Polyphosphate kinase as a nucleoside diphosphate kinase in Escherichia coli and Pseudomonas aeruginosa

Abstract

Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or over-abundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of polyphosphate kinase (PPK) to use inorganic polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E. coli; the activity was absent from the membrane fractions obtained from E. coli mutants lacking the ppk gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2-60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5' tetraphosphate.

Figure 1

NDK activity of purified E. coli PPK. The reaction mixture (10 μl) containing 29 ng of purified PPK, PPK buffer, 1 mM (d)NDP, and 0.1 mM [³²P]polyP was incubated at 37°C for 30 min. The TLC plate was developed with 1.5 M KH₂PO₄ (pH 3.5) and autoradiographed.

Figure 2

polyP-dependent NDK activity in the E. coli membrane fraction. The reaction mixture (10 μl) containing the membrane fraction, PPK buffer, 1 mM NDP, and 0.1 mM [³²P]polyP was incubated at 37°C for 40 min. E. coli membrane fractions from MG1655 (wild type; 1.6 μg) and Δppk ppx mutant (2.1 μg) were used. The TLC plates were developed with (A) 1.5 M KH₂PO₄ (pH 3.5) and (B) 0.75 M KH₂PO₄ (pH 3.5).

Figure 3

Autoradiogram of the products of PPK action on [³²P]polyP and GDP. Unlabeled GTP, guanosine 5′-diphosphate 3′-diphosphate (ppGpp), and ppppG were visualized by UV (outlined). The two-dimensional TLC analysis is described in Materials and Methods.

Figure 4

Degradation of ppppG by PPX. α-[³²P]ppppG (20 pmol) was digested by yeast PPX (500 units) at 37°C for 60 min. The TLC plate was developed with 1.5 M KH₂PO₄ (pH 3.5) and autoradiographed.

Figure 5

NDK activity of P. aeruginosa membranes. The reaction mixture (10 μl) containing P. aeruginosa membrane fraction (0.73 μg), PPK buffer, 1 mM NDP, and 0.1 mM [³²P]polyP was incubated at 37°C for 60 min. The TLC plate was developed with 0.75 M KH₂PO₄ (pH 3.5) and autoradiographed.