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 (NagB Ec ) 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 NagB Ec enzyme with that of the nonallosteric, B. subtilis homologue, NagB Bs . 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 NagB Ec and the N-acetylglucosamine-6P-sensitive transcriptional repressor, NagC. The genes for use of N-acetylglucosamine (GlcNAc) as a carbon source in Escherichia coli form a typical inducible regulon, controlled by the NagC transcription factor. The divergent operon nagE-nagBACD (Fig. 1) encodes the transporter (NagE) and the enzymes N-acetylglucosamine-6P (GlcNAc6P) deacetylase (NagA) and glucosamine-6P (GlcN6P) deaminase (NagB) for the conversion of GlcNAc6P to fructose-6P (F6P). NagD encodes a UMP phosphatase (UmpH) involved in pyrimidine homeostasis (1). The E. c...

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“…The kinetic values found for Amuc‐NagB are in line with previously characterized NagB‐like enzymes (Calcagno et al ., ), except for the absence of the need for an allosteric activator. In all cases, GlcNAc6P is described as an allosteric activator (Alvarez‐Anorve et al ., , ). Our results clearly show that the overexpressed Amuc‐NagB is not allosterically activated by GlcNAc6P (Table ).…”

Small RNA ‐binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli

“…For E. coli cells grown in the absence of amino sugars, intracellular GlcN6P concentrations in the range from 62 μM to 1.15 mM were reported and may increase up to ~10 mM when cells grow on amino sugars (Bennett et al , ; Alvarez‐Anorve et al , ). This concentration range fits well with our results indicating that 1–2 mM GlcN6P is necessary to elicit a response of RapZ in vitro (Figs F and E).…”

“…GlcN6P is the source of all amino sugar containing constituents of the cell wall and also of the outer membrane of Gram‐negative bacteria. Bacteria can procure GlcN6P from external amino sugars such as glucosamine (GlcN), which can be taken up and converted to GlcN6P (Alvarez‐Anorve et al , ). If not available, GlcN6P must be synthesized by GlmS (Milewski, ).…”

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“…Alvarez-Añorve et al [8] performed E. coli growth experiments, where the gene for the allosteric E. coli NagB was replaced with the gene of the non-allosteric B. subtilis NagB. No effect on growth rates, competitive fitness or any change in concentration of central metabolites was observed with the non-allosteric B. subtilis enzyme.…”

“…The inducer molecule for NagC is N-acetylglucosamine-6-phosphate (GlcNAc6P). Furthermore, GlcNAc6P acts a regulator at an enzymatic level, allosterically activating E. coli NagB, in what is hypothesised to be a feed-forward mechanism of sensing flux [8].…”