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Biochemical and Structural Characterization of Polyphosphate Kinase 2 from the Intracellular Pathogen Francisella tularensis.

DOI: 10.1042/BSR20150203 DOI Help
PMID: 26582818 PMID Help

Authors: L. E. Batten (University of Southampton) , A. Parnell (University of Southampton) , N. J. Wells (University of Southampton) , A. L. Murch (Dstl Porton Down) , P. C. F. Oyston (Dstl Porton Down) , P. L. Roach (University of Southampton)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Bioscience Reports

State: Published (Approved)
Published: November 2015

Open Access Open Access

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 antibacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the 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 nucleotides. The activity was also measured using 31P NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å resolution. The structure consists of a 6- 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

Journal Keywords: Polyphosphate; Kinase; Francisella Tularensis; X-Ray Crystallography; Enzyme Kinetics; Antibiotic Sensitivity Abbreviations: Ft; Francisella Tularensis Subsp. Tularensis Schu S4; Sm; Sinorhizobium Meliloti; Ppk; Polyphosphate Kinase.

Subject Areas: Biology and Bio-materials


Instruments: I02-Macromolecular Crystallography

Added On: 02/12/2015 14:22

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