Purification and characterization of polyphosphate kinase from Neisseria meningitidis

Abstract

The important human pathogens Neisseria meningitidis and Neisseria gonorrhoeae accumulate phosphate in the form of polyphosphate (A. Noegel and E. C. Gotschlich, J. Exp. Med. 157:2049-2060, 1983), and the localization of more than half of this long-chain polymer on the exterior of the cells suggests a function as a protective, capsule-like coating. To enable further genetic investigation of the role of polyphosphate in Neisseria spp., the enzyme polyphosphate kinase (PPK), which catalyzes the synthesis of polyphosphate from ATP, was purified from N. meningitidis BNCV. The activity is dependent on Mg2+ and phosphate or polyphosphate and is inhibited by ADP. The Km for ATP is 1.5 mM, and the turnover number is 47 phosphate residues per polypeptide per s. Analysis of PPK labelled with [gamma-32P]ATP indicates that the enzyme is phosphorylated during the reaction, probably at an arginine residue. N-terminal and two internal amino acid sequences were derived from the purified protein and will allow the design of synthetic oligonucleotides for cloning and genetic manipulation of the ppk gene.

Identification of genes involved in salt adaptation in the archaeonMethanosarcina mazeiGö1 using genome-wide gene expression profiling

“…It can serve as an ATP substitute and an energy source, as a phosphate reserve, as a chelator of metal ions, or as a buffer against alkali ions (Kornberg, 1995; Kulaev & Kulakovskaya, 2000). PolyP is also a component of the capsule of Neisseria species (Tinsley et al , 1993), and it is directly involved in the regulation of gene expression (Kusano & Ishihama, 1997; Kim et al , 1998; Rao & Kornberg, 1999). It was shown that polyP plays a role in the promoter selectivity control of RNA polymerase in E. coli cells grown at high salt concentrations during the stationary phase (Kusano & Ishihama, 1997).…”

Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates

“…The resulting homogenate was centrifuged at 25 000 g for 60 s at 4°C to remove cell debris. Recovery of polyP was achieved by combined precipitation with both cetyltrimethylammonium bromide (CTAB) detergent reagent (Tinsley et al 1993) and BaCl 2 (Rao et al 1985). CTAB reagent was prepared according to the method of Tinsley et al (1993) and contained 2AE5% CTAB, 0AE5 mol l )1 Tris, 0AE25 mol l )1 EDTA (pH 8AE0) and 0AE25 mol l )1 NaCl.…”

“…Recovery of polyP was achieved by combined precipitation with both cetyltrimethylammonium bromide (CTAB) detergent reagent (Tinsley et al 1993) and BaCl 2 (Rao et al 1985). CTAB reagent was prepared according to the method of Tinsley et al (1993) and contained 2AE5% CTAB, 0AE5 mol l )1 Tris, 0AE25 mol l )1 EDTA (pH 8AE0) and 0AE25 mol l )1 NaCl. To precipitate polyP from the assay system, equal volumes of CTAB reagent and assay mixture were combined and incubated for 10 min at 30°C under gentle agitation.…”

Discovery of potent polyphosphate kinase 1 (PPK1) inhibitors using structure‐based exploration of PPK1Pharmacophoric space coupled with docking analyses

“…1,4 PolyP is found to form capsule coatings linked to the cell membrane in Neisseria species. 5 In species like Helicobacter pylori, cytoplasmic polyP is present as compact particles easily spotted at the flagellar pole. 6 In addition, polyP has been found critical for attributes such as motility, quorum sensing, biofilm formation, resistance to oxidative, osmotic, heat, and alkaline stresses, and stationary-phase survival.…”