Cloning and characterization of Escherichia coli DUF299: a bifunctional ADP-dependent kinase--Pi-dependent pyrophosphorylase from bacteria

Abstract

Background: Phosphoenolpyruvate synthetase (PEPS; EC 2.7.9.2) catalyzes the synthesis of phosphoenolpyruvate from pyruvate in Escherichia coli when cells are grown on a three carbon source. It also catalyses the anabolic conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis. A bioinformatics search conducted following the successful cloning and expression of maize leaf pyruvate, orthophosphate dikinase regulatory protein (PDRP) revealed the presence of PDRP homologs in more than 300 bacterial species; the PDRP homolog was identified as DUF299.

Results: This paper describes the cloning and expression of both PEPS and DUF299 from E. coli and establishes that E. coli DUF299 catalyzes both the ADP-dependent inactivation and the Pi-dependent activation of PEPS.

Conclusion: This paper represents the first report of a bifunctional regulatory enzyme catalysing an ADP-dependent phosphorylation and a Pi-dependent pyrophosphorylation reaction in bacteria.

Figure 1

Phylogenetic relationships of bacterial and plant DUF299 proteins. The tree was constructed using a neighbour joining Poisson-corrected distance matrix method, with gaps distributed proportionally (MacVector, Accelrys).

Figure 2

Effect of pyruvate on the ADP-dependent inactivation of E. coli PEP synthetase. Purified PEPS and DUF299 were incubated in the presence of 0.65 units of PEPS, 0.5 mg DUF299, 25 mM Hepes-KOH, 5 mM MgCl₂, 5 mM DTT at pH 8.0. Inactivation was initiated by adding ADP and ATP to a final concentration of 2 and 0.1 mM, respectively. Pyruvate was added at the concentrations indicated. Experiments were conducted at least five times and the results presented in this figure are representative of the results obtained.

Figure 3

Effect of PEP pre-treatment on the ADP-dependent inactivation of PEPS. Purified PEPS was incubated with PEP (as shown by the square brackets) and the ATP removed by Sephadex G25 gel chromatography. The PEPS was then incubated in the presence of 25 mM Hepes-KOH, 5 mM MgCl₂, 5 mM DTT at pH 8.0. Additions as indicated were 2 mM ADP, 2 mM glucose (Glu), 5 units of hexokinase (HK) and 0.1 mM ATP. Experiments were conducted at least three times and the results presented in this figure are representative of the results obtained.

Figure 4

Scheme for the regulation of E. coli PEPS by ADP, pyruvate and ATP. The phosphorylation status of the catalytically-important histidine residue (His) and the regulatory threonine (Thr) residue of PEPS is shown and the location of the ATP, ADP and pyruvate is highlighted in boxes.