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Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis

Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis
Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis
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 ( 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 nucleotides. The activity was also measured using 31P NMR. Ft PPK2 has been crystallized and the structure determined to 2.23 Å resolution. The structure consists of a 6- stranded parallel ? sheet surrounded by 12 ? 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 ?FTT1564 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.
polyphosphate, kinase, francisella tularensis, x-ray crystallography, enzyme kinetics, antiobiotic sensitivity
0144-8463
1-35
Batten, Laura E.
5ad00360-5a0c-426c-bbf4-f274a25d7218
Parnell, Alice E.
a263bdb8-fa95-4137-aa82-7b3aaeb50ec0
Wells, Neil J.
86312185-007b-495b-86da-4e2e5b9b8025
Murch, Amber L.
23e3d7ca-bc11-4ddd-883d-92495b0ac434
Oyston, Petra C.F.
bba5dae5-b0ee-4733-b6bd-05866be64793
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9
Batten, Laura E.
5ad00360-5a0c-426c-bbf4-f274a25d7218
Parnell, Alice E.
a263bdb8-fa95-4137-aa82-7b3aaeb50ec0
Wells, Neil J.
86312185-007b-495b-86da-4e2e5b9b8025
Murch, Amber L.
23e3d7ca-bc11-4ddd-883d-92495b0ac434
Oyston, Petra C.F.
bba5dae5-b0ee-4733-b6bd-05866be64793
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9

Batten, Laura E., Parnell, Alice E., Wells, Neil J., Murch, Amber L., Oyston, Petra C.F. and Roach, Peter L. (2015) Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis. Bioscience Reports, 1-35. (PMID:26582818)

Record type: Article

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 ( 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 nucleotides. The activity was also measured using 31P NMR. Ft PPK2 has been crystallized and the structure determined to 2.23 Å resolution. The structure consists of a 6- stranded parallel ? sheet surrounded by 12 ? 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 ?FTT1564 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.

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Accepted/In Press date: 18 November 2015
e-pub ahead of print date: 18 November 2015
Keywords: polyphosphate, kinase, francisella tularensis, x-ray crystallography, enzyme kinetics, antiobiotic sensitivity

Identifiers

Local EPrints ID: 386858
URI: https://eprints.soton.ac.uk/id/eprint/386858
ISSN: 0144-8463
PURE UUID: 2576f9df-f4e5-42e3-93b4-a7e4e3853902
ORCID for Neil J. Wells: ORCID iD orcid.org/0000-0002-4607-5791
ORCID for Peter L. Roach: ORCID iD orcid.org/0000-0001-9880-2877

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Date deposited: 04 Feb 2016 16:44
Last modified: 14 Aug 2018 00:34

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