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

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