title: Allosteric Activation of Escherichia coli Glucosamine-6-Phosphate Deaminase (NagB) In Vivo Justified by Intracellular Amino Sugar Metabolite Concentrations.
abstract: We 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.
  
  
  
  Chitin, 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.
authors.affiliation: CNRS-UMR8261, Université Paris Diderot, Sorbonne Paris Cité, Institut de Biologie Physico-Chimique, Paris, France.|Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, CDMX, Mexico.
authors.forename: Laura I
authors.initials: LI
authors.lastname: Álvarez-Añorve
authors.affiliation: CNRS-UMR8261, Université Paris Diderot, Sorbonne Paris Cité, Institut de Biologie Physico-Chimique, Paris, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Gaugué
authors.affiliation: Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
authors.forename: Hannes
authors.initials: H
authors.lastname: Link
authors.affiliation: Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, CDMX, Mexico.
authors.forename: Jorge
authors.initials: J
authors.lastname: Marcos-Viquez
authors.affiliation: Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, CDMX, Mexico.
authors.forename: Dana M
authors.initials: DM
authors.lastname: Díaz-Jiménez
authors.affiliation: Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, CDMX, Mexico.
authors.forename: Sergio
authors.initials: S
authors.lastname: Zonszein
authors.affiliation: Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, CDMX, Mexico.
authors.forename: Ismael
authors.initials: I
authors.lastname: Bustos-Jaimes
authors.affiliation: Institut de Chimie des Substances Naturelles, ICSN-CNRS UPR2301, LabEx CEBA, Université Paris-Sud, Gif-sur-Yvette, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Schmitz-Afonso
authors.affiliation: Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, CDMX, Mexico.
authors.forename: Mario L
authors.initials: ML
authors.lastname: Calcagno
authors.affiliation: CNRS-UMR8261, Université Paris Diderot, Sorbonne Paris Cité, Institut de Biologie Physico-Chimique, Paris, France jackie.plumbridge@ibpc.fr.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Plumbridge
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D000606:Amino Sugars / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D009154:Mutation; D030781:Organisms, Genetically Modified; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000606:Amino Sugars; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C067862:nagC protein, E coli; C020582:glucosamine-6-phosphate isomerase; D019747:Aldose-Ketose Isomerases