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1.
Identification of a phosphoenolpyruvate:fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes. 总被引:5,自引:2,他引:3
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W J Mitchell J Reizer C Herring C Hoischen M H Saier Jr 《Journal of bacteriology》1993,175(9):2758-2761
Listeria monocytogenes is a gram-positive bacterium whose carbohydrate metabolic pathways are poorly understood. We provide evidence for an inducible phosphoenolpyruvate (PEP):fructose phosphotransferase system (PTS) in this pathogen. The system consists of enzyme I, HPr, and a fructose-specific enzyme II complex which generates fructose-1-phosphate as the cytoplasmic product of the PTS-catalyzed vectorial phosphorylation reaction. Fructose-1-phosphate kinase then converts the product of the PTS reaction to fructose-1,6-bisphosphate. HPr was shown to be phosphorylated by [32P]PEP and enzyme I as well as by [32P]ATP and a fructose-1,6-bisphosphate-activated HPr kinase like those found in other gram-positive bacteria. Enzyme I, HPr, and the enzyme II complex of the Listeria PTS exhibit enzymatic cross-reactivity with PTS enzyme constituents from Bacillus subtilis and Staphylococcus aureus. 相似文献
2.
Regulation of genes coding for enzyme constituents of the bacterial phosphotransferase system. 总被引:19,自引:9,他引:10
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Regulation of the synthesis of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system was systematically studied in wild-type and mutant strains of Salmonella typhimurium and Escherichia coli. The results suggest that enzyme I and HPr as well as the glucose-specific and the mannose-specific enzymes II are synthesized by a mechanism which depends on (i) cyclic adenosine monophosphate and its receptor protein; (ii) extracellular inducer; (iii) the sugar-specific enzyme II complex which recognizes the inducing sugar; and (iv) the general energy-coupling proteins of the phosphotransferase system, enzyme I and HPr. 相似文献
3.
Involvement of the histidine protein (HPr) of the phosphotransferase system in chemotactic signaling of Escherichia coli K-12. 总被引:5,自引:3,他引:2
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It is known that in mutants of Escherichia coli lacking the histidine protein (HPr) of the carbohydrate: phosphotransferase system, all substrates of the system can be taken up in the presence of the fructose-regulated HPr-like protein FPr (gene fruF). Although this protein fully substituted for HPr in transport and phosphorylation, we found that it was not able to complement efficiently for HPr in mediating chemotaxis toward phosphotransferase system substrates. Furthermore, transport activity and chemotaxis could be genetically dissected by the exchange of single amino acids in HPr. The results suggest a specific role of HPr in chemotactic signaling. We propose a possible link of signal transduction pathways for phosphotransferase system- and methyl chemotaxis protein-dependent substrates via HPr. 相似文献
4.
Garrett DS Seok YJ Peterkofsky A Gronenborn AM Clore GM 《Nature structural biology》1999,6(2):166-173
The solution structure of the first protein-protein complex of the bacterial phosphoenolpyruvate: sugar phosphotransferase system between the N-terminal domain of enzyme I (EIN) and the histidine-containing phosphocarrier protein HPr has been determined by NMR spectroscopy, including the use of residual dipolar couplings that provide long-range structural information. The complex between EIN and HPr is a classical example of surface complementarity, involving an essentially all helical interface, comprising helices 2, 2', 3 and 4 of the alpha-subdomain of EIN and helices 1 and 2 of HPr, that requires virtually no changes in conformation of the components relative to that in their respective free states. The specificity of the complex is dependent on the correct placement of both van der Waals and electrostatic contacts. The transition state can be formed with minimal changes in overall conformation, and is stabilized in favor of phosphorylated HPr, thereby accounting for the directionality of phosphoryl transfer. 相似文献
5.
P Reddy N Fredd-Kuldell E Liberman A Peterkofsky 《Protein expression and purification》1991,2(2-3):179-187
We present methods for the rapid, simple purification of Enzyme I, HPr, and Protein IIIGlc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) using plasmids overproducing gene products. The gene for HPr (ptsH) was cloned into the expression vector pKC30. A simple procedure was devised for the purification to homogeneity of this protein from extracts of heat-induced cells containing pKC30/ptsH recombinant clone. The genes for Enzyme I (ptsI) and Protein IIIGlc (crr) were cloned separately into the expression vector pRE1. Rapid purification procedures were developed for the isolation of homogeneous preparations of these two proteins from extracts of heat-induced cells containing pRE1/ptsI and pRE1/crr recombinants. From about 6 g of cells, these procedures yielded 100, 86, and 50 mg of Enzyme I, HPr, and Protein IIIGlc, respectively. The activity of the proteins purified by these methods was comparable to that of the proteins isolated by previously published less efficient procedures. 相似文献
6.
Characterization of mutant histidine-containing proteins of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium. 总被引:4,自引:1,他引:3
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Histidine-containing phosphocarrier protein (HPr) is common to all of the phosphoenolpyruvate:sugar phosphotransferase systems (PTS) in Escherichia coli and Salmonella typhimurium, except the fructose-specific PTS. Strains which lack HPr activity (ptsH) have been characterized in the past, and it has proved difficult to delineate between tight and leaky mutants. In this study four different parameters of ptsH strains were measured: in vitro sugar phosphorylation activity of the mutant HPr; detection of 32P-labeled P-HPr; ability of monoclonal antibodies to bind mutant HPr; and sensitivity of ptsH strains to fosfomycin. Tight ptsH strains could be defined; they were fosfomycin resistant and produced no HPr protein or completely inactive mutant HPr. All leaky ptsH strains were fosfomycin sensitive, usually produced normal amounts of mutant HPr protein, and had low but measurable activity, and HPr was detectable as a phosphoprotein. This indicates that the regulatory functions of the PTS require a very low level of HPr activity (about 1%). The antibodies used to detect mutant HPr in crude extracts were two monoclonal immunoglobulin G antibodies Jel42 and Jel44. Both antibodies, which have different pIs, inhibited PTS sugar phosphorylation assays, but the antibody-HPr complex could still be phosphorylated by enzyme I. Preliminary evidence suggests that the antibodies bind to two different epitopes which are in part located in a beta-sheet structure. 相似文献
7.
Interaction between IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system and glycerol kinase of Salmonella typhimurium. 总被引:16,自引:13,他引:3
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Purified IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system of Salmonella typhimurium inhibits glycerol kinase. Phosphorylation of IIIGlc via phosphoenolpyruvate, enzyme I, and HPr abolishes this inhibition. The glycerol facilitator is not inhibited by IIIGlc. It is proposed that regulation of glycerol metabolism by the phosphoenolpyruvate:sugar phosphotransferase system is at the level of glycerol kinase. 相似文献
8.
Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation. 总被引:13,自引:9,他引:4
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In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion. 相似文献
9.
Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria 总被引:30,自引:3,他引:27
Josef Deutscher Elke Küster Uta Bergstedt Véronique Charrier Wolfgang Hillen 《Molecular microbiology》1995,15(6):1049-1053
CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phosphocarrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity. 相似文献
10.
Presence of protein constituents of the gram-positive bacterial phosphotransferase regulatory system in Acholeplasma laidlawii. 总被引:2,自引:1,他引:1
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C Hoischen A Dijkstra S Rottem J Reizer M H Saier Jr 《Journal of bacteriology》1993,175(20):6599-6604
Acholeplasma species have been reported to lack a functional phosphoenolpyruvate:sugar phosphotransferase system (PTS). We show here that Acholeplasma laidlawii possesses activities of enzyme I, HPr, HPr(ser) kinase, and HPr(ser-P) phosphatase but lacks detectable activities of enzymes II of the PTS. HPr from this organism was purified, and the regulatory properties of the kinase and phosphatase were characterized and shown to differ from those of previously studied bacteria. The results suggest the presence of an incomplete PTS in A. laidlawii which has the potential to function in a unique regulatory capacity. 相似文献
11.
HPr polymorphism in oral streptococci is caused by the partial removal of the N-terminal methionine. 总被引:2,自引:0,他引:2
HPr is a small phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system involved in the transport and phosphorylation of sugars. It has recently been reported that streptococci possess two forms of HPr having identical biochemical properties. In this communication, we show by N-terminal amino-acid sequencing and by ion-spray mass spectroscopy that these two forms differ by the presence or the absence of the N-terminal methionine. 相似文献
12.
Seok YJ Koo BM Sondej M Peterkofsky A 《Journal of molecular microbiology and biotechnology》2001,3(3):385-393
Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients. One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS). Many proteins interact with and are regulated by PTS components in bacteria. Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS. HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown. The evolution of the allosteric regulation of E. coli GP by HPr is compared to that of other phosphorylases. 相似文献
13.
Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants. 总被引:2,自引:2,他引:0
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S Koch S L Sutrina L F Wu J Reizer K Schnetz B Rak M H Saier Jr 《Journal of bacteriology》1996,178(4):1126-1133
The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in HPr play important roles in controlling conformational aspects of HPr that influence (i) autophosphohydrolysis, (ii) the interaction of this protein with the sugar permeases of the bacterial phosphotransferase system, and (iii) catalysis of phosphoryl transfer to the IIA domains in these permeases. 相似文献
14.
1H and 31P nuclear magnetic resonance investigations of the phosphoprotein intermediate P-HPr and the parent molecule HPr of the E. coli phosphoenolpyruvate dependent phosphotransferase system (PTS) show that HPr can exist in two conformations. These conformations influence the protonation state of the reactive histidine residue, thereby determining the reaction pathway in the phosphoryl group transfer step. A general mechanism is proposed for the energy-coupling process in the PTS. 相似文献
15.
Peterkofsky A Wang G Garrett DS Lee BR Seok YJ Clore GM 《Journal of molecular microbiology and biotechnology》2001,3(3):347-354
The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes a collection of proteins that accomplish phosphoryl transfer from phosphoenolpyruvate (PEP) to a sugar in the course of transport. The soluble proteins of the glucose transport pathway also function as regulators of diverse systems. The mechanism of interaction of the phosphoryl carrier proteins with each other as well as with their regulation targets has been amenable to study by nuclear magnetic resonance (NMR) spectroscopy. The three-dimensional solution structures of the complexes between the N-terminal domain of enzyme I and HPr and between HPr and enzyme IIA(Glc) have been elucidated. An analysis of the binding interfaces of HPr with enzyme I, IIA(Glc) and glycogen phosphorylase revealed that a common surface on HPr is involved in all these interactions. Similarly, a common surface on IIA(Glc) interacts with HPr, IIB(Glc) and glycerol kinase. Thus, there is a common motif for the protein-protein interactions characteristic of the PTS. 相似文献
16.
CcpA-dependent carbon catabolite repression in bacteria. 总被引:2,自引:0,他引:2
17.
Properties of two sugar phosphate phosphatases from Streptococcus bovis and their potential involvement in inducer expulsion. 总被引:1,自引:1,他引:0
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Streptococcus bovis possesses two sugar phosphate phosphatases (Pases). Pase I is a soluble enzyme that is inhibited by the membrane fractions from lactose-grown cells and is insensitive to activation by S46D HPr, an analog of HPr(ser-P) of the sugar phosphotransferase system. Pase II is a membrane-associated enzyme that can be activated 10-fold by S46D HPr, and it appears to play a role in inducer expulsion. 相似文献
18.
The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon. 总被引:3,自引:3,他引:0
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J Stülke I Martin-Verstraete V Charrier A Klier J Deutscher G Rapoport 《Journal of bacteriology》1995,177(23):6928-6936
The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS. 相似文献
19.
Solution structure of the phosphoryl transfer complex between the signal transducing proteins HPr and IIA(glucose) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system
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The solution structure of the second protein-protein complex of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system, that between histidine-containing phosphocarrier protein (HPr) and glucose-specific enzyme IIA(Glucose) (IIA(Glc)), has been determined by NMR spectroscopy, including the use of dipolar couplings to provide long-range orientational information and newly developed rigid body minimization and constrained/restrained simulated annealing methods. A protruding convex surface on HPr interacts with a complementary concave depression on IIA(Glc). Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIA(Glc). Formation of the unphosphorylated complex, as well as the phosphorylated transition state, involves little or no change in the protein backbones, but there are conformational rearrangements of the interfacial side chains. Both HPr and IIA(Glc) recognize a variety of structurally diverse proteins. Comparisons with the structures of the enzyme I-HPr and IIA(Glc)-glycerol kinase complexes reveal how similar binding surfaces can be formed with underlying backbone scaffolds that are structurally dissimilar and highlight the role of redundancy and side chain conformational plasticity. 相似文献
20.
In Gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system can be phosphorylated at two distinct sites, His-15 and Ser-46. While the former phosphorylation is implicated in phosphoryl transfer to the incoming sugars, the latter serves regulatory purposes. In Bacillus subtilis, the two phosphorylation events are mutually exclusive. In contrast, doubly phosphorylated HPr is present in cell extracts of Mycoplasma pneumoniae. In this work, we studied the ability of the two single phosphorylated HPr species to accept a second phosphoryl group. Indeed, both Enzyme I and the HPr kinase/phosphorylase from M. pneumoniae are able to use phosphorylated HPr as a substrate. The formation of doubly phosphorylated HPr is substantially slower as compared to the phosphorylation of free HPr. However, the rate of formation of doubly phosphorylated HPr is sufficient to account for the amount of HPr(His approximately P)(Ser-P) detected in M. pneumoniae cells. 相似文献