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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 ? 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.

SUBMITTER: Mouilleron S

PROVIDER: S-EPMC3464560 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

Mouilleron Stéphane S Badet-Denisot Marie-Ange MA Pecqueur Ludovic L Madiona Karine K Assrir Nadine N Badet Bernard B Golinelli-Pimpaneau Béatrice B

The Journal of biological chemistry 20120731 41

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 ...[more]

PMID: 22851174

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Project description:UnlabelledWe have investigated the impact of growth on glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on cellular metabolism by quantifying glycolytic metabolites in Escherichia coli Growth on GlcNAc increased intracellular pools of both GlcNAc6P and GlcN6P 10- to 20-fold compared to growth on glucose. Growth on GlcN produced a 100-fold increase in GlcN6P but only a slight increase in GlcNAc6P. Changes to the amounts of downstream glycolytic intermediates were minor compared to growth on glucose. The enzyme glucosamine-6P deaminase (NagB) is required for growth on both GlcN and GlcNAc. It is an allosteric enzyme in E. coli, displaying sigmoid kinetics with respect to its substrate, GlcN6P, and is allosterically activated by GlcNAc6P. The high concentration of GlcN6P, accompanied by the small increase in GlcNAc6P, drives E. coli NagB (NagBEc) into its high activity state, as observed during growth on GlcN (L. I. Álvarez-Añorve, I. Bustos-Jaimes, M. L. Calcagno, and J. Plumbridge, J Bacteriol 191:6401-6407, 2009, http://dx.doi.org/10.1128/JB.00633-09). The slight increase in GlcNAc6P during growth on GlcN is insufficient to displace NagC, the GlcNAc6P-responsive repressor of the nag genes, from its binding sites, so there is only a small increase in nagB expression. We replaced the gene for the allosteric NagBEc enzyme with that of the nonallosteric, B. subtilis homologue, NagBBs We detected no effects on growth rates or competitive fitness on glucose or the amino sugars, nor did we detect any effect on the concentrations of central metabolites, thus demonstrating the robustness of amino sugar metabolism and leaving open the question of the role of allostery in the regulation of NagB.ImportanceChitin, the polymer of N-acetylglucosamine, is an abundant biomaterial, and both glucosamine and N-acetylglucosamine are valuable nutrients for bacteria. The amino sugars are components of numerous essential macromolecules, including bacterial peptidoglycan and mammalian glycosaminoglycans. Controlling the biosynthetic and degradative pathways of amino sugar metabolism is important in all organisms to avoid loss of nitrogen and energy via a futile cycle of synthesis and breakdown. The enzyme glucosamine-6P deaminase (NagB) is central to this control, and N-acetylglucosamine-6P is the key signaling molecule regulating amino sugar utilization in Escherichia coli Here, we investigate how the metabolic status of the bacteria impacts on the activity of NagBEc and the N-acetylglucosamine-6P-sensitive transcriptional repressor, NagC.

Dataset Information

Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

Publications

Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

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