IIA Glc Research Articles

Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAGlc

In a variety of bacteria, the phosphotransferase protein IIA(Glc) plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H(+), using a mechanism in which sugar- and H(+)-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA(Glc) (inducer exclusion). Here we report the thermodynamic features of the IIA(Glc)-LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA(Glc) binds to LacY with a Kd of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA(Glc) binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA(Glc) blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA(Glc) binding. The precise mechanism of the inhibition of LacY by IIA(Glc) elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA(Glc).

Insights into the Inhibitory Mechanisms of the Regulatory Protein IIAGlc on Melibiose Permease Activity

The phosphotransfer protein IIA(Glc) of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIA(Glc) regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIA(Glc) binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIA(Glc) directly binds to MelB of Salmonella typhimurium (MelB(St)) and Escherichia coli MelB (MelB(Ec)) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3-10 μM for MelB(St) and 25 μM for MelB(Ec). All of the binding is solely driven by favorable enthalpy forces. IIA(Glc) binding to MelB(St) in the absence or presence of melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIA(Glc)-bound MelB(St) exhibits a decreased binding affinity for melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease is involved in an induced fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIA(Glc) inhibits the induced fit process and restricts the conformational dynamics of MelB(St).

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)

Oligomeric interactions provide alternatives to direct steric modes of control of sugar kinase/actin/hsp70 superfamily functions by heterotropic allosteric effectors: Inhibition of E. coli glycerol kinase

Unlike those for monomeric superfamily members, heterotropic allosteric effectors of the tetrameric Escherichia coli glycerol kinase (EGK) bind to only one of the two domains that define the catalytic cleft and far from the active site. An R369A amino acid substitution removes oligomeric interactions of a novel mini domain-swap loop of one subunit with the catalytic site of another subunit, and an A65T substitution perturbs oligomeric interactions in a second interface. Linked-functions enzyme kinetics, analytical ultracentrifugation, and FRET are used to assess effects of these substitutions on the allosteric control of catalysis. Inhibition by phosphotransferase system protein IIA Glc is reduced by the R369A substitution, and inhibition by fructose 1,6-bisphosphate is abolished by the A65T substitution. The oligomeric interactions enable the heterotropic allosteric effectors to act on both domains and modulate the catalytic cleft closure despite binding to only one domain.

Amino acid substitutions in the sugar kinase/hsp70/actin superfamily conserved ATPase core of E. coli glycerol kinase modulate allosteric ligand affinity but do not alter allosteric coupling

IIA Glc, the glucose-specific phosphocarrier protein of the phospho enolpyruvate:glycose phosphotransferase system, is an allosteric inhibitor of Escherichia coli glycerol kinase. A linked-functions initial-velocity enzyme kinetics approach is used to define the MgATP–IIA Glc heterotropic allosteric interaction. The interaction is measured by the allosteric coupling constants Q and W, which describe the mutual effect of the ligands on binding affinity and the effect of the allosteric ligand on V max, respectively. Allosteric interactions between these ligands display K-type activation and V-type inhibition. The allosteric coupling constant Q is about 3, showing cooperative coupling such that each ligand increases the affinity for binding of the other. The allosteric coupling constant W is about 0.1, showing that the allosteric inhibition is partial such that binding of IIA Glc at saturation does not reduce V max to zero. E. coli glycerol kinase is a member of the sugar kinase/heat shock protein 70/actin superfamily, and an element of the superfamily conserved ATPase catalytic core was identified as part of the IIA Glc inhibition network because it is required to transplant IIA Glc allosteric control into a non-allosteric glycerol kinase [A.C. Pawlyk, D.W. Pettigrew, Proc. Natl. Acad. Sci. USA 99 (2002) 11115–11120]. Two of the amino acids at this locus of E. coli glycerol kinase are replaced with those from the non-allosteric enzyme to enable determination of its contributions to MgATP–IIA Glc allosteric coupling. The substitutions reduce the affinity for IIA Glc by about 5-fold without changing significantly the allosteric coupling constants Q and W. The insensitivity of the allosteric coupling constants to the substitutions may indicate that the allosteric network is robust or the locus is not an element of that network. These possibilities may arise from differences of E. coli glycerol kinase relative to other superfamily members with respect to oligomeric structure and location of the allosteric site in a single domain far from the catalytic site.

A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIA(Glc) (IIA(Glc))) mediates some of these regulatory phenomena. In this report, we characterize a novel IIA(Glc)-binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as "function unknown." FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIA(Glc), with the highest affinity of any protein thus far shown to interact with IIA(Glc). Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the gamma-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIA(Glc) regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA.

Effects of detergent alkyl chain length and chemical structure on the properties of a micelle-bound bacterial membrane targeting peptide

The effects of phospholipid or detergent chain length on the structure and translational diffusion coefficient of the membrane-targeting peptide corresponding to the N-terminal amphipathic sequence of Escherichia coli enzyme IIA Glc were investigated by nuclear magnetic resonance (NMR) spectroscopy. Three anionic phospholipids (dihexanoyl phosphatidylglycerol, dioctanoyl phosphatidylglycerol, and didecanoyl phosphatidylglycerol) and four lipid-mimicking anionic detergents (sodium hexanesulfonate, 2,2-dimethyl-silapentane-5-sulfonate, sodium nonanesulfonate, and sodium dodecylsulfate) were evaluated. In all cases, the cationic peptide adopts an amphipathic helical structure. While the chain length of the two-chain phospholipids has a negligible effect on the peptide conformation, the effect of chain length of those single-chain detergents on the helix length is more pronounced. The diffusion coefficients of the peptide/micelle complexes were found to correlate with the chain lengths of both the lipid and the detergent groups. Taken together, short-chain anionic phospholipids are proposed to be useful membrane-mimetic models for the structural elucidation of membrane-binding peptides such as cationic antimicrobial peptides. DSS does not form micelles by itself according to the diffusion coefficient data, but it does associate with this cationic peptide. Consequently, both DSS and its analog may be chosen as NMR chemical shift reference compounds depending on the nature of the biomolecules under investigation.

Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand.

Enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:glucose phosphotransferase system plays a direct role in regulating inducible transport systems. Dephosphorylated IIA(Glc) binds directly to lactose permease in a reaction that requires binding of a galactosidic substrate. A double-Cys mutation (Ile129 --> Cys/Lys131 --> Cys) was introduced into helix IV of the permease near the IIA(Glc) binding site in cytoplasmic loop IV/V and in the vicinity of the galactoside binding site at the interface of helices IV, V, and VIII. The mutant no longer requires galactoside for IIA(Glc) binding as demonstrated by both a [(125)I]IIA(Glc) binding assay and a newly developed fluorescence anisotropy assay. Further characterization of the mutant shows that it binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. The data are consistent with the interpretation that the double mutant is locked in an inward-facing conformation.

Why the Phosphotransferase System of Escherichia coli Escapes Diffusion Limitation

We calculated the implications of diffusion for the phosphoenolpyruvate:glucose phosphotransferase system (glucose-PTS) of Escherichia coli in silicon cells of various magnitudes. For a cell of bacterial size, diffusion limitation of glucose influx was negligible. Nevertheless, a significant concentration gradient for one of the enzyme species, nonphosphorylated IIA Glc, was found. This should have consequences because the phosphorylation state of IIA Glc is an important intracellular signal. For mammalian cell sizes we found significant diffusion limitation, as well as strong concentration gradients in many PTS components, and strong effects on glucose and energy signaling. We calculated that the PTS may sense both extracellular glucose and the intracellular free-energy state. We discuss i), that the effects of diffusion on cell function should prevent this highly effective bacterial system from functioning in eukaryotic cells, ii), that in the larger eukaryotic cell any similar chain of mobile group-transfer proteins can neither sustain the same volumetric flux as in bacteria nor transmit a signal far into the cell, and iii), that systems such as these may exhibit spatial differentiation in their sensitivity to different signals.

Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK 2) of Salmonella typhimurium

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK 2) and a periplasmic substrate binding protein. To further elucidate protein–protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK 2 complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V max of 1.25 μmol P i/min/mg and a K m of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA Glc, a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.

The regulation of Enzyme IIA(Glc) expression controls adenylate cyclase activity in Escherichia coli.

During the last few years, several genes, such as pap, bgl and flhDC, have been shown to be coregulated by the histone-like nucleoid-structuring (H-NS) protein and the cyclic AMP-catabolite activator protein (cAMP/CAP) complex, suggesting an interaction between both systems in the control of some cellular functions. In this study, the possible effect of H-NS on the cAMP level was investigated. In a CAP-deficient strain, the presence of an hns mutation results in a strong reduction in the amount of cAMP, due to a decrease in adenylate cyclase activity. This is caused by the reduced expression of crr, which encodes the Enzyme IIA(Glc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), from its specific P2 promoter. This leads to a twofold reduction in the global amount of Enzyme IIA(Glc), the adenylate cyclase activator, responsible for the decrease in adenylate cyclase activity observed in the hns crp strain.

Intein-mediated cyclization of a soluble and a membrane protein in vivo: function and stability

Cyclized subunits of the E. coli glucose transporter were produced in vivo by intein mediated trans-splicing. IIA Glc is a β-sandwich protein, IICB Glc spans the membrane eight times. Genes encoding the circularly permuted precursors U CΔ-IIA Glc-U NΔ and U CΔ-IICB Glc-U NΔ were assembled from DNA fragments encoding the 3′ and 5′ segments of the recA intein of M. tuberculosis and crr and ptsG of E. coli, respectively. A 20-residues long, Ala-Pro rich linker peptide and/or a histidine tag were used to join the native N- and C-termini in the cyclized proteins. The cyclized proteins complemented growth of glucose auxotrophic strains. Purified, cyclized IIA Glc and IICB Glc had 100 and 25%, respectively, of wild-type glucose phosphotransferase activity. They had an increased electrophoretic mobility, which decreased upon linearization of the proteins with chymotrypsin. Cyclized IIA Glc displayed increased stability against temperature and GuHCl-induced unfolding (75 vs. 70 °C; 1.52 vs. 1.05 M).

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB Glc subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB Glc are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB Glc and IIA Glc were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA Glc and IICB Glc had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA Glc displayed increased stability against temperature and GuHCl-induced unfolding.

Transport of glucose by a phosphoenolpyruvate:mannose phosphotransferase system in Pasteurella multocida

Pasteurella multicida was examined for glucose and mannose transport. P. multocida was shown to possess a phosphoenolpyruvate (PEP):mannose phosphotransferase system (PTS) that transports glucose as well as mannose and was functionally similar to the Escherichia coli mannose PTS. Phosphorylated proteins with molecular masses similar to those of E. coli mannose PTS proteins were visualized when incubated with 32P-PEP. The presence of an enzyme IIA Glc which could play an important role in regulation, as described in other Gram-negative bacteria, was detected. The enzymes of the pentose-phosphate pathway were present in P. multocida grown on glucose. The activity of 6-phosphofructokinase (the key enzyme of the Embden-Meyerhof pathway (EMP)), was very low in cell extracts, suggesting that EMP is not the major pathway for glucose catabolism.

Characterization of the presumptive phosphorylation sites of the Bacillus subtilis glucose permease by site-directed mutagenesis: implication in glucose transport and catabolite repression

Bacillus subtilis utilizes glucose as the preferred source of carbon and energy. Glucose is transported and concomitantly phosphorylated by the glucose permease (PtsG) of the phosphoenolpyruvate:sugar phosphotransferase system. The phosphate is transferred from enzyme I via HPr and domains IIA and IIB of the glucose permease to the sugar. In this study mutants affected in the putative phosphorylation sites of glucose permease were constructed and the effect on sugar transport and glucose repression tested. Phosphorylation of both domains IIA Glc and IIB Glc is required for efficient glucose transport and repression of β-xylosidase and the bglPH operon.

Functional interactions between proteins of the phosphoenolpyruvate:sugar phosphotransferase systems of Bacillus subtilis and Escherichia coli.

Proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus subtilis were overexpressed, purified to near homogeneity, and characterized. The proteins isolated include Enzyme I, HPr, the glucose-specific IIA domain of the glucose-specific Enzyme II (IIAglc), and the mannitol-specific IIA protein, IIAmtl. Site specific mutant proteins of IIAglc and HPr were also overexpressed and purified, and their properties were compared with those of the wild type proteins. These proteins and their phosphorylated derivatives were characterized with respect to their immunological cross-reactivities employing the Western blot technique and in terms of their migratory behavior during sodium dodecyl sulfate-gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. The interactions between homologous and heterologous Enzymes I and HPrs, between homologous and heterologous HPrs and the IIAglc proteins, and between homologous and heterologous IIAglc proteins and IIBCscr of B. subtilis as well as IICBglc of Escherichia coli were defined and compared kinetically. The mutant HPrs and IIAglc proteins were also characterized kinetically as PTS phosphocarrier proteins and/or as inhibitors of the phosphotransferase reactions of the PTS. These studies revealed that complexation of IIAglc with the mutant form of HPr in which serine 46 was replaced by aspartate (S46D) did not increase the rate of phosphoryl transfer from phospho Enzyme I to S46D HPr more than when IIAmtl was complexed to S46D HPr. These findings do not support a role for HPr(Ser-P) in the preferential utilization of one PTS carbohydrate relative to another. Functional analyses in E. coli established that IIAglc of B. subtilis can replace IIAglc of E. coli with respect both to sugar transport and to regulation of non-PTS permeases, catabolic enzymes, and adenylate cyclase. Site-specific mutations in histidyl residues 68 and 83 (H68A and H83A) inactivated IIAglc of B. subtilis with respect to phosphoryl transfer and its various regulatory roles.

Properties of phosphorylated protein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The phosphohydrolysis properties of the following phosphoprotein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) were investigated: enzyme I, HPr, and the IIAGlc domain of the glucose enzyme II of Bacillus subtilis; and IIAGlc (fast and slow forms) of Escherichia coli. The phosphohydrolysis properties were also studied for the site-directed mutant H68A of B. subtilis IIA Glc. Several conclusions were reached. (i) The phosphohydrolysis properties of the homologous phosphoprotein intermediates of B. subtilis and E. coli are similar. (ii) These properties deviate from those of isolated N delta 1- and N epsilon 2-phosphohistidine indicating the participation of neighbouring residues at the active sites of these proteins. (iii) The rates of phosphohydrolysis of the H68A mutant of B. subtilis IIAGlc were reduced compared with the wild-type protein, suggesting that both His-83 and His-68 are present at the active site of wild-type IIAGlc. (iv) The removal of seven N-terminal residues of E. coli IIAGlc reduced the rates of phosphohydrolysis between pH 5 and 8.