title: Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP.
abstract: The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK) catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK) with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP), was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123-170), which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent.
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland, USA.
authors.forename: Kap
authors.initials: K
authors.lastname: Lim
authors.forename: Liudmila
authors.initials: L
authors.lastname: Kulakova
authors.forename: Andrey
authors.initials: A
authors.lastname: Galkin
authors.forename: Osnat
authors.initials: O
authors.lastname: Herzberg
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D019343:Citric Acid / Q000737:chemistry*; D018360:Crystallography, X-Ray; D016829:Giardia lamblia / Q000201:enzymology*; D006860:Hydrogen Bonding; D008956:Models, Chemical; D008969:Molecular Sequence Data; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry*
chemical_list: D015800:Protozoan Proteins; D000266:Adenylyl Imidodiphosphate; D019343:Citric Acid; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase