Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP

• Published in: PloS one Vol. 8; no. 5; p. e64004
• Main Authors: Lim, Kap, Kulakova, Liudmila, Galkin, Andrey, Herzberg, Osnat
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20-05-2013; Public Library of Science (PLoS)
• Subjects: Acids; Adenine; Adenosine; Adenosine diphosphate; Adenosine monophosphate; Adenylyl Imidodiphosphate - chemistry; Amino Acid Sequence; Amino acids; Ammonium; AMP; Arginine; ATP; Binding sites; Biochemistry; Biology; Biosynthesis; Biotechnology; Carbamate kinase; Carbamates; Carbamoyl phosphate; Catalysis; Catalytic Domain; Citric acid; Citric Acid - chemistry; Coordination compounds; Crystal structure; Crystallography, X-Ray; Data collection; Drug development; Enzymes; Eukaryotes; Giardia; Giardia lamblia; Giardia lamblia - enzymology; Hydrogen Bonding; Kinases; Laboratories; Ligands; Materials Science; Models, Chemical; Molecular Sequence Data; Parasites; Phosphates; Phosphotransferases (Carboxyl Group Acceptor) - chemistry; Polyethylene glycol; Protein Binding; Protein structure; Protein Structure, Secondary; Proteins; Protozoan Proteins - chemistry; Reaction mechanisms; Residues; United States > US; Maryland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0064004

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.