title: Glucose-6-phosphate as a probe for the glucosamine-6-phosphate N-acetyltransferase Michaelis complex.
abstract: Glucosamine-6-phosphate N-acetyltransferase (GNA1) catalyses the N-acetylation of d-glucosamine-6-phosphate (GlcN-6P), using acetyl-CoA as an acetyl donor. The product GlcNAc-6P is an intermediate in the biosynthesis UDP-GlcNAc. GNA1 is part of the GCN5-related acetyl transferase family (GNATs), which employ a wide range of acceptor substrates. GNA1 has been genetically validated as an antifungal drug target. Detailed knowledge of the Michaelis complex and trajectory towards the transition state would facilitate rational design of inhibitors of GNA1 and other GNAT enzymes. Using the pseudo-substrate glucose-6-phosphate (Glc-6P) as a probe with GNA1 crystals, we have trapped the first GNAT (pseudo-)Michaelis complex, providing direct evidence for the nucleophilic attack of the substrate amine, and giving insight into the protonation of the thiolate leaving group.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, UK.
authors.forename: Ramon
authors.initials: R
authors.lastname: Hurtado-Guerrero
authors.forename: Olawale
authors.initials: O
authors.lastname: Raimi
authors.forename: Sharon
authors.initials: S
authors.lastname: Shepherd
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology; D002384:Catalysis; D018360:Crystallography, X-Ray; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D051081:Glucosamine 6-Phosphate N-Acetyltransferase / Q000737:chemistry* / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D013379:Substrate Specificity
chemical_list: C001293:glucosamine 6-phosphate; D019298:Glucose-6-Phosphate; D051081:Glucosamine 6-Phosphate N-Acetyltransferase; D005944:Glucosamine