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1.
InEnterobacteriaceae the nonphosphorylated form of IIAG1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIAGlc-Iike domain of the membrane-bound IIN-acetylglucosamine (IINag) of the PTS can replace IIAGlc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIAGlc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIAGlc-like domain, could not replace IIAGlc. Neither IINag nor LacS could replace IIAGlc in activation of adenylate cyclase.  相似文献   

2.
The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIALacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIAGlc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIALacS would be able to perform one or more functions associated with IIAGlc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIALacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIALacS was unable to restore glucose uptake in a IIAGlc-deficient strain, which is consistent with a very low rate of phosphorylation of IIALacS by phosphorylated HPr (HPr~P) from E. coli. With HPr~P from S. thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIALacS (k1 = 4.3 × 102 M−1 s−1) and dephosphorylation of IIALacS~P by HPr (k−1 = 1.1 × 103 M−1 s−1) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIAGlc and HPr from E. coli. This finding suggests that IIALacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIAGlc, we constructed a double mutant [IIALacS(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIALacS(I548E/G556D) by HPr~P increased (k1 = 4.0 × 103 M−1 s−1) and became nearly independent of the source of HPr~P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIALacS(I548E/G556D) was insufficient to complement IIAGlc in PTS-mediated glucose transport in E. coli. Both IIALacS and IIALacS(I548E/G556D) could replace IIAGlc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.  相似文献   

3.
In Streptococcus thermophilus, lactose is taken up by LacS, a transporter that comprises a membrane translocator domain and a hydrophilic regulatory domain homologous to the IIA proteins and protein domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The IIA domain of LacS (IIALacS) possesses a histidine residue that can be phosphorylated by HPr(His~P), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(His~P), HPr(Ser-P), and HPr(Ser-P)(His~P), in exponentially lactose-growing cells revealed that the doubly phosphorylated form of HPr represented 75% and 25% of the total HPr in S. thermophilus ATCC 19258 and S. thermophilus SMQ-301, respectively. Experiments conducted with [32P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIALacS) showed that IIALacS was reversibly phosphorylated by HPr(Ser-P)(His~P) at a rate similar to that measured with HPr(His~P). Sequence analysis of the IIALacS protein domains from several S. thermophilus strains indicated that they can be divided into two groups on the basis of their amino acid sequences. The amino acid sequence of IIALacS from group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIALacS from group II could also be phosphorylated by HPr(His~P) and HPr(Ser-P)(His~P), in vitro phosphorylation experiments were conducted with purified proteins from Streptococcus salivarius ATCC 25975, which possesses a IIALacS very similar to group II S. thermophilus IIALacS. The results indicated that S. salivarius IIALacS was phosphorylated by HPr(Ser-P)(His~P) at a higher rate than that observed with HPr(His~P). Our results suggest that the reversible phosphorylation of IIALacS in S. thermophilus is accomplished by HPr(Ser-P)(His~P) as well as by HPr(His~P).  相似文献   

4.
IIAGlc, the glucose-specific phosphocarrier protein of the phosphoenolpyruvate: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-IIAGlc 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 Vmax, 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 IIAGlc at saturation does not reduce Vmax 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 IIAGlc inhibition network because it is required to transplant IIAGlc 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-IIAGlc allosteric coupling. The substitutions reduce the affinity for IIAGlc 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.  相似文献   

5.
Kim YJ  Ryu Y  Koo BM  Lee NY  Chun SJ  Park SJ  Lee KH  Seok YJ 《FEBS letters》2010,584(22):4537-4544
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 IIAGlc of the PTS regulates the peptidase activity of a mammalian insulysin homolog in V. vulnificus. While interaction of IIAGlc with the insulysin homolog is independent of the phosphorylation state of IIAGlc, only unphosphorylated IIAGlc activates the insulysin homolog. Taken together, our results suggest that the V. vulnificus insulysin-IIAGlc 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)  相似文献   

6.
7.
The phosphotransfer protein IIAGlc 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 IIAGlc regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIAGlc binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIAGlc directly binds to MelB of Salmonella typhimurium (MelBSt) and Escherichia coli MelB (MelBEc) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3–10 μm for MelBSt and 25 μm for MelBEc. All of the binding is solely driven by favorable enthalpy forces. IIAGlc binding to MelBSt 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 IIAGlc-bound MelBSt 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 IIAGlc inhibits the induced fit process and restricts the conformational dynamics of MelBSt.  相似文献   

8.
Glucose is a universal energy source and a potent inducer of surface colonization for many microbial species. Highly efficient sugar assimilation pathways ensure successful competition for this preferred carbon source. One such pathway is the phosphoenolpyruvate phosphotransferase system (PTS), a multicomponent sugar transport system that phosphorylates the sugar as it enters the cell. Components required for transport of glucose through the PTS include enzyme I, histidine protein, enzyme IIAGlc, and enzyme IIBCGlc. In Escherichia coli, components of the PTS fulfill many regulatory roles, including regulation of nutrient scavenging and catabolism, chemotaxis, glycogen utilization, catabolite repression, and inducer exclusion. We previously observed that genes encoding the components of the Vibrio cholerae PTS were coregulated with the vps genes, which are required for synthesis of the biofilm matrix exopolysaccharide. In this work, we identify the PTS components required for transport of glucose and investigate the role of each of these components in regulation of biofilm formation. Our results establish a novel role for the phosphorylated form of enzyme I in specific regulation of biofilm-associated growth. As the PTS is highly conserved among bacteria, the enzyme I regulatory pathway may be relevant to a number of biofilm-based infections.  相似文献   

9.
Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat-and batch cultures were compared. Strain PP 799 transported and phosphorylated glucose via an intact PTS, while strain PP 952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP 799 and PP 952 exhibited similar Y Glc values. Assuming equal Y ATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc.Abbreviations PTS phosphoenolpyruvate: carbohydrate phosphotransferase system - HPr histidine-containing phosphocarrier protein - GalP galactose permease  相似文献   

10.
It is demonstrated here that in Escherichia coli, the phosphorylated form of the glucose-specific phosphocarrier protein IIAGlc of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIAGlc has no effect on the basal activity of adenylyl cyclase. To elucidate the specific role of IIAGlc phosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIAGlc were mutated by site-directed mutagenesis to glutamine and glutamate. Wild-type IIAGlc and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr~P) and were equally potent activators of adenylyl cyclase. Neither the H90Q nor the H90E mutant of IIAGlc was phosphorylated by HPr~P, and both failed to activate adenylyl cyclase. Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr~P, yet the H75E mutant of IIAGlc was a partial activator of adenylyl cyclase. The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase. This suggests that the H75E mutant was transiently phosphorylated by HPr~P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket. These results are discussed in the context of the proximity of H75 and H90 in the IIAGlc structure and the disposition of the negative charge in the modeled glutamate mutants.  相似文献   

11.
Our research group is studying the phosphotransferase system (PTS) of Streptomyces coelicolor, which, in other bacteria, is centrally involved in carbon source uptake and regulation. We have surveyed the public available S. coelicolor genome sequence produced by the ongoing genome sequencing project for pts gene homologues (http://www.sanger.ac.uk/Projects/S_coelicolor/). Three genes encoding homologues of the general PTS components enzyme I (ptsI), HPr (ptsH), and enzyme IIACrr (crr; IIAGlc-homologue) and six genes encoding homologues of sugar-specific PTS components were identified. The deduced primary sequences of the sugar-specific components shared significant similarities to PTS permeases of the mannitol/fructose family and of the glucose/sucrose family. A model is presented, in which possible functions of the novel described PTS homologues are discussed.  相似文献   

12.
13.
An isogenic pair of Escherichia coli strains lacking (pssA) and possessing (wild-type) the enzyme phosphatidylserine synthase was used to estimate the effects of the total lack of phosphatidylethanolamine (PE), the major phospholipid in E. coli membranes, on the activities of several sugar permeases (enzymes II) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The mutant exhibits greatly elevated levels of phosphatidylglycerol (PG), a lipid that has been reported to stimulate the in vitro activities of several PTS permeases. The activities, thermal stabilities, and detergent sensitivities of three PTS permeases, the glucose enzyme II (IIGlc), the mannose enzyme II (IIMan) and the mannitol enzyme II (IIMtl), were characterized. Western blot analyses revealed that the protein levels of IIGlc were not appreciably altered by the loss of PE. In the pssA mutant, IIGlc and IIMan activities were depressed both in vivo and in vitro, with the in vivo transport activities being depressed much more than the in vitro phosphorylation activities. IIMtl also exhibited depressed transport activity in vivo but showed normal phosphorylation activities in vitro. IIMan and IIGlc exhibited greater thermal lability in the pssA mutant membranes than in the wild-type membranes, but IIMtl showed enhanced thermal stability. All three enzymes were activated by exposure to TritonX100 (0.4%) or deoxycholate (0.2%) and inhibited by SDS (0.1%), but IIMtl was the least affected. IIMan and, to a lesser degree, IIGlc were more sensitive to detergent treatments in the pssA mutant membranes than in the wild-type membranes while IIMtl showed no differential effect. The results suggest that all three PTS permeases exhibit strong phospholipid dependencies for transport activity in vivo but much weaker and differential dependencies for phosphorylation activities in vitro, with IIMan exhibiting the greatest and IIMtl the least dependency. The effects of lipid composition on thermal sensitivities and detergent activation responses paralleled the effects on in vitro phosphorylation activities. These results together with those previously published suggest that, while the in vivo transport activities of all PTS enzymes II require an appropriate anionic to zwitterionic phospholipid balance, the in vitro phosphorylation activities of these same enzymes show much weaker and differential dependencies. Alteration of the phospholipid composition of the membrane thus allows functional dissection of transport from the phosphorylation activities of PTS enzyme complexes.  相似文献   

14.
Phosphoenolpyruvate (PEP)-dependent phosphorylation experiments have indicated that the grampositive bacteriumStaphylococcus carnosus possesses an EIICBA fusion protein specific for glucose. Here we report the cloning of a 7 kb genomic DNA fragment containing two genes,glcA andglcB, coding for the glucose-specific PTS transporters EIIGlc1 and EIIGlc2 which are 69% identical. The translation products derived from the nucleotide sequence consist of 675 and 692 amino acid residues and have calculated molecular weights of 73 025 and 75 256, respectively. Both genes can be stably maintained inEscherichia coli cells and restore the ability to ferment glucose toptsG deletion mutants ofE. coli. This demonstrates the ability of the PTS proteins HPr and/or EIIAGlc of a gram-negative organism (E. coli) to phosphorylate an EIICBAGlc from a gram-positive organism (S. carnosus).  相似文献   

15.
16.
17.
The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK2) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK2 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 Vmax of 1.25 μmol Pi/min/mg and a Km of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIAGlc, 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.  相似文献   

18.
19.
Enzyme IIA and HPr are central regulatory proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase (PTS) system. Three-dimensional structures of the glucose enzyme IIA domain (IIAglc) and HPr of Bacillus subtilis and Escherichia coli have been studied by both X-ray crystallography and Nuclear Magnetic Resonance (NMR) Spectroscopy. Phosphorylation of HPr of B. subtilis and IIAglc of E. coli have also been characterized by NMR spectroscopy. In addition, the binding interfaces of B. subtilis HPr and IIAglc have been identified from backbone chemical shift changes. This paper reviews these recent advances in the understanding of the three-dimensional structures of HPr and IIAglc and their interaction with each other. © 1993 Wiley-Liss, Inc.  相似文献   

20.
The utilization of maltose by Clostridium acetobutylicum ATCC 824 was investigated. Glucose was used preferentially to maltose, when both substrates were present in the medium. Maltose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on maltose, but not glucose or sucrose, as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that the specificity for maltose is contained entirely within the membrane in this organism. A putative gene system for the maltose PTS was identified (from the C. acetobutylicum ATCC 824 genome sequence), encoding an enzyme IIMal and a maltose 6-phosphate hydrolase. Journal of Industrial Microbiology & Biotechnology (2001) 27, 298–306. Received 12 September 2000/ Accepted in revised form 30 November 2000  相似文献   

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