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Two Crystal Structures of Escherichia coli N-Acetyl-l-Glutamate Kinase Demonstrate the Cycling between Open and Closed Conformations
Authors:Fernando Gil-Ortiz  María L Fernández-Murga  Vicente Rubio
Institution:1 Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas (IBV-CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), Jaime Roig 11, Valencia 46010, Spain
2 Instituto de Biología Molecular de Barcelona-Consejo Superior de Investigaciones Científicas (IBMB-CSIC) and Institute for Research in Biomedicine, Josep Samitier 1-5, Parc Cientific, Barcelona 08028, Spain
Abstract:N-Acetyl-l-glutamate kinase (NAGK), the paradigm enzyme of the amino acid kinase family, catalyzes the second step of arginine biosynthesis. Although substrate binding and catalysis were clarified by the determination of four crystal structures of the homodimeric Escherichia coli enzyme (EcNAGK), we now determine 2 Å resolution crystal structures of EcNAGK free from substrates or complexed with the product N-acetyl-l-glutamyl-5-phosphate (NAGP) and with sulfate, which reveal a novel, very open NAGK conformation to which substrates would associate and from which products would dissociate. In this conformation, the C-domain, which hosts most of the nucleotide site, rotates ∼ 24°-28° away from the N-domain, which hosts the acetylglutamate site, whereas the empty ATP site also exhibits some changes. One sulfate is found binding in the region where the β-phosphate of ATP normally binds, suggesting that ATP is first anchored to the β-phosphate site, before perfect binding by induced fit, triggering the shift to the closed conformation. In contrast, the acetylglutamate site is always well formed, although its β-hairpin lid is found here to be mobile, being closed only in the subunit of the EcNAGK-NAGP complex that binds NAGP most strongly. Lid closure appears to increase the affinity for acetylglutamate/NAGP and to stabilize the closed enzyme conformation via lid-C-domain contacts. Our finding of NAGP bound to the open conformation confirms that this product dissociates from the open enzyme form and allows reconstruction of the active center in the ternary complex with both products, delineating the final steps of the reaction, which is shown here by site-directed mutagenesis to involve centrally the invariant residue Gly11.
Keywords:NAGK  acetylglutamate kinase  EcNAGK  Escherichia coli acetylglutamate kinase  AMPPNP  adenylyl-imidodiphosphate  NAGK-apo  crystal form of acetylglutamate kinase free from substrates  NAGK-NAGP  l-glutamyl-5-phosphate and sulfate" target="_blank">crystal form of acetylglutamate kinase bound to N-acetyl-l-glutamyl-5-phosphate and sulfate  l-NAG" target="_blank">NAG and l-NAG  l-glutamate" target="_blank">N-acetyl-l-glutamate  d-NAG" target="_blank">d-NAG  d-glutamate" target="_blank">N-acetyl-d-glutamate  NAGP  l-glutamyl-5-phosphate" target="_blank">N-acetyl-l-glutamyl-5-phosphate  PaNAGK  Pseudomonas aeruginosa acetylglutamate kinase  TmNAGK  Thermotoga maritima acetylglutamate kinase  AtNAGK  Arabidopsis thaliana acetylglutamate kinase  PEG  polyethylene glycol
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