Allosteric Regulation of Glucosamine-6-Phosphate Deaminase (NagB) and Growth ofEscherichia colion Glucosamine

Abstract

Growth on N-acetylglucosamine (GlcNAc) produces intracellular N-acetylglucosamine-6-phosphate (GlcNAc6P), which affects the regulation of the catabolism of amino sugars in Escherichia coli in two ways. First, GlcNAc6P is the inducing signal for the NagC repressor, and thus it increases the expression of the enzymes of the nagE-nagBACD operon. Second, it is the allosteric activator of glucosamine-6P (GlcN6P) deaminase, NagB, and thus increases the catalytic capacity of this key enzyme in the metabolism of amino sugars. We showed previously that both the level of expression of the nagB gene and the transport of glucosamine were limiting the growth rate on GlcN (L. I. Álvarez-Añorve et al., J. Bacteriol. 187:2974-2982, 2005). We were unable to conclude if the lack of allosteric activation of wild-type NagB was also contributing to the slower growth rate on GlcN. Using a single-copy plasmid, with a constitutive promoter, we have separated the effects of GlcNAc6P on the NagB protein level and on deaminase activity. We show that over a range of intracellular NagB concentrations it is the quantity of the substrate, GlcN6P, which is limiting growth rather than the concentration of the allosteric activator, GlcNAc6P. On the other hand, the F174A mutant of NagB, which requires higher concentrations of GlcNAc6P for activity in vitro, grew better on GlcN in the presence of GlcNAc6P. However, wild-type NagB behaves as if it is already fully allosterically activated during growth on GlcN, and we present evidence suggesting that sufficient GlcNAc6P for allosteric activation is derived from the recycling of peptidoglycan.Amino sugars are widely distributed in nature and are valuable nutrients to most organisms. The most commonly found amino sugars are glucosamine (GlcN) and N-acetylglucosamine (GlcNAc). Their most abundant source is chitin, the high-molecular-weight polymer composed of 1-4 ␤-linked GlcNAc residues found in the cell walls of fungi and the exoskeletons of crustaceans and other arthropods. Amino sugars are both carbon and nitrogen sources but are also essential components of subcellular structures like the peptidoglycan (PG) of bacterial cell walls and the high-molecular-weight glycosaminoglycans and glycoproteins of the extracellular matrix of higher eukaryotes. Enteric Escherichia coli bacteria are likely to encounter host-derived amino sugars during their normal life cycle. The amino sugars, once taken up by the bacteria, can be used both to synthesize the PG and lipopolysaccharides of its cell wall and as an energy source.We previously investigated why E. coli grows more slowly on glucosamine than on N-acetylglucosamine. Growth on GlcNAc requires the nagE-or manXYZ-encoded phosphotransferase (PTS) transporters, which produce N-acetylglucosamine-6-phosphate (GlcNAc6P), and the two genes nagA and nagB, which encode GlcNAc6P deacetylase and GlcN6P deaminase, respectively, needed for the metabolism of GlcNAc6P (Fig. 1). Growth on GlcN requires the manXYZ-encoded transporter, producing GlcN6P, and the nagB gene pro...

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

“…The enzyme under these conditions is operating at between 50 and 90% of its maximum rate. These values are consistent with the high NagB activity deduced for the enzyme during growth on GlcN (22).…”

“…The nagE-BA genes, controlled by the GlcNAc6P-sensitive NagC repressor, are partially induced by growth on GlcN (22,30), This low-level induction supports the idea that some GlcNAc6P is formed during growth on GlcN but also implies that it is only present in a low concentration.…”

“…The gamA and nagB Bs alleles were also introduced by P1 transduction into MC-B52 (a ⌬nagEBACD::Tc strain carrying a nagB-lacZ fusion on a lysogen) (22), with selection for growth on GlcNAc and screening for tetracycline sensitivity. The ␤-galactosidase activities were measured as described previously (22).…”

“…In this case there was no effect of GlcNAc6P on the quantity of the deaminase, but we still could not show any contribution of the allosteric activation of wild-type E. coli NagB to growth on either GlcN or GlcNAc. In fact, the enzyme behaved as if it was fully active during growth on either sugar (22).…”

NagR Differentially Regulates the Expression of the glmS and nagAB Genes Required for Amino Sugar Metabolism by Streptococcus mutans

“…The repressor activity of NagC depends on its binding to palindromic dre (for DasR responsive element) (9,10) sites in the promoter regions of the target genes. When GlcNAc is present in the environment, intracellular GlcNAc-6-P accumulates and activates the expression of genes for GlcNAc and GlcN catabolism by disrupting the interaction between NagC and dre sites (5,(11)(12)(13). In the Gram-positive bacterium Bacillus subtilis, a GntR/HutC-type regulator, designated NagR (formerly YvoA), binds to nonhomologous operator sites, also called dre (14,15), to control the transcription of nagA, nagB, and nagP, which codes for a GlcNAc-specific PTS permease.…”