title: Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation.
abstract: N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.
authors.affiliation: Freie Universität Berlin, Institut für Chemie und Biochemie-Kristallographie, Takustrasse 6, 14195 Berlin-Dahlem, Germany.
authors.forename: Wilhelm A
authors.initials: WA
authors.lastname: Weihofen
authors.forename: Markus
authors.initials: M
authors.lastname: Berger
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Wolfram
authors.initials: W
authors.lastname: Saenger
authors.forename: Stephan
authors.initials: S
authors.lastname: Hinderlich
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002861:Chromobacterium / Q000201:enzymology; D019281:Dimerization; D004789:Enzyme Activation; D005643:Fucose / Q000031:analogs & derivatives* / Q000737:chemistry; D005947:Glucose / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D016966:Porphyromonas gingivalis / Q000201:enzymology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005643:Fucose; C059377:N-acetylfucosamine; D000244:Adenosine Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C018716:N-acetylglucosamine kinase; D005947:Glucose