title: Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.
abstract: Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme.
authors.affiliation: Department of Pharmaceutical Sciences, University of Kentucky, College of Pharmacy, 789 S. Limestone St., Lexington, KY 40536, United States.
authors.forename: Chathurada S
authors.initials: CS
authors.lastname: Gajadeera
authors.affiliation: Department of Chemistry, University of Kentucky, 505 Rose St., Lexington, KY 40506, United States.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, University of Kentucky, 505 Rose St., Lexington, KY 40506, United States. Electronic address: yinan.wei@uky.edu.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Department of Pharmaceutical Sciences, University of Kentucky, College of Pharmacy, 789 S. Limestone St., Lexington, KY 40536, United States. Electronic address: oleg.tsodikov@uky.edu.
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Tsodikov
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D043564:Inorganic Pyrophosphatase / Q000737:chemistry*; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D010710:Phosphates; D008345:Manganese; D043564:Inorganic Pyrophosphatase
keywords: Enzyme; Hydrolase; Novel drug target; Phosphatase; Phosphate metabolism; Pyrophosphorolysis