by 3'-dATP.Biochem. J. 2015; 469: 367-374Crossref PubMed Scopus (13) Google Scholar, 24Mehne F.M. Schroder-Tittmann K. Eijlander R.T. Herzberg C. Hewitt L. Kaever V. Lewis R.J. Kuipers O.P. Tittmann K. Stülke J. Control of the diadenylate cyclase CdaS in Bacillus subtilis: An autoinhibitory domain limits cyclic di-AMP production.J. Biol. Chem. 2014; 289: 21098-21107Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). CdaA (also referred to as DacA in some bacteria) is a membrane-bound cyclase with three predicted N-terminal transmembrane helices and a cytoplasmic catalytic DAC domain. CdaA (DacA) is the "housekeeping" c-di-AMP cyclase in Firmicutes, as it is conserved and often the sole c-di-AMP cyclase in this phylum (25Fahmi T. Port G.C. Cho K.H. c-di-AMP: An essential molecule in the signaling pathways that regulate the viability and virulence of Gram-positive bacteria.Genes (Basel). 2017; 8: 197Crossref PubMed Scopus (54) Google Scholar, 26Corrigan R.M. Gründling A. Cyclic di-AMP: Another second messenger enters the fray.Nat. Rev. Microbiol. 2013; 11: 513-524Crossref PubMed Scopus (255) Google Scholar). The two other B. subtilis c-di-AMP cyclases, DisA and CdaS, are soluble proteins, not as widely distributed among bacteria and have more specialized functions, with DisA involved in controlling DNA-repair processes during sporulation or spore outgrowth and CdaS also specifically expressed during the sporulation process (22Zheng C. Ma Y. Wang X. Xie Y. Ali M.K. He J. Functional analysis of the sporulation-specific diadenylate cyclase CdaS in Bacillus thuringiensis.Front. Microbiol. 2015; 6: 908Crossref PubMed Scopus (24) Google Scholar, 23Müller M. Deimling T. Hopfner K.P. Witte G. Structural analysis of the diadenylate cyclase reaction of DNA-integrity scanning protein A (DisA) and its inhibition by 3'-dATP.Biochem. J. 2015; 469: 367-374Crossref PubMed Scopus (13) Google Scholar, 27Torres R. Carrasco B. Gandara C. Baidya A.K. Ben-Yehuda S. Alonso J.C. Bacillus subtilis DisA regulates RecA-mediated DNA strand exchange.Nucleic Acids Res. 2019; 47: 5141-5154Crossref PubMed Scopus (16) Google Scholar). While there are no publications on the 3D-structures of the B. subtilis c-di-AMP cyclases, structures are available for the cytoplasmic enzymatic domains of the L. monocytogenes and S. aureus CdaA/DacA homologs (21Rosenberg J. Dickmanns A. Neumann P. Gunka K. Arens J. Kaever V. Stülke J. Ficner R. Commichau F.M. Structural and biochemical analysis of the essential diadenylate cyclase CdaA from Listeria monocytogenes.J. Biol. Chem. 2015; 290: 6596-6606Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 28Tosi T. Hoshiga F. Millership C. Singh R. Eldrid C. Patin D. Mengin-Lecreulx D. Thalassinos K. Freemont P. Gründling A. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.PLoS Pathog. 2019; 15e1007537Crossref PubMed Scopus (21) Google Scholar), the DisA homolog from Thermotoga maritima (3Witte G. Hartung S. Buttner K. Hopfner K.P. Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.Mol. Cell. 2008; 30: 167-178Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), and the CdaS homolog from Bacillus cereus (PDB 2FB5). These studies revealed that DAC domains have a mixed αß-fold, with highly conserved DGA and RHR amino acid motifs required for ligand binding (3Witte G. Hartung S. Buttner K. Hopfner K.P. Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.Mol. Cell. 2008; 30: 167-178Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 21Rosenberg J. Dickmanns A. Neumann P. Gunka K. Arens J. Kaever V. Stülke J. Ficner R. Commichau F.M. Structural and biochemical analysis of the essential diadenylate cyclase CdaA from Listeria monocytogenes.J. Biol. Chem. 2015; 290: 6596-6606Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 29Gundlach J. Mehne F.M. Herzberg C. Kampf J. Valerius O. Kaever V. Stülke J. An essential poison: Synthesis and degradation of cyclic di-AMP in Bacillus subtilis.J. Bacteriol. 2015; 197: 3265-3274Crossref PubMed Scopus (80) Google Scholar). Formation of c-di-AMP requires a head-to-head conformation of two DAC domains. This was first demonstrated in the crystal structure of DisA, a protein that forms an octamer with four DAC domain dimers in the active head-to-head conformation (3Witte G. Hartung S. Buttner K. Hopfner K.P. Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.Mol. Cell. 2008; 30: 167-178Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). While the L. monocytogenes and S. aureus CdaA/DacA catalytic domains and the CdaS protein, were also present as dimers and hexamers, respectively, they were in an inactive conformation. These proteins therefore either need to rearrange or more likely form higher oligomers in order to yield active enzymes with DAC domains in the head-to-head dimer conformation (24Mehne F.M. Schroder-Tittmann K. Eijlander R.T. Herzberg C. Hewitt L. Kaever V. Lewis R.J. Kuipers O.P. Tittmann K. Stülke J. Control of the diadenylate cyclase CdaS in Bacillus subtilis: An autoinhibitory domain limits cyclic di-AMP production.J. Biol. Chem. 2014; 289: 21098-21107Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 28Tosi T. Hoshiga F. Millership C. Singh R. Eldrid C. Patin D. Mengin-Lecreulx D. Thalassinos K. Freemont P. Gründling A. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.PLoS Pathog. 2019; 15e1007537Crossref PubMed Scopus (21) Google Scholar). Recently, another structure of the cytoplasmic catalytic domain of the L. monocytogenes CdaA enzyme (Δ100CdaA) has been reported with a c-di-AMP bound between two monomers, which based on the crystal cell packing were arranged in an active dimer of dimer configuration (30Heidemann J.L. Neumann P. Dickmanns A. Ficner R. Crystal structures of the c-di-AMP-synthesizing enzyme CdaA.J. Biol. Chem. 2019; 294: 10463-10470Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). These findings are consistent with the idea that CdaA (DacA) enzymes will need to form higher oligomers to achieve an active enzyme configuration. Hence, factors influencing the ability of c-di-AMP cyclases to rearrange into an active conformation or to form higher oligomers will be able to regulate the activity of these enzymes. The genetic arrangement and operon structure coding for the "housekeeping" c-di-AMP cyclase CdaA (DacA) is conserved in Firmicutes (29Gundlach J. Mehne F.M. Herzberg C. Kampf J. Valerius O. Kaever V. Stülke J. An essential poison: Synthesis and degradation of cyclic di-AMP in Bacillus subtilis.J. Bacteriol. 2015; 197: 3265-3274Crossref PubMed Scopus (80) Google Scholar, 31Zhu Y. Pham T.H. Nhiep T.H. Vu N.M. Marcellin E. Chakrabortti A. Wang Y. Waanders J. Lo R. Huston W.M. Bansal N. Nielsen L.K. Liang Z.X. Turner M.S. Cyclic-di-AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis.Mol. Microbiol. 2016; 99: 1015-1027Crossref PubMed Scopus (43) Google Scholar). Two genes, coding for the membrane-linked CdaA regulator CdaR (also named YbbR in some bacteria) and cytoplasmically located peptidoglycan precursor synthesis enzyme GlmM, are found downstream and in an operon with cdaA (29Gundlach J. Mehne F.M. Herzberg C. Kampf J. Valerius O. Kaever V. Stülke J. An essential poison: Synthesis and degradation of cyclic di-AMP in Bacillus subtilis.J. Bacteriol. 2015; 197: 3265-3274Crossref PubMed Scopus (80) Google Scholar, 31Zhu Y. Pham T.H. Nhiep T.H. Vu N.M. Marcellin E. Chakrabortti A. Wang Y. Waanders J. Lo R. Huston W.M. Bansal N. Nielsen L.K. Liang Z.X. Turner M.S. Cyclic-di-AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis.Mol. Microbiol. 2016; 99: 1015-1027Crossref PubMed Scopus (43) Google Scholar). Through recent studies in B. subtilis, L. monocytogenes, Lactococcus lactis, and S. aureus, it is has become apparent that these three genes are not only cotranscribed but that the encoded proteins also form a complex and that CdaR and GlmM can regulate the activity of the c-di-AMP cyclase CdaA (29Gundlach J. Mehne F.M. Herzberg C. Kampf J. Valerius O. Kaever V. Stülke J. An essential poison: Synthesis and degradation of cyclic di-AMP in Bacillus subtilis.J. Bacteriol. 2015; 197: 3265-3274Crossref PubMed Scopus (80) Google Scholar, 32Pham T.H. Liang Z.X. Marcellin E. Turner M.S. Replenishing the cyclic-di-AMP pool: Regulation of diadenylate cyclase activity in bacteria.Curr. Genet. 2016; 62: 731-738Crossref PubMed Scopus (23) Google Scholar, 33Gibhardt J. Heidemann J.L. Bremenkamp R. Rosenberg J. Seifert R. Kaever V. Ficner R. Commichau F.M. An extracytoplasmic protein and a moonlighting enzyme modulate synthesis of c-di-AMP in Listeria monocytogenes.Environ. Microbiol. 2020; 22: 2771-2791Crossref PubMed Scopus (10) Google Scholar). While CdaR has been reported to function as both an activator and a repressor of CdaA depending on the growth conditions, GlmM has been shown to be a potent inhibitor of the cyclase (17Mehne F.M. Gunka K. Eilers H. Herzberg C. Kaever V. Stülke J. Cyclic di-AMP homeostasis in Bacillus subtilis: Both lack and high level accumulation of the nucleotide are detrimental for cell growth.J. Biol. Chem. 2013; 288: 2004-2017Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 29Gundlach J. Mehne F.M. Herzberg C. Kampf J. Valerius O. Kaever V. Stülke J. An essential poison: Synthesis and degradation of cyclic di-AMP in Bacillus subtilis.J. Bacteriol. 2015; 197: 3265-3274Crossref PubMed Scopus (80) Google Scholar, 31Zhu Y. Pham T.H. Nhiep T.H. Vu N.M. Marcellin E. Chakrabortti A. Wang Y. Waanders J. Lo R. Huston W.M. Bansal N. Nielsen L.K. Liang Z.X. Turner M.S. Cyclic-di-AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis.Mol. Microbiol. 2016; 99: 1015-1027Crossref PubMed Scopus (43) Google Scholar, 32Pham T.H. Liang Z.X. Marcellin E. Turner M.S. Replenishing the cyclic-di-AMP pool: Regulation of diadenylate cyclase activity in bacteria.Curr. Genet. 2016; 62: 731-738Crossref PubMed Scopus (23) Google Scholar, 34Rismondo J. Gibhardt J. Rosenberg J. Kaever V. Halbedel S. Commichau F.M. Phenotypes associated with the essential diadenylate cyclase CdaA and its potential regulator CdaR in the human pathogen Listeria monocytogenes.J. Bacteriol. 2016; 198: 416-426Crossref PubMed Scopus (27) Google Scholar). However, the molecular mechanisms on how the CdaA cyclase activity is regulated by these proteins are not yet known, and this was further investigated as part of this study. GlmM is a phosphoglucomutase enzyme catalyzing the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is subsequently used to produce the essential peptidoglycan precursor UDP-N-acetyl-glucosamine (35Barreteau H. Kovac A. Boniface A. Sova M. Gobec S. Blanot D. Cytoplasmic steps of peptidoglycan biosynthesis.FEMS Microbiol. Rev. 2008; 32: 168-207Crossref PubMed Scopus (466) Google Scholar). In B. subtilis, L. monocytogenes, and L. lactis, a protein–protein interaction between CdaA and GlmM has been detected using bacterial two-hybrid assays performed in Escherichia coli (29Gundlach J. Mehne F.M. Herzberg C. Kampf J. Valerius O. Kaever V. Stülke J. An essential poison: Synthesis and degradation of cyclic di-AMP in Bacillus subtilis.J. Bacteriol. 2015; 197: 3265-3274Crossref PubMed Scopus (80) Google Scholar, 31Zhu Y. Pham T.H. Nhiep T.H. Vu N.M. Marcellin E. Chakrabortti A. Wang Y. Waanders J. Lo R. Huston W.M. Bansal N. Nielsen L.K. Liang Z.X. Turner M.S. Cyclic-di-AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis.Mol. Microbiol. 2016; 99: 1015-1027Crossref PubMed Scopus (43) Google Scholar, 33Gibhardt J. Heidemann J.L. Bremenkamp R. Rosenberg J. Seifert R. Kaever V. Ficner R. Commichau F.M. An extracytoplasmic protein and a moonlighting enzyme modulate synthesis of c-di-AMP in Listeria monocytogenes.Environ. Microbiol. 2020; 22: 2771-2791Crossref PubMed Scopus (10) Google Scholar). In B. subtilis this interaction has been further confirmed by in vivo protein cross-linking and pull-down assays (29Gundlach J. Mehne F.M. Herzberg C. Kampf J. Valerius O. Kaever V. Stülke J. An essential poison: Synthesis and degradation of cyclic di-AMP in Bacillus subtilis.J. Bacteriol. 2015; 197: 3265-3274Crossref PubMed Scopus (80) Google Scholar) and in L. monocytogenes by the coelution of purified proteins (33Gibhardt J. Heidemann J.L. Bremenkamp R. Rosenberg J. Seifert R. Kaever V. Ficner R. Commichau F.M. An extracytoplasmic protein and a moonlighting enzyme modulate synthesis of c-di-AMP in Listeria monocytogenes.Environ. Microbiol. 2020; 22: 2771-2791Crossref PubMed Scopus (10) Google Scholar). The first evidence that GlmM serves as negative regulator of CdaA/DacA activity came from an L. lactis strain that produces a GlmM variant that is thought to form a stronger interaction with CdaA; this strain produces lower cellular c-di-AMP levels than the bacteria expressing wild-type GlmM (31Zhu Y. Pham T.H. Nhiep T.H. Vu N.M. Marcellin E. Chakrabortti A. Wang Y. Waanders J. Lo R. Huston W.M. Bansal N. Nielsen L.K. Liang Z.X. Turner M.S. Cyclic-di-AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis.Mol. Microbiol. 2016; 99: 1015-1027Crossref PubMed Scopus (43) Google Scholar). Furthermore, the activity of the soluble recombinant S. aureus DacA catalytic domain (DacACD) could be blocked almost completely by the addition of purified GlmM protein in in vitro assays, and the recombinant proteins were shown to form a stable complex that could be purified via size-exclusion chromatography (28Tosi T. Hoshiga F. Millership C. Singh R. Eldrid C. Patin D. Mengin-Lecreulx D. Thalassinos K. Freemont P. Gründling A. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.PLoS Pathog. 2019; 15e1007537Crossref PubMed Scopus (21) Google Scholar). On the other hand, the activity of GlmM was not affected by the interaction with DacACD (28Tosi T. Hoshiga F. Millership C. Singh R. Eldrid C. Patin D. Mengin-Lecreulx D. Thalassinos K. Freemont P. Gründling A. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.PLoS Pathog. 2019; 15e1007537Crossref PubMed Scopus (21) Google Scholar). Additional mass spectrometry and small-angle X-ray scattering (SAXS) analyses suggested that the complex is composed of a DacACD dimer and a GlmM dimer (28Tosi T. Hoshiga F. Millership C. Singh R. Eldrid C. Patin D. Mengin-Lecreulx D. Thalassinos K. Freemont P. Gründling A. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.PLoS Pathog. 2019; 15e1007537Crossref PubMed Scopus (21) Google Scholar). Crystal structures of the individual S. aureus DacACD and GlmM dimers revealed that the S. aureus DacACD protein assumed an "inactive" dimer conformation. GlmM had the typical four-domain fold of phosphoglucomutases with a flexible C-terminal domain 4 and the dimer was "M-shaped," characteristic for this class of enzymes (28Tosi T. Hoshiga F. Millership C. Singh R. Eldrid C. Patin D. Mengin-Lecreulx D. Thalassinos K. Freemont P. Gründling A. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.PLoS Pathog. 2019; 15e1007537Crossref PubMed Scopus (21) Google Scholar). However, a high-resolution structure of the complex could not be obtained, and only a model for the complex could be proposed by fitting the individual DacACD and GlmM dimer structures into the SAXS envelope (28Tosi T. Hoshiga F. Millership C. Singh R. Eldrid C. Patin D. Mengin-Lecreulx D. Thalassinos K. Freemont P. Gründling A. Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.PLoS Pathog. 2019; 15e1007537Crossref PubMed Scopus (21) Google Scholar). Based on this, a model was proposed in which GlmM could potentially block the activity of the DacACD cyclase by preventing the formation of higher oligomers. Here, we set out to provide atomic resolution information on the CdaA:GlmM complex to gain insight into the molecular mechanism of how GlmM can control the activity of the c-di-AMP cyclase enzyme. Using the purified B. subtilis CdaA catalytic domain (CdaACD) and purified full-length GlmM or the truncated GlmMF369 variant lacking the flexible C-terminal domain 4, we show that the two proteins form a stable complex in vitro and that GlmM and GlmMF369 are potent inhibits of the cyclase. Crystal structures of the B. subtilis CdaACD cyclase, the GlmM phosphoglucomutase, and the CdaACD:GlmMF369 complex were obtained, revealing dimer conformations of the individual proteins as well as a dimer of dimer conformation in the complex structure. More importantly, from the complex structure the mechanism by which binding of GlmM inhibits the cyclase activity become
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