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.
Highlights
E. coli glucosamine-6-phosphate synthase (GlmS) is active as a dimer
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 E. coli C1A-GlmS mutant was crystallized in the presence of fructose 6-phosphate (Fru6P) and Gln, cyclic glucose 6-phosphate (Glc6P) and Glu were observed at the synthase and glutaminase catalytic sites, respectively
Summary
E. coli GlmS is active as a dimer. Results: The dimer is in equilibrium with a hexameric state that is catalytically inactive in vitro. 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. 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. 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 there-
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