title: Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis.
abstract: The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using (31)P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Batten
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
authors.forename: Alice E
authors.initials: AE
authors.lastname: Parnell
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Wells
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K. Defence Science and Technology Laboratory, Porton Down, Salisbury, SP4 OJQ, U.K.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Murch
authors.affiliation: Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, U.K. Defence Science and Technology Laboratory, Porton Down, Salisbury, SP4 OJQ, U.K.
authors.forename: Petra C F
authors.initials: PC
authors.lastname: Oyston
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K. Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, U.K. plr2@soton.ac.uk.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Roach
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D005604:Francisella tularensis / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase
keywords: Francisella tularensis; X-ray crystallography; antibiotic sensitivity; enzyme kinetics; kinase; polyphosphate