Enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: protein-protein and protein-phospholipid interactions

The mannitol-specific enzyme II (EII), purified free of phospholipid, exhibits a concentration dependence in its specific activity with P-HPr and mannitol as the donor and acceptor substrates, respectively. This concentration dependence, previously observed only in the case of mannitol----mannitol phosphate exchange reaction, indicates that an oligomeric form of the enzyme is responsible for catalyzing the phosphorylation reaction (P-HPr + mannitol----mannitol-P + HPr) as well as the exchange reaction. Kinetic analysis revealed that the monomeric enzyme has a much lower specific activity than the associated species. The specific activity can be increased by raising the steady-state level of phosphorylation of EII and also by adding phospholipid, demonstrating that phosphorylation and the binding of phospholipid facilitate the association process. Kinetic measurements and fluorescence energy transfer measurements demonstrate a strong preference of EII for phospholipids with specific head group and fatty acid composition.     

Staphylococcal lactose phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis on the lacE gene gives evidence that a cysteine residue is responsible for phosphorylation

The lactose-specific phosphocarrier protein enzyme II of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacE gene in order to replace the histidine residues 245, 274 and 510 and the cysteine residue 476 of the amino acid sequence with a serine residue. The wild-type and mutant genes were expressed in Escherichia coli and the gene products were characterized in different in vitro test systems. In vitro phosphorylation studies on mutant derivatives of the lactose-specific enzyme II led to the conclusion that cysteine residue 476 is the active-site for phosphorylation of this enzyme II by a phospho-enzyme III of the same sugar specificity. A cysteine residue phosphorylated intermediate was first postulated for the mannitol-specific enzyme II of E. coli and studies performed independently concerning the lactose-specific enzyme II of Lactobacillus casei are in agreement with the above results.     

Details of mannitol transport in Escherichia coli elucidated by site-specific mutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system

The mannitol transport protein (EIImtl) carries out translocation with concomitant phosphorylation of mannitol from the periplasm to the cytoplasm, at the expense of phosphoenolpyruvate (PEP). The phosphoryl group which is needed for this group translocation is sequentially transferred from PEP via two phosphorylation sites, located exclusively on the C-terminal cytoplasmic domain, to mannitol. Oligonucleotide-directed mutagenesis was used to investigate the precise role of these sites in phosphoryl group transfer, by producing specific amino acid substitutions. The first phosphorylation site, His-554 (P1), was replaced by Ala, which renders the EII-H554A completely inactive in PEP-dependent mannitol phosphorylation, but not in mannitol/mannitol 1-phosphate exchange. The P2 site mutant, EII-C384S, was inactive both in the mannitol phosphorylation reaction and in the exchange reaction, due to replacement of the essential Cys-384 by Ser. Although EII-H554A and EII-C384S were both catalytically inactive in the PEP-dependent phosphorylation, EII-C384S was able to restore up to 55% of the wild-type mannitol phosphorylation activity with the EII-H554A mutant, indicating a direct phosphotransfer between two subunits. These phosphorylation data together with the data obtained from mannitol/mannitol phosphate exchange kinetics, after mixing EII-H554A and EII-C384S, indicated the formation of functionally stable heterodimers, which consist of an EII-H554A and an EII-C384S monomer.     

Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: purification and characterization of the phosphocarrier protein.

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble enzyme I, and a soluble phosphocarrier protein, HPr. The HPr has been purified to homogeneity by a combination of ammonium sulfate precipitations, gel filtration and diethylaminoethyl, carboxymethyl Bio-Gel A, and hydroxylapatite column chromatography. The purified protein is relatively heat stable (ca. 50% activity survives 30 min of boiling) and has a molecular weight of ca. 10,000 (determined by sodium dodecyl sulfate-gel electrophoresis and amino acid analysis). It contains a single histidine residue per molecule and can be totally inactivated by photooxidation with Rose Bengal dye. Although the mycoplasma HPr is very similar to that of Escherichia coli, it shows no significant association with antiserum produced against E. coli HPr.     

Pleiotropic function of phosphoenolpyruvate-dependent phosphotransferase system in bacteria. Report 1

Modern data (collected mainly in the 1998-2001 studies) about the transport of carbohydrates in bacteria, about the regulation of utilization of sugars via the glycolytic pathway as well as about the regulation of transformation of pyruvat into the products of secondary metabolism and of tricarboxylic acid cycle are presented in the survey. Issues, related with the regulation of synthesis of enzymes involved in the last mentioned process, are discussed in detail. Besides, the key pathways pertaining to the regulation of synthesis and activity of adenylate cyclase; elimination of the inductor in the gram-negative bacteria and entry of phage lambda DNA into E. coli are described. As for the gram-positive bacteria, properties of their main components (involved in catabolic repression), i.e. HPr, K/P, CcpA, CCpB and CcpC, cre, are presented. The mechanisms of catabolic repressions and of catabolic activation in bacteria are in the focus of attention. Finally, issues related with the structural organization of PTS as well as molecular-and-biological aspects of the interaction of proteins of the mentioned system are considered in the survey.     

Sugar permeases of the bacterial phosphoenolpyruvate-dependent phosphotransferase system: sequence comparisons

The amino acyl sequences of eight permeases (enzymes II and enzyme II-III pairs) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) have been analyzed. All systems show similar sizes, and six of these systems exhibit the same molecular weight +/- 2%. Several exhibit sequence homology. Characteristic NH2-terminal and COOH-terminal sequences were found. The NH2-terminal leader sequences are believed to function in targeting of the permeases to the membrane, whereas the characteristic COOH-terminal sequences are postulated to mediate interaction with the energy-coupling protein phospho HPr. One of the systems, the one specific for mannose, exhibits distinctive characteristics. A pair of probable phosphorylation sites was detected in each of the five most similar systems, those specific for beta-glucosides, sucrose, glucose, N-acetylglucosamine, and mannitol. One of the two equivalent phosphorylation sites (proposed phosphorylation site 1) was located approximately 80 residues from the COOH terminus of each system. The other site (proposed phosphorylation site 2) was located approximately 440 residues from the COOH termini of the glucose and N-acetylglucosamine systems, approximately 320 residues from the COOH termini of the beta-glucoside and sucrose systems, and 381 residues from the COOH terminus of the mannitol system. Intragenic rearrangement during evolutionary history may account for the different positions of phosphorylation sites 2 in the different PTS permeases. More extensive intragenic rearrangements may have given rise to entirely different positions of phosphorylation in the glucitol, mannose, and lactose systems. A single, internal amphipathic alpha-helix with characteristic features was found in each of seven of the eight enzymes II. The lactose-specific enzyme III of Staphylococcus aureus was unique in possessing a COOH-terminal amphipathic alpha-helix rich in basic amino acyl residues. Possible functions for these amphipathic segments are discussed.     

Enzyme IIMtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

The cysteines of the membrane-bound mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity [Roossien, F. F., & Robillard, G. T. (1984) Biochemistry 23, 211-215]. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [14C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing.     

Bacterial phosphoenolpyruvate-dependent phosphotransferase system. Mechanism of the transmembrane sugar translocation and phosphorylation

The phosphoryl-group transfer from PHPr to glucose or alpha-methylglucose and from glucose 6-phosphate to these same sugars catalyzed by membrane-bound EIIBGlc of the bacterial phosphoenolpyruvate-dependent phosphotransferase system has been studied in vitro. Kinetic measurements revealed that both the phosphorylation reaction and the exchange reaction proceed according to a ping-pong mechanism in which a phosphorylated membrane-bound enzyme II acts as an obligatory intermediate. The occurrence of a phospho-IIBGlc/IIIGlc has been physically demonstrated by the production of a glucose 6-phosphate burst from membranes phosphorylated by phosphoenolpyruvate, HPr, and EI. The observation of similar second-order rate constants for the production of sugar phosphate starting with different phosphoryl-group donors confirms the catalytic relevance of the phosphoenzyme IIBGlc intermediate. The in vitro results, together with data published by other investigators, have led to a model describing sugar phosphorylation and transport in vivo.     

Dimeric enzyme IImtl of the E. coli phosphoenolpyruvate-dependent phosphotransferase system. Cross-linking studies with bifunctional sulfhydryl reagents

The occurrence of intermolecular dithiols on EIImtl has been studied with a number of thiol-specific cross-linking reagents. The reaction of EIImtl with bifunctional maleimide derivatives inactivates the enzyme. At the same time the enzyme is irreversibly cross-linked to a dimeric species. Under optimal conditions 50% of the protein is cross-linked upon reaction with the dimaleimides. The enzyme is also cross-linked under oxidizing conditions in the presence of CuCl2, presumably by oxidizing an intermolecular dithiol to a disulfide. This oxidation can be reversed by the addition of the reducing agent dithiothreitol. The reaction of phosphorylated EIImtl with the same sulfhydryl-specific bifunctional reagents does not lead to any cross-linked product. The results are discussed in terms of the association state of the purified protein and the distribution of its thiol groups.     

The pleiotropic function of the phosphoenolpyruvate-dependent phosphotransferase system in bacteria. Communication II

The structure and function of regulators and anti-terminators is under discussion in gram-positive bacteria. The regulators of lichen and levan operons (LiR and LevR) as well as the implementation of both gram-positive and negative regulations of operons by them are in the focus of attention. Po-independent termination is regarded by the example of the regulatory activity for the utilization systems of glucose (GlcT) beta-glucosides (LicT), sucrose (low-efficiency system SacY-SacX) and of glycerin (GlcP). Changes in the functional activity of the above systems, which are dependent on a condition of anti-terminators (phosphorylated or dephosphorylated forms and an ability to demirelize etc.) are regarded from the viewpoint of a possibility of occurrence of catabolic repression.     

Genetic and biochemical characterization of the phosphoenolpyruvate:glucose/mannose phosphotransferase system of Streptococcus thermophilus

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB(Man), two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB(Man), IIC(Man), IID(Man), and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB(Man) as well as membrane fragments containing IIC(Man) and IID(Man). These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB(Man).     

Isolation of IIIGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system of Salmonella typhimurium.

We report a procedure for the isolation of IIIglc of Salmonella typhimurium, a protein component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. IIIGlc is a soluble protein with a molecular weight of 21,000, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein is involved in the phosphoenolpyruvate-dependent phosphorylation of methyl alpha-glucoside in vitro. Its affinity for octyl-Sepharose may be an indication of the partial hydrophobic nature of IIIGlc. A specific antiserum against purified IIIGlc was prepared. Growth on different carbon sources did not affect the synthesis of IIIGlc, as determined by quantitative immunoelectrophoresis. Mutations which lower the adenosine 3',5'-phosphate level, such as cya and pts, do not alter the IIIGlc level. The closely related enteric bacteria Escherichia coli and Klebsiella aerogenes contain a protein factor which is closely related to IIIGlc of S. typhimurium, whereas Staphylococcus aureus does not.     

Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: glucose-negative mutant and regulation of intracellular cyclic AMP.

Glucose-negative mutants of Mycoplasma capricolum were selected for growth on fructose in the presence of the toxic glucose analog alpha-methyl-D-glucopyranoside. The mutants are defective in the phosphoenolpyruvate:sugar phosphotransferase system for glucose. One mutant, pts-4, was studied in detail. It lacks the glucose-specific, membrane-bound enzyme II, IIGlc, as well as the general, low-molecular-weight, phosphocarrier protein, HPr. In place of the latter, however, it has a fructose-specific protein, HPrFru. Consistent with these changes, the mutant lost the ability to grow on glucosamine and maltose but retained its ability to grow on sucrose. In the glucose-negative mutant, glucose did not regulate the intracellular concentration of cyclic AMP. The intracellular concentration of cyclic AMP in M. capricolum is regulated by the presence of metabolizable sugars. In the wild-type, both glucose and fructose reduced the intracellular concentration of cyclic AMP; however, in the glucose-negative mutant, glucose no longer regulated the intracellular level of cyclic AMP.     

Molecular analysis of the phosphoenolpyruvate-dependent l-sorbose: phosphotransferase system from Klebsiella pneumoniae and of its multidomain structure

We have cloned a 3.4 kb DNA fragment from the chromosome of Klebsiella pneumoniae that codes for a phosphoenolpyruvate-dependent l-sorbose: phosphotransferase system (PTS). The cloned fragment was sequenced and four open reading frames coding for 135 (sorF), 164 (sorB), 266 (sorA) and 274 (sorM) amino acids, respectively, were found. The corresponding proteins could be detected in a T7 overexpression system, which yielded molecular masses of about 14000 for SorF, 19000 for SorB, 25000 for SorA and 27000 for SorM. SorF and SorB have all the characteristics of soluble and intracellular proteins in accordance with their functions as EIIA^Sor and EIIB^Sor domains of the l-sorbose PTS. SorA and SorM, by contrast, are strongly hydrophobic, membrane-bound proteins with two to five putative transmembrane helices that alternate with a series of hydrophilic loops. They correspond to domains EIIC^Sor and EIID^Sor. The four proteins of the l-sorbose PTS resemble closely (27%–60%) the four subunits of a d-fructose PTS (EIIA^Lev, EIIB^Lev, EIIC^Lev, and EIID^Lev) from Bacillus subtilis and the three subunits of the d-mannose PTS (EIIA,B^Man, EIIC^Man, and EIID^Man) from Escherichia coli K-12. The three systems constitute a new PTS family, and sequence comparisons revealed highly conserved structures for the membranebound proteins. A consensus sequence for the membrane proteins was used to postulate a model for their integration into the membrane.     

Nucleotide sequence of the lipase gene lip2 from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues

The lip2 gene from the antarctic psychotroph Moraxella TA144 was sequenced. The primary structure of the Lip2 preprotein deduced from the nucleotide sequence is composed of 433 amino acids with a predicted Mr of 47,222. This enzyme contains a Ser-centered consensus sequence and a conserved His-Gly dipeptide found in most lipase amino-terminal domains. These sequences are involved in the lipase active site conformation since substitution of the conserved Ser or His residues by Ala and Gln, respectively, results in the loss of both lipase and esterase activities. Structural factors that would allow proper enzyme flexibility at low temperatures are discussed. It is suggested that only subtle changes in the primary structure of these psychrotrophic enzymes can account for their ability to catalyze lipolysis at temperatures close to 0 degrees C.