title: Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.
abstract: The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Mouilleron
authors.forename: Marie-Ange
authors.initials: MA
authors.lastname: Badet-Denisot
authors.forename: Ludovic
authors.initials: L
authors.lastname: Pecqueur
authors.forename: Karine
authors.initials: K
authors.lastname: Madiona
authors.forename: Nadine
authors.initials: N
authors.lastname: Assrir
authors.forename: Bernard
authors.initials: B
authors.lastname: Badet
authors.forename: Béatrice
authors.initials: B
authors.lastname: Golinelli-Pimpaneau
mesh_terms: D000494:Allosteric Regulation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)