Allosteric Activation of Escherichia coli Glucosamine-6-Phosphate Deaminase (NagB) In Vivo Justified by Intracellular Amino Sugar Metabolite Concentrations.

UNLABELLED: 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. IMPORTANCE: 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.

The tertiary origin of the allosteric activation of E. coli glucosamine-6-phosphate deaminase studied by sol-gel nanoencapsulation of its T conformer.

The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase from Escherichia coli (EcGNPDA) as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P). We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the background of the known crystallographic structures of T and R EcGNPDA conformers. These results are consistent with the postulates of the Tertiary Two-States (TTS) model.

Swi/SNF-GCN5-dependent chromatin remodelling determines induced expression of GDH3, one of the paralogous genes responsible for ammonium assimilation and glutamate biosynthesis in Saccharomyces cerevisiae.

It is accepted that Saccharomyces cerevisiae genome arose from complete duplication of eight ancestral chromosomes; functionally normal ploidy was recovered because of the massive loss of 90% of duplicated genes. There is evidence that indicates that part of this selective conservation of gene pairs is compelling to yeast facultative metabolism. As an example, the duplicated NADP-glutamate dehydrogenase pathway has been maintained because of the differential expression of the paralogous GDH1 and GDH3 genes, and the biochemical specialization of the enzymes they encode. The present work has been aimed to the understanding of the regulatory mechanisms that modulate GDH3 transcriptional activation. Our results show that GDH3 expression is repressed in glucose-grown cultures, as opposed to what has been observed for GDH1, and induced under respiratory conditions, or under stationary phase. Although GDH3 pertains to the nitrogen metabolic network, and its expression is Gln3p-regulated, complete derepression is ultimately determined by the carbon source through the action of the SAGA and SWI/SNF chromatin remodelling complexes. GDH3 carbon-mediated regulation is over-imposed to that exerted by the nitrogen source, highlighting the fact that operation of facultative metabolism requires strict control of enzymes, like Gdh3p, involved in biosynthetic pathways that use tricarboxylic acid cycle intermediates.