A polyphosphate kinase (PPK2) widely conserved in bacteria

Abstract

Synthesis of inorganic polyphosphate (poly P) from the terminal phosphate of ATP is catalyzed reversibly by poly P kinase (PPK, now designated PPK1) initially isolated from Escherichia coli. PPK1 is highly conserved in many bacteria, including some of the major pathogens such as Pseudomonas aeruginosa. In a null mutant of P. aeruginosa lacking ppk1, we have discovered a previously uncharacterized PPK activity (designated PPK2) distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by poly P. The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, poly P synthesis from GTP. The gene encoding PPK2 (ppk2) was identified from the amino acid sequence of the protein purified near 1,000-fold, to homogeneity. The 5'-end is 177 bp upstream of the annotated genome sequence of a "conserved hypothetical protein"; ppk2 (1,074 bp) encodes a protein of 357 aa with a molecular mass of 40.8 kDa. Sequences homologous to PPK2 are present in two other proteins in P. aeruginosa, in two Archaea, and in 32 other bacteria (almost all with PPK1 as well); these include rhizobia, cyanobacteria, Streptomyces, and several pathogenic species. Distinctive features of the poly P-driven nucleoside diphosphate kinase activity and structural aspects of PPK2 are among the subjects of an accompanying report.

Fig. 1.

SDS/PAGE analysis of purified PPK2. Samples were precipitated with 0.015% deoxycholate-5% trichloroacetic acid (18). Proteins were visualized by Coomassie blue staining. Samples of two different purification operations (A and B), both with nearly the same specific activity of 5.5 × 10⁶ units/mg, were applied as indicated. M, protein molecular mass markers (Precision Protein Standards, Bio-Rad).

Fig. 2.

Processivity of PPK2 poly P synthesis. A 25-μl assay mixture contained 56 ng of PPK2. After incubation at 37°C for the indicated times, the products were separated by PAGE (20% gel) containing 7 M urea and visualized by PhosphorImager (Molecular Dynamics); the [γ-³²P]GTP and poly P₇₅₀ migrated on the gel as indicated.

Fig. 3.

Chain lengths of poly P synthesized by PPKs. PPK2 of P. aeruginosa (Pa; 140 ng); PPK1 of P. aeruginosa (Pa; 120 ng); PPK1 of E. coli (Ec; 29 ng). After 45 min at 37°C, the products were separated by PAGE (20% gel) containing 7 M urea and visualized by PhosphorImager (Molecular Dynamics); the positions to which [γ-³²P]ATP and [γ-³²P]GTP migrated on the gel are indicated.

Fig. 4.

Nucleotide (ppk2) and the predicted amino acid (PPK2) sequences of P. aeruginosa. The nucleotide sequence (5′ to 3′) of the noncoding strand starts at nucleotide 1 and terminates at 1074. A possible Shine–Delgarno sequence (AGGAGT) for translation initiation starting at −11 bp is marked by a wavy line. The amino-terminal amino acid sequence determined from purified PPK2 (underlined) agrees with the deduced amino acid sequence from the start codon. The incorrect original annotation of the start codon at nucleotide 178 (GTG) is shaded. The P-loop sequence is marked by a dotted line, and the conserved amino acids are shaded. Three aspartyl-prolyl (D-P) bonds are identified in boxes.

Fig. 5.

Alignment of PPK2 and two homologs in P. aeruginosa. The identical amino acids are shaded in solid black. Hom1 is homolog 1 (hypothetical protein PA2428, GenBank accession no. NP_251118); Hom2 is homolog 2 (hypothetical protein PA3455, GenBank accession no. NP_252145).