Enzyme I-Ntr from Escherichia coli - A novel enzyme of the phosphoenolpyruvate-dependent phosphotransferase system exhibiting strict specificity for its phosphoryl acceptor, NPr

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) pho sphorylates sugars and regulates cellular metabolic processes using a phosp horyl transfer chain including the general energy coupling proteins, Enzyme I (EI) and HPr as well as the sugar-specific Enzyme II complexes. Analysis of the Escherichia coil genome has revealed the presence of 5 paralogues o f EI and 5 paralogues of HPr, most of unknown function. The ptsP gene encod es an EI paralogue designated Enzyme I-nitrogen (EINtr), and two genes loca ted in the rpoN operon encode PTS protein paralogues, NPr and IIANtr both i mplicated in the regulation of sigma(54) activity. The ptsP gene was polyme rase chain reaction amplified from the E. coil chromosome and cloned into a n overexpression vector allowing the overproduction and purification of EIN tr. EINtr was shown to phosphorylate NPr in vitro using either a [P-32]PEP- dependent protein phosphorylation assay or a quantitative sugar phosphoryla tion assay. EINtr phosphorylated NPr but not HPr, whereas Enzyme I exhibite d a strong preference for HPr, These two pairs of proteins (EINtr/NPr and E I/HPr) thus exhibit little cross-reactivity. Phosphoryl transfer from PEP t o NPr catalyzed by EINtr has a pH optimum of 8.0 is dependent on Mg2+ is st imulated by high ionic strength, and exhibits two K-m values for NPr (2 and 10 mu M) possibly because of negative cooperativity. The results suggest t hat E. coil possesses at least two distinct PTS phosphoryl transfer chains, EINtr --> NPr --> IIA(Ntr) and EI --> HPr --> IIA(sugar). Sequence compari sons allow prediction of residues likely to be important for specificity. T his is the first report demonstrating specificity at the level of the energ y coupling proteins of the PTS.