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J Biol Chem, Vol. 274, Issue 37, 26185-26191, September 10, 1999

Enzyme I^Ntr from Escherichia coli
A NOVEL ENZYME OF THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM EXHIBITING STRICT SPECIFICITY FOR ITS PHOSPHORYL ACCEPTOR, NPr

From the Department of Biology, University of California at San Diego, La Jolla, California 92093-0116

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) phosphorylates sugars and regulates cellular metabolic processes using a phosphoryl 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 coli genome has revealed the presence of 5 paralogues of EI and 5 paralogues of HPr, most of unknown function. The ptsP gene encodes an EI paralogue designated Enzyme I^nitrogen (EI^Ntr), and two genes located in the rpoN operon encode PTS protein paralogues, NPr and IIA^Ntr, both implicated in the regulation of ⁵⁴ activity. The ptsP gene was polymerase chain reaction amplified from the E. coli chromosome and cloned into an overexpression vector allowing the overproduction and purification of EI^Ntr. EI^Ntr was shown to phosphorylate NPr in vitro using either a [³²P]PEP-dependent protein phosphorylation assay or a quantitative sugar phosphorylation assay. EI^Ntr phosphorylated NPr but not HPr, whereas Enzyme I exhibited a strong preference for HPr. These two pairs of proteins (EI^Ntr/NPr and EI/HPr) thus exhibit little cross-reactivity. Phosphoryl transfer from PEP to NPr catalyzed by EI^Ntr has a pH optimum of 8.0, is dependent on Mg²⁺, is stimulated by high ionic strength, and exhibits two K_m values for NPr (2 and 10 µM) possibly because of negative cooperativity. The results suggest that E. coli possesses at least two distinct PTS phosphoryl transfer chains, EI^Ntr  NPr  IIA^Ntr and EI  HPr  IIA^sugar. Sequence comparisons allow prediction of residues likely to be important for specificity. This is the first report demonstrating specificity at the level of the energy coupling proteins of the PTS.

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