Molecular cloning and characterization of murine and human N‐acetylglucosamine kinase

Abstract

N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity.The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level. Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present.Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.

Comparative Genomics and Experimental Characterization of N-Acetylglucosamine Utilization Pathway of Shewanella oneidensis

“…The k cat of GlcNAc kinase is 14.1 s Ϫ1 (Table 3). The apparent K m value for GlcNAc (0.24 mM) at a saturating concentration of ATP (1.2 mM) is comparable with the respective values reported for the enzymes from Candida albicans (0.37 mM) or from mouse (0.20 mM) (47,53). To our knowledge, this is the first documented report of bacterial GlcNAc kinase functionally replacing GlcNAc phosphorylation by PTS system, such as in the NAG pathway of E. coli.…”

“…Therefore, it was deemed a candidate for the missing GlcNAc kinase role (named here NagK-I). This functional prediction was supported by significant sequence similarity (ϳ24%) with the recently characterized mammalian GlcNAc kinase (47). Although the observed homology alone would not be sufficient to assign the sugar specificity of the respective bacterial enzymes, taken together with the genome and functional context analysis, it provided strong evidence, sufficient for experimental testing as described below.…”

Substrate Specificity of the Sialic Acid Biosynthetic Pathway

“…Indeed, a bacterial homologue has been shown to be quite permissive with respect to the N-acyl position of the substrate (33). Additionally, GlcNAc 6-kinase can utilize ManNAc as a substrate, albeit with 100-fold lower efficiency than GlcNAc, providing an alternative route to ManNAc-6-phosphate from ManNAc (34).…”

Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway

“…These results suggested that the weak metabolic labeling by Ac 4 GlcNAz was due to an inefficient step in the GlcNAc salvage pathway upstream of UDP-GlcNAz biosynthesis. The GlcNAc salvage pathway consists of three enzymes: a GlcNAc kinase (NAGK) (22), a GlcNAc-6-phosphate mutase (AGM1) (23), and a UDP-GlcNAc pyrophosphorylase (AGX1 or AGX2, splicevariant isoforms) (24) (Fig. 1A).…”