Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life? - PubMed (original) (raw)

. 2002 Oct 15;99(21):13442-7.

doi: 10.1073/pnas.212410399. Epub 2002 Oct 1.

Sandrine Poncet, Anne Galinier, Vicente Monedero, Sonia Fieulaine, Joël Janin, Sylvie Nessler, José Antonio Marquez, Klaus Scheffzek, Sonja Hasenbein, Wolfgang Hengstenberg, Josef Deutscher

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Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life?

Ivan Mijakovic et al. Proc Natl Acad Sci U S A. 2002.

Abstract

In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes ( approximately 10% of their genome) by acting as catabolite corepressor. HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate. We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate. HprK/P, which binds phosphate at the same site as the beta phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr. HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho-phosphorolysis mechanism. This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr. Growth of Bacillus subtilis on glucose increased intracellular pyrophosphate to concentrations ( approximately 6 mM), which in in vitro tests allowed efficient pyrophosphate-dependent HPr phosphorylation. To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM. In B. subtilis, this might be achieved by YvoE. This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with hprK.

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Figures

Figure 1

Figure 1

[32P]P-Ser-HPr dephosphorylation by B. subtilis HprK/P. [32P]P-Ser-HPr dephosphorylation was carried out as described in Materials and Methods. The reaction products were separated by TLC with 0.3 M potassium phosphate, pH 7.4. Radioactive standards for pyrophosphate, phosphate, and P-Ser-HPr (lanes 1–3, respectively); 10 μM [32P]P-Ser-HPr incubated with HprK/P for 30, 2, 5, 10, 20, and 30 min (lanes 4–9, respectively) in the absence (lane 4) or in the presence (lanes 5–9) of 5 mM phosphate; lane 10, 0.1 mM [32P]P-Ser-HPr was incubated for 15 min with HprK/P in the presence of 20 μM phosphate, and lane 11, same experiment as in lane 10 except that before adding HprK/P the sample was preincubated for 10 min with maltose and maltose phosphorylase. PPi, pyrophosphate.

Figure 2

Figure 2

Identification of the [32P]P-Ser-HPr dephosphorylation product. The reaction products of HprK/P-catalyzed [32P]P-Ser-HPr dephosphorylation were separated by TLC using 0.3 M KH2PO4 as solvent. Radioactive standards for pyrophosphate and P-Ser-HPr (lanes 1 and 4, respectively); 1-[32P]FBP formed from fructose-6-P and either [32P]pyrophosphate or [γ-32P]ATP (lanes 2 and 3, respectively). Dephosphorylation of [32P]P-Ser-HPr with B. subtilis HprK/P was carried out in the presence of fructose-6-P (lane 5), pyrophosphate-dependent phosphofructokinase (lane 6), and fructose-6-P and pyrophosphate-dependent phosphofructokinase (lane 7).

Figure 3

Figure 3

Pyrophosphate-dependent HprK/P-catalyzed phosphorylation of HPr. (A) HPr was phosphorylated with B. subtilis HprK/P and [32P]pyrophosphate at various concentrations (0.1, 0.2, 0.4, 1, 2 ,and 4 mM for lanes 1–6, respectively). The samples were separated on a 12.5% polyacrylamide gel containing 0.1% SDS. (B and C) HPr was phosphorylated with B. subtilis HprK/P in the absence (lanes 1–5) or presence (lanes 6–9) of 5 mM FBP with varying concentrations of ATP (B) or pyrophosphate (C), and samples were separated on nondenaturing polyacrylamide gels.

Figure 4

Figure 4

YvoE-catalyzed hydrolysis of pyrophosphate. [32P]Pyrophosphate (10 μM) was incubated with 0, 2.5, 25, and 50 nM YvoE (lanes 1–4, respectively). Phosphorolysis of 0.1 mM [32P]P-Ser-HP by HprK/P in the absence of YvoE (lane 5) and in the presence of 250 nM YvoE (lane 6). Samples were separated by TLC.

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