Enzyme I: the gateway to the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

Regulatory aspects of the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) are reviewed. The structure and conformational stability of the first protein (enzyme I) of the PTS, as well as the requirement for enzyme I to dimerize for autophosphorylation by PEP in the presence of MgCl2 are discussed.     

Crystal structure of the phosphoenolpyruvate-binding enzyme I-domain from the Thermoanaerobacter tengcongensis PEP: sugar phosphotransferase system (PTS)

Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal protein-binding domain (EIN) and a C-terminal PEP-binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here, we report the 1.82A crystal structure of the homodimeric EIC domain from Thermoanaerobacter tengcongensis, a saccharolytic eubacterium that grows optimally at 75 degrees C. EIC folds into a (betaalpha)(8) barrel with three large helical insertions between beta2/alpha2, beta3/alpha3 and beta6/alpha6. The large amphipathic dimer interface buries 3750A(2) of accessible surface area per monomer. A comparison with pyruvate phosphate dikinase (PPDK) reveals that the active-site residues in the empty PEP-binding site of EIC and in the liganded PEP-binding site of PPDK have almost identical conformations, pointing to a rigid structure of the active site. In silico models of EIC in complex with the Z and E-isomers of chloro-PEP provide a rational explanation for their difference as substrates and inhibitors of EI. The EIC domain exhibits 54% amino acid sequence identity with Escherichia coli and 60% with Bacillus subtilis EIC, has the same amino acid composition but contains additional salt-bridges and a more complex salt-bridge network than the homology model of E.coli EIC. The easy crystallization of EIC suggests that T.tengcongensis can serve as source for stable homologs of mesophilic proteins that are too labile for crystallization.     

Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189: implications for phosphorylation effects

The amino terminal domain of enzyme I (residues 1-258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, deltaH approximately 140 +/- 10 (160) kcal mol(-1) and deltaCp approximately 2.7 (3.3) kcal K(-1) mol(-1). Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) degrees C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO3(2-) due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm approximately 66-68 degrees C). This possibly arises from an ion pair interaction between the gamma-carboxyl of Glu189 and the epsilon-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K(A)' = 3 +/- 1 x 10(5) M(-1) for EIN and approximately 1.2 x 10(5) M(-1) for EI.     

Structural characterization of the PTS IIA and IIB proteins associated with pneumococcal fucose utilization

Streptococcus pneumoniae harbors a significant number of transporters, including phosphotransferase (PTS) systems, allowing the bacterium to utilize a number of different carbohydrates for metabolic and other purposes. The genes encoding for one PTS transport system in particular (EII^fuc) are found within a fucose utilization operon in S. pneumoniae TIGR4. Here, we report the three‐dimensional structures of IIA^fuc and IIB^fuc providing evidence that this PTS system belongs to the EII^man family. Additionally, the predicted metabolic pathway for this distinctive fucose utilization system suggests that EII^fuc transports the H‐disaccharide blood group antigen, which would represent a novel PTS transporter specificity. Proteins 2017; 85:963–968. © 2016 Wiley Periodicals, Inc.     

Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli

The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(2+)) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 degrees C, 5 mM pyruvate (with 2 mM Mg(2+)) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(2+) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to < 5 x 10(5) or 3 x 10(7) M(-1), respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold. With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-1). The binding of PEP to EI(H189A) is synergistic with that of Mg(2+). Thus, physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-enzyme I.     

Cloning, sequencing and expression in Escherichia coli of the ptsI gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system from Streptococcus salivarius.

We present the cloning and sequencing of the ptsI gene, encoding enzyme I (EI) of the phosphoenolpyruvate (PEP): sugar phosphotransferase (PTS) transport system from Streptococcus salivarius. The ptsI gene corresponds to an open reading frame of 1731 nucleotides, which translates into a putative 577-amino acid (aa) protein with a M(r) of 62,948 and a pI of 4.49. The EI was produced in Escherichia coli under the control of its own promoter located immediately upstream of ptsI, a situation never previously reported for any other gene coding for an EI. The deduced aa sequence of the S. salivarius EI shows a high degree of similarity with the E. coli EI and the EI moiety of the multiphosphoryl transfer protein from Rhodobacter capsulatus. The S. salivarius EI also shares a highly conserved aa cluster with a non-PTS protein, the maize pyruvate:orthophosphate dikinase. The conserved cluster is located in a domain which is hypothesized to be the PEP-binding site.     

Defining the epitope region of a peptide from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system able to bind to the enzyme I

The bacterial PEP:sugar PTS consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the HPr by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr^9-30, spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His-15) and the first α-helix of HPr of Streptomyces coelicolor, HPr^sc. By using fluorescence and circular dichroism, we first determined qualitatively that HPr^sc and HPr^9-30 did bind to EI^sc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr^9-30 and HPr^sc for EI^sc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr^9-30 was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EI^sc and HPr^sc is enthalpy driven and in other species is entropy driven; further, the affinity of HPr^sc for EI^sc was smaller than in other species. However, the affinity of HPr^9-30 for EI^sc was only moderately lower than that of EI^sc for HPr^sc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS.     

Purification and characterization of the fructose-inducible HPr-like protein, FPr, and the fructose-specific enzyme III of the phosphoenolpyruvate: sugar phosphotransferase system of Salmonella typhimurium

The proteins comprising the fructose-specific phosphoenolpyruvate:sugar phosphotransferase system were investigated using a strain of Salmonella typhimurium which lacks the general phosphotransferase system proteins, HPr and Enzyme I, synthesizes the fructose phosphotransferase system proteins, FPr, Enzyme IIfru, Enzyme IIIfru, and fructose-1-phosphate kinase, constitutively, and expresses the Enzyme I-like protein Enzyme I. Enzyme I activity was found in the cytoplasmic fraction, Enzyme IIfru in the membrane fraction, and FPr and Enzyme IIIfru activities were distributed between the two fractions. Extraction of membranes with butanol and urea led to quantitative release of the membrane-associated Enzyme IIIfru and FPr activities, while Enzyme IIfru remained with the membranes. FPr was purified to homogeneity using ion exchange chromatography, gel filtration, and reversed phase high pressure liquid chromatography (HPLC), and its amino acid composition and N-terminal sequence were determined. A complex of FPr and Enzyme IIIfru (Mr 50,000) was also purified to near homogeneity using ion exchange chromatography, gel filtration, and chromatography on hydroxylapatite. When the purified complex was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was visualized as two protein bands with mobilities corresponding to molecular weights of about 40,000 (Enzyme IIIfru) and 9,000 (FPr). Neither the FPr and Enzyme IIIfru activities nor the proteins represented by these two bands separated during the above chromatography steps or using any of several other techniques, including reversed phase HPLC, indicating a very tight association. Active Enzyme IIIfru free of FPr was never isolated or observed. The proteins could be separated in denatured form by gel filtration in the presence of guanidine HCl or urea. Free FPr and the FPr-Enzyme IIIfru complex were characterized, and the properties of free and complexed FPr were compared to those of HPr.     

Tautomeric state and pKa of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy. The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Ndelta1-H tautomer, and its Nepsilon2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.     

Phosphoproteins and the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIImannose, IIIfructose, IIIglucitol, and the phosphorylation of enzyme IImannitol and enzyme IIN-acetylglucosamine

Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [32P]phosphoenolpyruvate or [gamma 32P]ATP have been resolved and detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Simple techniques were found such that distinctions could be made between phosphoproteins containing acid-labile or stable phosphoamino acids and between N1-P-histidine and N3-P-histidine. Phosphoproteins were found to be primarily formed from phosphoenolpyruvate, but because of an efficient phosphoexchange, ATP also led to the formation of the major phosphoenolpyruvate-dependent phosphoproteins. These proteins had the following apparent subunit molecular weights: 65,000, 65,000, 62,000, 48,000, 40,000, 33,000, 25,000, 20,000, 14,000, 13,000, 9,000, 8,000. Major ATP-dependent phosphoproteins were detected with apparent subunit molecular weights of 75,000, 46,000, 30,000, and 15,000. Other minor phosphoproteins were detected. The phosphorylation of the 48,000- and 25,000-MW proteins by phosphoenolpyruvate was independent of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme IIN-acetylglucosamine (membrane bound; MW = 65,000); enzyme IImannitol (membrane bound; MW = 62,000); IIIfructose (soluble; MW = 40,000); IIImannose (partially membrane associated; MW = 33,000); IIIglucose (soluble; MW = 20,000); IIIglucitol (soluble; MW = 13-14,000); HPr (soluble; MW = 9,000); FPr (fructose induced HPr-like protein (soluble; MW = 8,000). HPr and FPr are phosphorylated on the N-1 position of a histidyl residue while all the others appear to be phosphorylated on an N-3 position of a histidyl residue. These studies identify some previously unknown proteins of the PTS and show the phosphorylation of others, which although previously known, had not been shown to be phosphoproteins.     

A mammalian insulysin homolog is regulated by enzyme IIA(Glc) of the glucose transport system in Vibrio vulnificus

Vibrio vulnificus is an opportunistic human pathogen that causes severe infections in susceptible individuals. While the components of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) have been shown to regulate numerous targets, little such information is available for the V. vulnificus PTS. Here we show that enzyme IIA^Glc of the PTS regulates the peptidase activity of a mammalian insulysin homolog in V. vulnificus. While interaction of IIA^Glc with the insulysin homolog is independent of the phosphorylation state of IIA^Glc, only unphosphorylated IIA^Glc activates the insulysin homolog. Taken together, our results suggest that the V. vulnificus insulysin-IIA^Glc complex plays a role in survival in the host by sensing glucose.

Structured summary

MINT-8045996: IIA glu (uniprotkb:Q7MBY2) binds (MI:0407) to vIDE (uniprotkb:Q7MIS6) by pull down (MI:0096)MINT-8045817, MINT-8045967: IIA glu (uniprotkb:Q7MBY2) physically interacts (MI:0915) with vIDE (uniprotkb:Q7MIS6) by pull down (MI:0096)