The N-Acetyl-d-Glucosamine Kinase of Escherichia coli and Its Role in Murein Recycling

In 1966, Asensio and Ruiz-Amil reported the properties of GlcNAc kinase activity in Escherichia coli and purified the enzyme about 150-fold (1). No further work on the enzyme has been reported, and the nagK gene has not been identified heretofore. The metabolism of GlcNAc and d-glucosamine (GlcN) has been studied for decades. The divergently transcribed nagE and nagBACD operons are involved in the uptake and degradation of the amino sugars. When E. coli grows on GlcNAc-containing medium, GlcNAc is transported by a phosphotransferase system, either NagE or ManXYZ, to generate GlcNAc-6-phosphate (GlcNAc-6-P) (26). GlcNAc-6-P is converted by NagA deacetylase to GlcN-6-P (38). GlcN-6-P enters either the glycolysis pathway via conversion to fructose-6-P by the NagB deaminase (38) or the biosynthesis pathways for murein and lipopolysaccharide by conversion to UDP-GlcNAc by GlmM and GlmU (15-17) (Fig. ​(Fig.1).1). Hence, NagK is not required for the metabolism of GlcNAc derived from the medium. The other proteins encoded by the nag gene cluster are NagC, a repressor controlling the expression of nagE and the nagBACD operon (24), and NagD, a putative hydrolase, whose activity is not essential for the metabolism of the amino sugars. FIG. 1. Scheme of murein recycling and metabolism of GlcNAc. UDP-GlcNAc is the main cytoplasmic precursor of murein and lipopolysaccharide. AmpG is a specific permease for muropeptides. NagE and ManXYZ are phosphoenolpyruvate-dependent phosphotransferases. IM, ... However, significant amounts of nonphosphorylated GlcNAc are released in the cytoplasm via the murein recycling pathway (4, 37) (Fig. ​(Fig.1).1). Over half of side-wall murein is broken down each generation by lytic transglycosylases and endopeptidases (7, 9, 11). The principal degradation product is the anhydro-muropeptide GlcNAc-β-1,4-anhydro-N-acetylmuramyl(anhMurNAc)-l-alanyl-γ-d-glutamyl-meso-diaminopimelyl-d-alanine. Muropeptides are imported into the cytoplasm via a specific permease, AmpG (5, 11), and thereafter are cleaved into GlcNAc, anhMurNAc, murein tripeptide, and d-alanine by the combined actions of the NagZ protein (β-N-acetylglucosaminidase), the AmpD protein (anhMurNAc-l-Ala amidase), and the LdcA protein (dl-carboxypeptidase) (4, 10, 12, 33, 37). Murein tripeptide reenters the murein synthesis pathway following direct ligation to UDP-MurNAc by Mpl (18). To a lesser extent, murein tripeptide is cleaved by MpaA, YcjJ, and PepD to yield the individual amino acids (30, 36). Interestingly, the roles of all eight of these enzymes, except PepD, are concerned exclusively with the recycling process. Recently, both cytoplasmic GlcNAc and anhMurNAc derived from cell wall recycling were shown to be reused (23). Cytoplasmic GlcNAc must be phosphorylated to enter the known metabolic pathway of GlcNAc-6-P (Fig. ​(Fig.1).1). Though one possibility for utilization of murein-derived GlcNAc is for GlcNAc to be exported and then taken up by a specific phosphotransferase system, it was reasonable to assume that NagK is responsible for the phosphorylation of murein-derived GlcNAc. In this communication, the nagK (ycfX) gene is identified, and NagK is characterized and shown to be the only GlcNAc kinase expressed in E. coli to phosphorylate GlcNAc in the cytoplasm. On the basis of the observation that GlcNAc does not accumulate in a nagK nagEBACD mutant, we suggest that an unknown pathway must exist to utilize GlcNAc in the absence of NagK and NagA and that the pathway may be regulated by GlcNAc-6-P.