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Enzyme INtr from Escherichia coli

1999; Elsevier BV; Volume: 274; Issue: 37 Linguagem: Inglês

10.1074/jbc.274.37.26185

1083-351X

Ralf Rabus, Jonathan Reizer, Ian T. Paulsen, Milton H. Saier,

RNA and protein synthesis mechanisms

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 Initrogen(EINtr), and two genes located in the rpoN operon encode PTS protein paralogues, NPr and IIANtr, both implicated in the regulation of ς54 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 EINtr. EINtr was shown to phosphorylate NPr in vitro using either a [32P]PEP-dependent protein phosphorylation assay or a quantitative sugar phosphorylation assay. EINtr phosphorylated NPr but not HPr, whereas Enzyme I exhibited a strong preference for HPr. These two pairs of proteins (EINtr/NPr and EI/HPr) thus exhibit little cross-reactivity. Phosphoryl transfer from PEP to NPr catalyzed by EINtr has a pH optimum of 8.0, is dependent on Mg2+, is stimulated by high ionic strength, and exhibits two Km 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, EINtr → NPr → IIANtr and EI → HPr → IIAsugar. 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. 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 Initrogen(EINtr), and two genes located in the rpoN operon encode PTS protein paralogues, NPr and IIANtr, both implicated in the regulation of ς54 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 EINtr. EINtr was shown to phosphorylate NPr in vitro using either a [32P]PEP-dependent protein phosphorylation assay or a quantitative sugar phosphorylation assay. EINtr phosphorylated NPr but not HPr, whereas Enzyme I exhibited a strong preference for HPr. These two pairs of proteins (EINtr/NPr and EI/HPr) thus exhibit little cross-reactivity. Phosphoryl transfer from PEP to NPr catalyzed by EINtr has a pH optimum of 8.0, is dependent on Mg2+, is stimulated by high ionic strength, and exhibits two Km 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, EINtr → NPr → IIANtr and EI → HPr → IIAsugar. 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. phosphoenolpyruvate phosphoenolpyruvate:sugar phosphotransferase system dithiothreitol 4-morpholinepropanesulfonic acid EI, Enzyme I The bacterial phosphoenolpyruvate(PEP)1:sugar phosphotransferase system (PTS) is a complex protein system that mediates uptake and concomitant phosphorylation of carbohydrates (1Kundig W. Gosh S. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1067-1074Crossref PubMed Scopus (329) Google Scholar, 2Meadow N.D. Fox D.K. Roseman S. Annu. Rev. Biochem. 1990; 59: 497-542Crossref PubMed Scopus (302) Google Scholar, 3Postma P.W Lengeler J.W Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar). Characterized PTS proteins include the cytoplasmic Enzyme I and HPr, which lack sugar specificity, and membranous Enzyme II complexes, each specific for one or a few sugars. The latter complexes usually consist of three proteins or protein domains that are designated IIA, IIB, and IIC (4Saier Jr., M.H. Reizer J. J. Bacteriol. 1992; 174: 1433-1438Crossref PubMed Google Scholar). The phosphoryl relay proceeds sequentially from PEP to Enzyme I, HPr, IIA, IIB, and finally to the incoming sugar that is transported across the membrane and concomitantly phosphorylated by IIC. The PTS is present in a wide variety of Gram-positive and Gram-negative bacteria, but PTS protein homologues have not been found in archaea or eukaryotes. In addition to its primary functions in sugar transport, sugar phosphorylation, and chemoreception, the PTS is involved in regulatory processes such as catabolite repression and inducer exclusion (5Saier Jr., M.H. Microbiol. Rev. 1989; 53: 108-120Google Scholar, 6Saier Jr., M.H. Reizer J. Mol. Microbiol. 1994; 13: 755-764Crossref PubMed Scopus (171) Google Scholar). Novel PTS proteins, NPr and IIANtr (paralogues of HPr and IIAFru, respectively), are encoded by the npr and ptsN genes, respectively, localized to the rpoN operon of Escherichia coli, which also encodes the nitrogen-related ς factor, ς54 (7Merrick M.J. Mol. Microbiol. 1993; 10: 903-909Crossref PubMed Scopus (341) Google Scholar, 8Merrick M.J. Taylor M. Saier Jr., M.H. Reizer J. Nitrogen Fixation: Fundamentals and Applications.in: Tikhonovich I.A. Provorov N.A. Romanov V.I. Newton W.E. Proceedings of the 10th International Congress on Nitrogen Fixation. Kluwer Academic Publishers, London1995: 189-194Google Scholar, 9Jones D.H.A. Franklin F.C.H. Thomas C.M. Microbiology (Read.). 1994; 140: 1035-1043Crossref PubMed Scopus (42) Google Scholar, 10Powell B.S. Court D.L. Inada T. Nakamura Y. Michotey V. Cui X. Reizer A. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1995; 270: 4822-4839Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar).ptsN deletion mutants lacking IIANtr exhibit a growth defect in the presence of an organic nitrogen source and a sugar or tricarboxylic acid cycle intermediate. Protein phosphorylation involving NPr and IIANtr was suggested to function in linking carbon and nitrogen metabolism, and IIANtr has also been implicated in the regulation of the essential GTPase, Era, which appears to function in cell cycle progression and the initiation of cell division (10Powell B.S. Court D.L. Inada T. Nakamura Y. Michotey V. Cui X. Reizer A. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1995; 270: 4822-4839Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 11Britton R.A. Powell B.S. Dasgupta S. Sun Q. Margolin W. Lupski J.R. Court D.L. Mol. Microbiol. 1998; 27: 739-750Crossref PubMed Scopus (109) Google Scholar). IIANtr homologues have been identified in numerous Gram-negative bacteria (10Powell B.S. Court D.L. Inada T. Nakamura Y. Michotey V. Cui X. Reizer A. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1995; 270: 4822-4839Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), and a link between the ptsN gene and nitrogen regulation has been suggested for Rhizobium etli (12Michiels J. Van Soom T. D'Hooghe I. Dombrecht B. Benhassine T. De Wilde P. Vanderleyden J. J. Bacteriol. 1998; 180: 1729-1740Crossref PubMed Google Scholar), Pseudomonas aeruginosa (13Jin S. Ishimoto K. Lory S. J. Bacteriol. 1994; 176: 1316-1322Crossref PubMed Google Scholar), and Klebsiella pneumoniae (14Merrick M.J. Coppard J.R. Mol. Microbiol. 1989; 3: 1765-1775Crossref PubMed Scopus (83) Google Scholar). The crystal structure of Enzyme IIANtr has recently been determined (15Bordo D. van Monfort R.L. Pijning T. Kalk K.H. Reizer J. Saier Jr., M.H. Dijkstra B.W. J. Mol. Biol. 1998; 279: 245-255Crossref PubMed Scopus (31) Google Scholar). Analysis of the E. coli genome revealed a gene, ptsP, encoding Enzyme INtr (EINtr), consisting of 2 domains, an N-terminal domain of 127 amino acids homologous to the N-terminal "sensory" domain of the NifA protein of Azotobacter vinelandii (16Austin S. Buck M. Cannon W. Eydmann T. Dixon R. J. Bacteriol. 1994; 176: 3460-3465Crossref PubMed Scopus (65) Google Scholar) and a C-terminal domain of 578 amino acids homologous to all currently sequenced enzymes I. EINtr was suggested to serve a sensory function linking carbon and nitrogen metabolism (17Reizer J. Reizer A. Merrick M.J. Plunkett III, G. Rose D.J. Saier Jr., M.H. Gene (Amst.). 1996; 181: 103-108Crossref PubMed Scopus (67) Google Scholar). A mutation in the orthologous EINtr-encoding ptsP gene of A. vinelandii resulted in impaired metabolism of poly-β-hydroxybutyrate as well as diminished respiratory protection of nitrogenase under carbon-limiting conditions (18Segura D. Espin G. J. Bacteriol. 1998; 180: 4790-4798Crossref PubMed Google Scholar). In the present study we report the cloning and overexpression of the ptsP gene from E. coli, the purification of EINtr, and the characterization of its biochemical activities. The results of these studies establish for the first time the existence of parallel but independent PTS phosphoryl transfer chains in which distinct Enzyme I paralogues exhibit specificity for their cognate HPr paralogues. Residues are identified that may account for this specificity. Salmonella typhimurium strain SB2950(ΔcysKptsHIcrrΔtrpB223) has been described (19Cordaro J.C. Roseman S. J. Bacteriol. 1972; 112: 17-29Crossref PubMed Google Scholar). The cloning vector pCR2.1TM was from Invitrogen (San Diego, CA). Plasmid pJRENtr used for overexpression of EINtr is described below. Purified ppGpp was kindly provided by Mike Cashel (National Institutes of Health, Bethesda, MD). [U-14C]d-Mannitol (specific activity 32 mCi/mmol) and [γ-32P]ATP (specific activity >4000 Ci/mmol) were obtained from ICN (Costa Mesa, CA). Other chemicals were of analytical grade. A DNA fragment containing the ptsP gene encoding EINtrwas amplified by polymerase chain reaction using E. coli genomic DNA. The top strand primer (Ntr1) contained a Nde I site within the initiation codon (underlined) of ptsP, 5′-ACACGAATTCCATATGCTCACTCGCCTGCGCGAAATAG-3′. The bottom strand (Ntr2) contained a Sal I site (underlined) 5′-CTAAGTCGACATGATCCGCGCTATAACCCTCCGCGAA-3′. Amplification was performed with a Hybaid thermal reactor (Hybaid Ltd., Teddigton, Middlessex, United Kingdom) in a reaction mixture containing Taq DNA polymerase, 100 mm Tris/HCl, pH 8.8, 15 mm MgCl2, and 250 mm KCl (Stratagene, La Jolla, CA) in a total volume of 100 μl. The amplification mixture was overlaid with 50 μl of mineral oil and subjected to 30 cycles of amplification as follows: 1-min denaturation at 94 °C, 1-min annealing at 55 °C, and 2-min extension at 72 °C. The polymerase chain reaction-amplified DNA was ligated to pCR2.1TM (Invitrogen), and the Nde I-Sal I fragment encompassing the complete ptsP gene was then excised from this plasmid and cloned between the Nde I and Sal I sites of the overexpression vector pROEXTM-1 (Life Technologies, Inc.) to create the plasmid pJRENtr. Cloning of ptsP was confirmed by nucleotide sequencing using polymerase chain reaction dye terminator sequencing on an ABI 373 sequencer (Applied Biosystems) with a series of oligonucleotides specific for ptsP. Synthetic oligonucleotides were obtained commercially from either GenSet (La Jolla, CA) or Research Genetics, Inc. (Huntsville, AL). E. coli DH10B was transformed with the expression vector pJRENtr encoding EINtr. DH10B cells bearing the overexpression plasmid were grown overnight at 37 °C in LB containing ampicillin (100 μg/ml) and diluted 50–100-fold into fresh LB medium containing the same concentration of ampicillin. When the optical density of the culture growing at 37 °C reached an A600 of 0.5–1.0, isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 0.1 mm, and incubation was continued for an additional 3 h. Cells were collected by centrifugation, resuspended in TP buffer (50 mm Tris/HCl, pH 7.4, containing 0.2 mm phenylmethylsulfonyl fluoride), and ruptured as described previously (20Reizer J. Sutrina S.L. Saier Jr., M.H. Stewart G.C. Peterkofsky A. Reddy P. EMBO J. 1989; 8: 2111-2120Crossref PubMed Scopus (94) Google Scholar). The crude extract was centrifuged for 20 min at 16,000 rpm (SS34 rotor, RC5-centrifuge; Sorvall-DuPont, Wilmington, DE), and the overexpressed EINtr, which was found primarily in the pellet, was solubilized with 6 m guanidinium hydrochloride. Following centrifugation for 20 min at 16,000 rpm (SS34 rotor, RC5-centrifuge), the supernatant containing the overexpressed protein was applied to a nickel-nitrilotriacetic acid resin (Novagen, Inc., Madison, WI) column equilibrated with 20 mm Tris/HCl (pH 7.9) containing 0.5m NaCl and 6 m guanidinium hydrochloride. The column was washed with 10 volumes of 20 mm Tris/HCl, pH 7.9, containing 0.5 m NaCl, 20 mm imidazole, and 6 m guanidinium hydrochloride, and EINtrwas eluted with 50 mm Tris/HCl buffer, pH 7.9, containing 1m NaCl, 0.3 m imidazole, 10% glycerol, and 6m guanidinium hydrochloride. The fraction containing EINtr was then dialyzed against 50 mm Tris/HCl buffer, pH 7.5, containing 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm EDTA, 1 mm dithiothreitol, and 10% glycerol. E. coli EI, HPr, and NPr were overproduced and purified as described previously (10Powell B.S. Court D.L. Inada T. Nakamura Y. Michotey V. Cui X. Reizer A. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1995; 270: 4822-4839Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 21Reizer J. Reizer A. Saier Jr., M.H. Jacobson G.R. Protein Sci. 1992; 1: 722-726Crossref PubMed Scopus (63) Google Scholar,22Reizer J. Sutrina S.L. Wu L.F. Deutscher J. Reddy P. Saier Jr., M.H. J. Biol. Chem. 1992; 267: 9158-9169Abstract Full Text PDF PubMed Google Scholar). [32P]PEP was synthesized from [γ-32P]ATP using phosphoenolpyruvate carboxykinase from E. coli (23Mattoo R.L. Waygood E.B. Anal. Biochem. 1983; 128: 245-249Crossref PubMed Scopus (36) Google Scholar). [32P]PEP was separated from [γ-32P]ATP and [32P]Pi by ion-exchange chromatography on AG-1-X8 bicarbonate resin (analytical grade anion exchange resin, 20–50 mesh, chloride form (Bio-Rad)). Protein phosphorylation reactions were modified after Powell et al. (10Powell B.S. Court D.L. Inada T. Nakamura Y. Michotey V. Cui X. Reizer A. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1995; 270: 4822-4839Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The EINtr-specific phosphorylation reaction (at 37 °C for 15 min; 20 μl final volume) contained 250 mm HEPES, pH 8.0, 2 mm dithiothreitol, 5 mm MgCl2, 0.125 mm[32P]PEP (1.2 × 105 counts/min/nmol), 0.6 μg of EINtr, and 10 μg of NPr. The EI-specific phosphorylation reaction (at 37 °C for 15 min, 20 μl final volume) contained 50 mm Tris/HCl, pH 7.2, 2 mmdithiothreitol, 5 mm MgCl2, 0.125 mm [32P]PEP (1.2 × 105counts/min/nmol), 0.6 μg of EI, and 1.25 μg of HPr. Proteins were separated by SDS-polyacrylamide gel electrophoresis as described previously (20Reizer J. Sutrina S.L. Saier Jr., M.H. Stewart G.C. Peterkofsky A. Reddy P. EMBO J. 1989; 8: 2111-2120Crossref PubMed Scopus (94) Google Scholar, 24Reizer J. Novotny M.J. Hengstenberg W. Saier Jr., M.H. J. Bacteriol. 1984; 160: 333-340Crossref PubMed Google Scholar). Proteins labeled with [32P]PEP were detected by autoradiography as described (24Reizer J. Novotny M.J. Hengstenberg W. Saier Jr., M.H. J. Bacteriol. 1984; 160: 333-340Crossref PubMed Google Scholar). Concentrations of soluble PTS proteins, EINtr, EI, NPr, and HPr, were determined according to the method described by Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216334) Google Scholar). Protein concentrations in butanol-urea-extracted membranes were measured using the DC Protein Assay as described by the manufacturer (Bio-Rad). Bovine plasma γ globulin was used as a standard. Membrane samples and standards were boiled for 5 min in the presence of 1% SDS or N-octyl-3-d-glucopyranoside prior to the addition of the reagents. Membranes containing high levels of Enzyme IICBAMtl, used for assaying EI and EINtr, were prepared from strain SB2950, which lacks EI, HPr, and IIAGlc (26Waygood E.B. Meadow N.D. Roseman S. Anal. Biochem. 1979; 95: 293-304Crossref PubMed Scopus (66) Google Scholar). Batch cultures (5 liters) of strain SB2950 were grown overnight in nutrient broth (Difco). Cultures were centrifuged at 16,000 × g (GSA rotor, RC-5 centrifuge) for 5–10 min at 4 °C, washed in mineral medium (50 mm potassium phosphate buffer, pH 7.5, containing 15 mm(NH4)2SO4 and 1.7 mmMgSO4), and resuspended in 25 ml of 50 mmpotassium phosphate buffer, pH 7.5, containing 2 mm DTT, 1 mm EDTA, and 10 μg/ml DNase I. The cells were ruptured at 10,000 p.s.i. in a French pressure cell, and intact cells and cell debris were removed by centrifugation at 14,500 × g (SS34 rotor, RC-5 centrifuge) for 10 min at 4 °C. Membranes were recovered from the supernatant by centrifugation at 100,000 ×g (Ti rotor, L7–65 Ultracentrifuge, Beckman Instruments) and resuspended in a few ml of 50 mm potassium phosphate buffer, pH 7.5, containing 2 mm DTT and 1 mmEDTA. These membranes were extracted with urea and 1-butanol (27Kundig W. Roseman S. J. Biol. Chem. 1971; 246: 1407-1418Abstract Full Text PDF PubMed Google Scholar). Urea (480 mg/ml) was added and dissolved by stirring on ice. Then 1-butanol (40 μl/ml) was added, and this solution was gently stirred for 2 h on ice. Membranes were recovered by centrifugation at 100,000 ×g (Ti rotor) for 4 h at 4 °C. Extracted membranes from 5 liters of stationary phase cells were resuspended in 10 ml of 25 mm Tris/HCl, pH 7.5, containing 1 mm DTT and 1 mm EDTA to a final protein concentration of 11–32 mg/ml and transferred to boiled dialysis tubes (MWCO: 12–14.000, Spectrum Medical Industries Inc., Houston, TX). Membranes were dialyzed 3 times for 8 h against the same buffer and were then aliquoted in small plastic vials for storage at −20 °C. Extraction of peripheral proteins from the membranes eliminated background sugar phosphorylation activity. The [32P]PEP-dependent protein phosphorylation assay described above does not provide a rate but instead represents an equilibrium situation. To allow estimation of relative rates of phosphoryl transfer via various PTS proteins, a quantitative assay was required. We found that phosphorylation of [14C]mannitol (or another PTS sugar) could be used for this purpose although the rate of phosphoryl transfer involving EINtr and NPr was very low relative to that involving EI and HPr. [14C]Mannitol was selected as the phosphoryl acceptor because this sugar is PTS-specific, and no phosphatase cleaving the product ([14C]mannitol-1-phosphate) is known. The standard assay for PTS sugar phosphorylation employs membranes isolated from disrupted E. coli cells that overproduce several Enzyme II complexes because of a pts operon deletion (28Rephaeli A.W. Saier Jr., M.H. J. Bacteriol. 1980; 141: 658-663Crossref PubMed Google Scholar). Because the inverted membrane preparations are "leaky," protein components and sugar phosphate products are not compartmentalized. Thus, this assay provides a useful measure of relative rates of overall phosphoryl transfer employing EINtr and NPr as well as EI and HPr, even though the former reaction is not physiologically significant. The sugar phosphorylation assay used was modified from the method described by Kundig and Roseman (27Kundig W. Roseman S. J. Biol. Chem. 1971; 246: 1407-1418Abstract Full Text PDF PubMed Google Scholar) as required by the exceptionally low activity of EINtr in this assay. The reaction mixture (50 μl) generally consisted of 0.1 μg of EINtr, 5 μg of NPr, 5–10 μl of extracted membranes, 10 mm PEP, 10 mm MgCl2, 25 mm KF, 2.5 mm DTT, 250 mm HEPES or potassium phosphate, pH 8.0, and 1.5 μm[U-14C]d-mannitol (specific activity 32 mCi/mmol). The reaction mixture was usually preincubated for 1 h in the absence of [U-14C]d-mannitol, and the reaction was started by the addition of the labeled sugar. After incubation (usually at 37 °C for 2 h), the reaction was stopped by the addition of 1 ml of ice-cold deionized water. The sugar phosphorylation assay for EI was in general carried out in 50 mm HEPES buffer, pH 8.0, containing 3 ng of EI, 0.1 μg of HPr, 10 μl of extracted membrane, 10 mm PEP, 10 mm MgCl2, 25 mm KF, 2.5 mm DTT, and 1.5 μm[U-14C]d-mannitol (specific activity 32 mCi/mmol). EINtr or EI was present in rate-limiting amounts, whereas all other components of the assay mixtures were present in excess. [U-14C]d-Mannitol-phosphate was separated from the nonphosphorylated [U-14C]d-mannitol by ion exchange chromatography. The reaction mixture was transferred to Poly-Prep® chromatography columns (0.8 × 4 cm, Bio-Rad) containing analytical grade Bio-Rad anion exchange resin (AG 1-X2, 50–100 mesh, chloride form, Bio-Rad) (27Kundig W. Roseman S. J. Biol. Chem. 1971; 246: 1407-1418Abstract Full Text PDF PubMed Google Scholar). Nonphosphorylated [U-14C]d-mannitol was washed from the columns by the addition of three 10-ml aliquots of water. [U-14C]d-Mannitol-phosphate was then eluted with three 3-ml aliquots of 1 mm LiCl and collected in liquid scintillation vials. After the addition of 9 ml of Bio SafeII counting mixture (Research Products International Corp., Mount Prospect, CA), the radioactivity was measured. To study the properties of EINtr, we developed a sugar phosphorylation assay coupling phosphoryl transfer from PEP via purified EINtr, NPr, and Enzyme IICBAMtl to [14C]mannitol (see "Materials and Methods"). Under the condition described by Kundig and Roseman (27Kundig W. Roseman S. J. Biol. Chem. 1971; 246: 1407-1418Abstract Full Text PDF PubMed Google Scholar) (20 mm potassium phosphate buffer, pH 7.5), EINtr showed only marginal activity, but activity was greatly enhanced using 250 mm potassium phosphate, pH 8.0. Potassium phosphate buffer could be replaced by HEPES but not by MOPS, TES, TRIZMA, or Tris. The HEPES-based buffer system was used when precipitation of cations by phosphate was problematic. The pH range for EINtr was examined using the phosphate-buffered assay system. In contrast to Enzyme I, which has a pH optimum of 7.0–7.5 (29Waygood E.B. Steeves T. Can. J. Biochem. 1979; 58: 40-48Crossref Scopus (53) Google Scholar, 30Saier Jr., M.H. Schmidt M.R. Lin P. J. Biol. Chem. 1980; 255: 8579-8584Abstract Full Text PDF PubMed Google Scholar), EINtr activity was optimal at pH 8.0 (Fig. 1). The dependence of EINtr activity on divalent cations was tested with the HEPES-buffered system (31Weigel N. Kukuruzinska M.A. Nakazawa A. Waygood E.B. Roseman S. J. Biol. Chem. 1982; 257: 14477-14491Abstract Full Text PDF PubMed Google Scholar, 32Weigel N. Waygood E.B. Kukuruzinska M.A. Nakazawa A. Roseman S. J. Biol. Chem. 1982; 257: 14461-14469Abstract Full Text PDF PubMed Google Scholar). EINtr required Mg2+ with saturation being observed at a concentration of about 2 mm (Fig. 2). Mn2+ allowed activity of EINtr only at low concentrations (Fig. 2). In the presence of low concentrations of Co2+ or Ni2+, activity could be measured, but at concentrations above 0.2 mm, strong inhibition was observed.Figure 2Dependence of EINtr activity on the concentration of Mg2+. Activity of EINtr was determined using the sugar phosphorylation assay as described under "Materials and Methods." HEPES, pH 8.0, was used as the buffer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous reports on the Mg2+ dependence of EI have shown that the divalent cation is required for the PEP-dependent autophosphorylation of EI but not for phosphoryl transfer between EI and HPr (31Weigel N. Kukuruzinska M.A. Nakazawa A. Waygood E.B. Roseman S. J. Biol. Chem. 1982; 257: 14477-14491Abstract Full Text PDF PubMed Google Scholar, 32Weigel N. Waygood E.B. Kukuruzinska M.A. Nakazawa A. Roseman S. J. Biol. Chem. 1982; 257: 14461-14469Abstract Full Text PDF PubMed Google Scholar). The dependence of EINtr activity on the concentration of PEP was examined using the [14C]mannitol phosphorylation assay in phosphate buffer with three different concentrations of NPr (Fig. 3). Regardless of the concentration of NPr used, EINtr proved to be saturated at PEP concentrations higher than 10 mm. The data shown in Fig. 3 A were converted to a double reciprocal plot (1/v versus 1/S, Fig. 3 B). As can be seen, a single straight line could not be drawn through the data obtained with any one NPr concentration, but they could be fitted to two distinct lines with differing slopes. This feature is characteristic of an enzyme population that either consists of two distinct enzyme species with differing kinetic properties or of a single enzyme species exhibiting the property of negative cooperativity. The y intercepts (1/Vmax apparent) shown in Fig. 3 B were plotted versus 1/NPr for the two parts of the curves (Fig. 3 C). Straight lines were observed in each case, and these lines extrapolated to give Vmax and Km values, respectively, of 1 μmol of [14C]mannitol phosphate/mg of EINtr/min and 10 μm for the high velocity, low affinity curve, and 0.3 μmol of [14C]mannitol phosphate/mg of EINtr/min and 2 μm for the low velocity, high affinity curve. Analysis of time courses for EINtractivity showed that the enzyme exhibited a lag phase of 30–40 min under our standard assay conditions (Fig.4). When the otherwise complete reaction mixtures were preincubated for 1 h without either [U-14C]d-mannitol or the extracted membranes, no lag for sugar phosphorylation was observed (Fig. 4). However, the absence of extracted membranes during preincubation yielded lower activity than when mannitol was omitted, suggesting that the membranes might facilitate EINtr-NPr association. In contrast, when either EINtr or NPr was absent during the preincubation, a lag was still observed. Preincubation of EINtr with NPr was therefore required for optimal activity, suggesting that a slow association of these two proteins accounts for the lag phase observed in the control sample. Selective phosphoryl transfer by EI and EINtrcould be demonstrated using the 32P-protein phosphorylation assay. NPr was found to be a specific protein substrate of EINtr as the latter could not phosphorylate HPr (Fig.5 A, lanes 3 and 4). In contrast, Enzyme I was found to be specific for HPr and could barely phosphorylate NPr (Fig. 5 A, lanes 1 and 2). These observations were confirmed using the [14C]mannitol phosphorylation assay. In a previous report, a regulatory function was suggested for the N-terminal domain of EINtr (17Reizer J. Reizer A. Merrick M.J. Plunkett III, G. Rose D.J. Saier Jr., M.H. Gene (Amst.). 1996; 181: 103-108Crossref PubMed Scopus (67) Google Scholar). We employed the sugar phosphorylation assay to screen various compounds for effects on the NPr-dependent phosphoryl transfer activity of EINtr. The following salts increased the activity of EINtr when added to the potassium phosphate (50 mm, pH 8.0) -buffered reaction mixture: (NH4)2SO4, NH4Cl, Na2SO4, NaCl, K2SO4, KCl, and LiCl (tested in a concentration range of 10–400 mm). This demonstrated that EINtr activity requires high ionic strength, but no evidence for a specific effect by any one ionic species was obtained. Other compounds (concentrations between 0.1 and 10 mm) tested in the presence of 20 mm Mg2+ (to avoid Mg2+ limitation because of chelation) were l-glutamine,l-glutamate, α-ketoglutarate, ATP, ADP, AMP, GTP, GDP, GMP, UDP, ppGpp, and adenosine 3′-phosphate 5′-phophosulfate. Only GDP and ppGpp at concentrations of 5 mm or higher had effects on the activity of EINtr (50–60% inhibition at 10 mm). The inhibitory effect of GDP was confirmed using the [32P]PEP-dependent protein phosphorylation assay. EINtr-dependent phosphorylation of NPr was significantly reduced although the amount of phosphorylated Enzyme INtr remained similar (Fig. 5 B, lanes 1 and 2). Although phosphorylation of EI was not affected, the inhibition of NPr phosphorylation is not likely to be of physiological significance. Cyclic AMP and cyclic GMP were without effect in the concentration range 0.01–1 mm. A number of redox cofactors were examined with respect to potential regulatory effects. The following compounds were tested in the concentration range of 1–10 mm: NAD, NADH, NADP, NADPH, and FAD. Of these compounds, only FAD was inhibitory (50% with 1 mm FAD). EI was similarly inhibited. EINtr resembles the classical EI except for the N-terminal NifA-like putative sensory domain of EINtr. Both enzymes catalyze PEP-dependent phosphoryl transfer to an HPr-like protein in a pH-, salt-, and Mg2+- or Mn2+-dependent process (30Saier Jr., M.H. Schmidt M.R. Lin P. J. Biol. Chem. 1980; 255: 8579-8584Abstract Full Text PDF PubMed Google Scholar, 33Hoving H. Koning J.H. Robillard G.T. Biochemistry. 1982; 21: 3128-3136Crossref PubMed Scopus (9) Google Scholar, 34Kukuruzinska M.A. Harrington W.F. Roseman S. J. Biol. Chem. 1982; 257: 14470-14476Abstract Full Text PDF PubMed Google Scholar). The effects of ionic strength may be because of stabilization of the dimeric forms of these enzymes, and Mg2+ may similarly promote association (35Misset O. Brouwer M. Robillard G.T. Biochemistry. 1980; 19: 883-890Crossref PubMed Scopus (44) Google Scholar, 36Han M.K. Knutson J.R. Roseman S. Brand L. J. Biol. Chem. 1990; 265: 1996-2003Abstract Full Text PDF PubMed Google Scholar, 37Chauvin F. Brand L. Roseman S. J. Biol. Chem. 1994; 269: 20263-20269Abstract Full Text PDF PubMed Google Scholar). However, EINtr exhibits biphasic kinetics with respect to PEP concentration, whereas EI exhibits monophasic kinetics; EINtr exhibits essentially absolute specificity for NPr, whereas EI could phosphorylate NPr at a rate that was only a small fraction of that at which it phosphorylates HPr. The turnover number (38Segel I.H. Enzyme kinetics. Behaviour and analysis of rapid equilibrium and steady-state enzyme systems. John Wiley & Sons, New York1975Google Scholar) for EINtr using the sugar phosphorylation assay proved to be 0.8 pmol/pmol EINtr/min, whereas that for EI is 1.34 nmol/pmol EI/min (39Mattoo R.L. Waygood E.B. Can. J. Biochem. 1982; 61: 29-37Google Scholar). This 1000-fold difference explains, in part, why EI is absolutely required for sugar phosphorylation under in vivo conditions. ptsI deletion mutants can be mutated so as to express an EI paralogue that can phosphorylate HPr (40Chin A.M. Sutrina S. Feldheim D.A. Saier Jr., M.H. J. Bacteriol. 1987; 169: 894-896Crossref PubMed Google Scholar), but preliminary evidence suggests that this enzyme is not EINtr. 2R. Rabus, J. Reizer, I. Paulsen, and M. H. Saier, Jr., unpublished results. The nearly absolute specificity of EINtr for NPr and of EI for HPr provides the first evidence that two different EIs in a single organism exhibit specificity at the level of their phosphoryl acceptor PTS proteins. This finding clearly leads to both functional and mechanistic predictions. EINtr does not appear to function in sugar phosphorylation and may function exclusively in regulation, possibly controlling the activities of NPr and IIANtr. These latter proteins are encoded within the rpoN operon of E. coli and have been implicated in the regulation of ς54-dependent transcriptional initiation of genes concerned with organic nitrogen utilization (10Powell B.S. Court D.L. Inada T. Nakamura Y. Michotey V. Cui X. Reizer A. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1995; 270: 4822-4839Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Moreover, in A. vinelandii, the EINtr-encoding ptsP gene has been shown to play a role in poly-β-hydroxybutyrate metabolism and respiratory protection of nitrogenase under carbon-limiting conditions (18Segura D. Espin G. J. Bacteriol. 1998; 180: 4790-4798Crossref PubMed Google Scholar). In K. pneumoniae, P. aeruginosa, and R. etli, IIANtr has been shown to function in capacities similar to those demonstrated for E. coli (12Michiels J. Van Soom T. D'Hooghe I. Dombrecht B. Benhassine T. De Wilde P. Vanderleyden J. J. Bacteriol. 1998; 180: 1729-1740Crossref PubMed Google Scholar, 13Jin S. Ishimoto K. Lory S. J. Bacteriol. 1994; 176: 1316-1322Crossref PubMed Google Scholar, 14Merrick M.J. Coppard J.R. Mol. Microbiol. 1989; 3: 1765-1775Crossref PubMed Scopus (83) Google Scholar, 41Du Y. Holtel A. Reizer J. Saier Jr., M.H. Res. Microbiol. 1996; 147: 129-132Crossref PubMed Scopus (21) Google Scholar). EINtr, NPr, and IIANtr have all been identified in the fully sequenced genome of P. aeruginosa. 3J. Reizer and M. H. Saier, Jr., manuscript in preparation. As for E. coli, these Pseudomonas proteins presumably comprise a phosphoryl transfer chain that functions in parallel with EI, HPr, and various IIAsugar proteins with entirely different physiological consequences (see Fig. 7). Phylogenetic data have shown that NPr is a distant homolog of HPr (10Powell B.S. Court D.L. Inada T. Nakamura Y. Michotey V. Cui X. Reizer A. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1995; 270: 4822-4839Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), whereas EINtr is a distant homolog of EI (17Reizer J. Reizer A. Merrick M.J. Plunkett III, G. Rose D.J. Saier Jr., M.H. Gene (Amst.). 1996; 181: 103-108Crossref PubMed Scopus (67) Google Scholar). The mechanistic implications of our biochemical observations are that specific residues in EI versus EINtr and/or HPr versus NPr must control the interactions and phosphoryl transfer reactions between these proteins. Alignments of two segments of all sequenced EINtrs (Fig.6 A, top) with representative EIs (Fig. 6 A, bottom) as well as of recognized NPrs (Fig. 6 B, top) and HPrs (Fig.6 B, bottom) revealed such candidate residues. Residues that are fully conserved among all homologous proteins are presented in bold print, whereas residues that are fully conserved in one group, but of a different type in the other group, are shaded. In comparing EINtr with EI, we found that residues conserved in one of these two sets of proteins but not the other were scattered unevenly throughout the alignment. The greatest abundance of such residues was found to immediately surround the active site histidine (Fig. 6 A, top), a region of catalytic importance. This fact is particularly significant as the active site region is well conserved among either the EIs or the EINtrs. The second region of the multiple alignment exhibiting a high frequency of residues conserved in only one set of these two proteins occurred far downstream of the active site histidine in a region of unknown function (Fig. 6 A, bottom). Neither of these two regions is at the EI-HPr interface (42Garrett D.S. Seok Y.-J. Peterkofsky A. Gronenborn A. Clore G.M. Nat. Struct. Biol. 1999; 6: 166-173Crossref PubMed Scopus (208) Google Scholar). In the region that defines the EI-HPr interaction surface, two residues were markedly different in the EIs versus the EINtrs. These residues are the (A/G)H dipeptide in the EIs, which corresponds to the (V/L)Y dipeptide in the EINtrs. Fig. 6 B shows the full alignment of NPr sequences with HPr sequences. As for the EIs, the greatest abundance of residues fully conserved in the NPrs, but of a different nature in the HPrs, surrounds the active site histidine. These dissimilar residues may control relative rates of phosphoryl transfer. A second region exhibiting this characteristic is found C-terminal to the regulatory serine (43Saier Jr., M.H. Chauvaux S. Cook G.M. Deutscher J. Paulsen I.T. Reizer J. Ye J.-J. Microbiology (Read.). 1996; 142: 217-230Crossref PubMed Scopus (206) Google Scholar). Both of these regions comprise interaction sites with EI (42Garrett D.S. Seok Y.-J. Peterkofsky A. Gronenborn A. Clore G.M. Nat. Struct. Biol. 1999; 6: 166-173Crossref PubMed Scopus (208) Google Scholar). Of possible significance to the specificity of EINtr for NPr is the A XX M sequence in the NPrs that is lacking in the HPrs (positions 49–52 in the E. coli HPr). The C termini of NPrs additionally possess a fully conserved N XX FDE sequence that is not found in any of the HPrs. These observations provide a guide for site-directed mutagenic studies aimed at defining their functional differences. The presence of a NifA protein-like putative sensory domain in EINtr led to the postulate that this domain might sense the availability of a ligand that could control the phosphoryl transfer activity of the enzyme. Our search for such a small compound has been unsuccessful until now. Thus, GDP exerted an inhibitory effect but only at superphysiological concentrations. FAD also exerted an inhibitory effect at superphysiological concentrations, but because this effect was shared with EI, it cannot be attributed to the presence of the N-terminal NifA-like domain. It is possible that we have not identified the correct effector molecule, but it is equally possible that the effector we seek is a macromolecule such as the PII nitrogen regulatory protein (44Magasanik B. Neidhardt F.C. Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology. 2nd Ed. 1. ASM Press, Washington, D. C.1996: 1344-1356Google Scholar) rather than a small metabolite. Aravind and Ponting (45Aravind L. Ponting C.P. Trends Biochem. Sci. 1997; 22: 458-459Abstract Full Text PDF PubMed Scopus (488) Google Scholar) have noted that the NifA domain is homologous to a family of so-called GAF domains found in a variety of signal-transducing proteins, but the significance of this observation to EINtr function is not known. In summary, we have found that EINtr exhibits virtually absolute specificity for NPr, whereas EI is highly specific for HPr (Figs. 5 and 7). This finding demonstrates for the first time that a single organism can possess two complete and independent PEP-dependent phosphoryl transfer chains, which presumably serve completely different physiological functions. The fact that E. coli possesses 5 paralogues of both EI and HPr (46Reizer J. Saier Jr., M.H. Curr. Opin. Struct. Biol. 1997; 7: 407-415Crossref PubMed Scopus (68) Google Scholar) now leads to the possibility of multiple parallel and independently functioning phosphoryl transfer chains, each playing a different physiological role in the cell. Determination of the physiological functions of these PTS phosphoryl transfer chains provides exciting challenges for the future. We thank Mike Cashel for providing us with purified ppGpp as well as Joy Garg, Don Jack, Peter Jähn, and Karin Strecker for computational and technical assistance and Milda Simonaitis for manuscript assistance.