Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis. Issue 1 (10th February 2016)

• Record Type: Journal Article
• Authors: Batten, Laura E.; Parnell, Alice E.; Wells, Neil J.; Murch, Amber L.; Oyston, Petra C. F.; Roach, Peter L.
• Is Part Of: Bioscience reports. Volume 36:Issue 1(2016)
• Publication Date: 2016-02-10
• Subjects: antibiotic sensitivity -- enzyme kinetics -- Francisella tularensis -- kinase -- polyphosphate -- X-ray crystallography; Molecular biology -- Periodicals; Cytology -- Periodicals; 572.8
• DOI: 10.1042/BSR20150203
• Languages: English
• ISSNs: 0144-8463

Abstract

Abstract : The polyphosphate kinase 2 (PPK2) from the intracellular pathogen Francisella tularensis has been characterized by a range of biochemical methods and X-ray crystallography. The antibiotic sensitivity of a deletion mutant lacking the gene encoding PPK2 is also reported. 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 ( Ft PPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of Ft PPK2 was found to include purine but not pyrimidine nts. The activity was also measured using 31 P-NMR. Ft PPK2 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.