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PubMed2005

Evolution of the bacterial phosphotransferase system: from carriers and enzymes to group translocators.

The bacterial phosphotransferase system (PTS) is a structurally and functionally complex system with a surprising evolutionary history. The substrate-recognizing protein constituents of the PTS (Enzymes II) derive from at least four independent sources. Some of the non-PTS precursor constituents have been identified, and evolutionary pathways taken have been proposed. Our analyses suggest that two of these independently evolving systems are still in transition, not yet having acquired the full-fledged characteristics of PTS Enzyme II complexes. The work described provides detailed insight into the process of catalytic protein evolution.

Bacteria↗

PubMed2002

The glucose-specific carrier of the Escherichia coli phosphotransferase system.

Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate-sugar phosphotransferase system (PTS). 2',3'-Epoxypropyl beta-d-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates. They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc-specific but structurally different transporter. 3a is the only analogue that inhibits EIIGlc by binding to the high-affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro, methyl 6,7-anhydro-d,l-glycero-alpha-d-gluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl alpha-d-glucopyranoside (1a) and methyl alpha-d-mannopyranoside (1c), methyl 6-deoxy-6-isothiocyanato-alpha-d-glucopyranoside (1e), beta-d-glucopyranosyl isothiocyanate (3c) and beta-d-glucopyranosyl phenyl isothiocyanate (3d). Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421. Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose-induced conformational changes in the dimeric transporter. Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells. This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.

Binding Sites↗

PubMed2000

Inorganic polyphosphate kinase and adenylate kinase participate in the polyphosphate:AMP phosphotransferase activity of Escherichia coli.

Polyphosphate kinase (PPK), responsible for the processive synthesis of inorganic polyphosphate (polyP) from ATP in Escherichia coli, can transfer in reverse the terminal phosphate residue of polyP to ADP to yield ATP. PolyP also serves as a donor in a polyP:AMP phosphotransferase (PAP) activity observed in extracts of Acinetobacter johnsonii and Myxococcus xanthus. We have found that overexpression of the gene encoding PPK results in a large enhancement of PAP activity in E. coli. The PAP activity requires both PPK and adenylate kinase in equimolar amounts. PPK and adenylate kinase form a complex in the presence of polyphosphate. We discuss a phosphotransfer mechanism that involves both enzymes and enables polyP to be a phospho-donor to AMP.

Adenylate Kinase↗

PubMed2004

Subcellular distribution of enzyme I of the Escherichia coli phosphoenolpyruvate:glycose phosphotransferase system depends on growth conditions.

The phosphoenolpyruvate:glycose phosphotransferase system (PTS) participates in important functions in the bacterial cell, including the phosphorylation/uptake of PTS sugars. Enzyme I (EI), the first protein of the PTS complex, accepts the phosphoryl group from phosphoenolpyruvate, which is then transferred through a chain of proteins to the sugar. In these studies, a mutant GFP, enhanced yellow fluorescent protein (YFP), was linked to the N terminus of EI, giving Y-EI. Y-EI was active both in vitro (>/=90% compared with EI) and in vivo. Unexpectedly, the subcellular distribution of Y-EI varied significantly. Three types of fluorescence were observed: (i) diffuse (dispersed throughout the cell), (ii) punctate (concentrated in numerous discrete spots throughout the cell), and (iii) polar (at one or both ends of the cell). Cells from dense colonies grown on agar plates with LB broth or synthetic (Neidhardt) medium showed primarily bipolar or punctate fluorescence. In liquid culture, under carefully defined carbon-limiting growth conditions [ribose (non-PTS), mannitol (PTS sugar), or dl-lactate], cellular levels of enzymatically active Y-EI remain essentially constant for each carbon source, but fluorescence distribution depends on C source, cell density, growth phase, and apparently on "conditioned medium." Fluorescence was diffuse during exponential growth on LB or ribose/Neidhardt medium. On ribose they became punctate in the stationary phase, reverting to diffuse when more ribose was added. In LB, both Y-EI and a nonphosphorylatable mutant, H189Q-Y-EI, showed a diffuse fluorescence during growth, but, shortly after the addition of isopropyl beta-d-thiogalactopyranoside, Y-EI became bipolar; H189Q-Y-EI did not. The functions of EI sequestration remain to be determined.

Cell Count↗

PubMed2002

Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes.

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3' end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg(2+). The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.

Anti-HIV Agents↗

PubMed1974

Participation of N1-oxide derivatives of adenine nucleotides in the phosphotransferase activity of liver mitochondria.

The modified adenine nucleotides ATP-NO, ADP-NO, and AMP-NO were tested as potential substrates and/or inhibitors of mitochondrial phosphotransferases. ADP-NO is not recognized by the translocase system located in the inner mitochondrial membrane; however, it is rapidly phosphorylated to ATP-NO in the outer compartment of mitochondria, by way of the nucleosidediphosphate kinase (EC 2.7.4.6) reaction, provided there is sufficient ATP in the mitochondria. AMP-NO is not phosphorylated by liver mitochondria to the corresponding nucleoside diphosphate; it cannot serve as substrate for adenylate kinase (EC 2.7.4.3). ATP-NO and ADP-NO, however, are substrates of this enzyme. The apparent equilibrium constant for the reaction, ADP-NO + ADP right harpoon over left harpoon ATP-NO + AMP, of 0.908 at pH 7.4 and 5 mM Mg(2+) is significantly higher than that of the reaction with natural nucleotides. Although adenosine N(1)-oxide is easily phosphorylated to AMP-NO by adenosine kinase [Schnebli et al. (1967) J. Biol. Chem. 242, 1997-2004], the formation of corresponding nucleoside triphosphate in vivo seems also to be limited by adenylate kinase; adenosine N(1)-oxide cannot replace adenosine in restoring the normal ATP level in ethionine-treated rats.

Adenine Nucleotides↗

PubMed1974

Phosphotransferase-system enzymes as chemoreceptors for certain sugars in Escherichia coli chemotaxis.

For D-glucose and analogs there are two distinct phosphotransferase enzymes II with different specificities. Transport and chemotaxis were studied in E. coli mutants having only one or the other of these two enzymes. It was found that the transport specificity of a given enzyme II correlates with taxis specificity, and mutational loss of an enzyme II abolishes taxis toward only those sugars which it alone transports. Although enzyme I and the phosphate-carrier protein are required for full D-glucose taxis, it could not be determined if phosphorylation and transport are also required.

Bacterial Proteins↗

PubMed1975

Interaction of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system with adenylate cyclase of Escherichia coli.

Transient repression by glucose of induced enzyme synthesis involves lowering of intracellular cAMP levels. This glucose effect is partially explained by a glucose inhibition of adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase(cyclizing)]. Since the phosphoenolpyruvate:sugar phosphotransferase system has been implicated in repression phenomena, an investigation was made of adenylate cyclase activity in mutants of that transport system. The results suggest that glucose phosphorylation is not necessary for inhibition of adenylate cyclase since an HPr mutant retained sensitivity to glucose inhibition. The results also suggest that adenylate cyclase activity requires the presence of Enzyme I in a phosphorylated form and that adenylate cyclase activity may be regulated by a phosphorylation-dephosphorylation mechanism.

Adenylyl Cyclases↗

PubMed1977

Nucleotide phosphotransferase of Escherichia coli: purification by affinity chromatography.

Improved extraction and purification procedures permit the isolation from Escherichia coli W cells of much larger quantities and of more highly purified preparations of nucleotide phosphotransferase. Of various affinity resins tested for efficiency of purification, columns of agarose/5'-AMP (AGAMP), type 3, proved the best. In this way a 300- to 450-fold purification of the enzyme was achieved in a few steps. The enzyme, which, as reported before, transfers organically bound phosphate to the 2' or 3' hydroxyls of nucleosides and nucleotides, was tested in its behavior toward a series of ribonucleosidonucleotides, namely, CpC, ApA, CpA, and ApC. All were phosphate acceptors, but a detailed comparative study of adenosine and cytidine, 5'-AMP and 5'-CMP, and ApA and ApC revealed peculiar specificities in the relative distribution of the phosphorylated products.

Chromatography, Affinity↗

PubMed1982

Molecular cloning of the crr gene and evidence that it is the structural gene for IIIGlc, a phosphocarrier protein of the bacterial phosphotransferase system.

Sugar substrates of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) normally prevent bacterial cells from utilizing sugars that are not substrates of this system (diauxic growth, "the glucose effect"). We have previously shown that this type of PTS-mediated repression can be completely reversed by a single mutation, designated crr. Two lines of evidence are presented in this report showing that crr is the structural gene for IIIGlc, one of the proteins of the PTS. First, homogeneous IIIGlc was isolated from wild-type and a crr- mutant of Salmonella typhimurium, and the proteins were compared. The preparations of IIIGlc were indistinguishable except as follows: IIIGlc from the mutant showed only 2-3% of the activity of the wild-type IIIGlc in its ability to act as a phosphocarrier protein in the in vitro phosphorylation of methyl alpha-glucoside. In addition, under certain conditions, the two proteins exhibited different behavior on gel filtration columns and in polyacrylamide gel electrophoresis. The second line of evidence was obtained by cloning the Escherichia coli crr gene, which has an estimated minimum length of 0.6 kilobase, into a high-copy-number plasmid as part of a 1.3-kilobase fragment. The plasmid transforms E. coli crr- to crr+ strains and simultaneously directs the synthesis of IIIGlc.

Carrier Proteins↗

PubMed1986

Pel, the protein that permits lambda DNA penetration of Escherichia coli, is encoded by a gene in ptsM and is required for mannose utilization by the phosphotransferase system.

Mannose uptake and phosphorylation in Escherichia coli is catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The mannose-specific complex of the PTS, designated IIMan, comprises lipid and two membrane proteins, II-AMan and II-BMan. The proteins are encoded by ptsM, located at approximately equal to 40 minutes on the E. coli chromosome. A different genetic marker, pel, maps with ptsM, and is required for lambda DNA penetration of the cytoplasmic membrane. Earlier studies suggested that both pel function and II-BMan are encoded by the same gene, while a different gene (also in ptsM) encodes II-AMan. In the present studies, a ptsM clone, pCS13, was isolated from an E. coli HindIII gene bank in pBR322 and restored both mannose termentation and pel+ function to ptsM mutants defective in II-BMan. Subclones of pCS13 show that two distinct genes, manY and manZ, encode the pel+ function and the II-BMan protein, respectively; each gene may have its own promoter; whereas the protein encoded by manY (Pel) alone seems sufficient for lambda sensitivity, all three gene products are required for mannose fermentation, transport of the mannose analogue 2-deoxyglucose, and phosphorylation of the latter by cytoplasmic membranes. Thus, Pel is required for function of the IIMan complex. The efficiency of the complex may depend on the ratio of Pel to IIMan.

Bacterial Proteins↗

PubMed1987

Sequence homologies between proteins of bacterial phosphoenolpyruvate-dependent sugar phosphotransferase systems: identification of possible phosphate-carrying histidine residues.

The DNA sequences for some of the genes involved in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) of Escherichia coli and Salmonella typhimurium have been reported. Comparison of the deduced amino acid sequences of enzyme IIBgi, enzyme IIMtl, and enzyme IIGlc/enzyme IIIGlc, which catalyze the uptake and concomitant phosphorylation of beta-glucosides, mannitol, and glucose, respectively, reveals considerable sequence homology. In particular, the carboxyl-terminal region of enzyme IIBgl is so homologous to the whole of enzyme IIIGlc as to suggest a common function. We postulate that His-547 of enzyme IIBgl receives a phosphate group directly from the cytoplasmic protein HPr and transfers this phosphate to His-306 located in the amino-terminal half of enzyme IIBgl. This latter histidine is conserved in enzyme IIBgl and enzyme IIGlc and, in both proteins, occurs in a region that shows homology with the His-15 region of HPr, which is known to act as the phosphate carrier. An equivalent histidine residue, His-195, is also present in enzyme IIMtl, although here the flanking sequence is different. None of these specified histidine residues is likely to be buried within the membrane.

Amino Acid Sequence↗

PubMed1987

II-BGlc, a glucose receptor of the bacterial phosphotransferase system: molecular cloning of ptsG and purification of the receptor from an overproducing strain of Escherichia coli.

The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) consists of interacting cytoplasmic and membrane proteins that catalyze the phosphorylation and translocation of sugar substrates across the cell membrane. One PTS protein, II-BGlc, is the membrane receptor specific for glucose and methyl D-glucopyranosides; the protein has been purified to homogeneity from Salmonella typhimurium [Erni, B., Trachsel, H., Postma, P. & Rosenbusch, J. (1982) J. Biol. Chem. 257, 13726-13730]. In the present experiments, the Escherichia coli ptsG locus, which encodes II-BGlc, was isolated from a transducing phage library and subcloned into plasmid vectors. The resulting plasmids complement the following phenotypic defects of ptsG mutants: growth on glucose, uptake and phosphorylation of methyl alpha-D-glucoside, and repression of the utilization of non-PTS sugars by methyl alpha-glucoside. The transformed cells overproduce II-BGlc 4- to 10-fold, and a Mr 43,000 polypeptide was synthesized from the plasmids in an in vitro transcription/translation system. The E. coli and S. typhimurium II-BGlc proteins differ in their physical properties, and a modified, three-step purification procedure was developed for isolating the E. coli protein.

Biological Transport↗

PubMed1989

Localization to the inner surface of the cytoplasmic membrane by immunoelectron microscopy of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

The phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli constitutes a major pathway for sugar translocation. It is composed of integral membrane proteins (enzyme II components) that recognize specific extracellular sugars as well as phosphocarrier proteins, one of which is called enzyme I. While enzyme I plays a role in energizing the enzyme II for sugar transfer, its precise cellular distribution had not previously been defined. This study was designed to elucidate the cellular location of this protein by immunoelectron microscopy. Enzyme I antibody bound to E. coli cryosections was visualized with protein A-gold. The gold particles in sections of wild-type E. coli were found primarily associated with the surface of the inner membrane. A strain of E. coli harboring a plasmid encoding the gene for enzyme I was also tested for its distribution of enzyme I. Consistent with the biochemically established overproduction of enzyme I, this strain showed an approximately 80-fold higher density of gold particles per unit cell volume than the wild-type cells. The substantial overproduction of immunoreactive enzyme I was associated with a significant (approximately 20-fold) increase in the amount of that protein bound to the inner membrane. In addition, a substantial fraction of the total enzyme I accumulated within a 60-nm-wide zone in the vicinity of the inner membrane. A model to explain the zonal distribution of enzyme I under conditions of overexpression of the protein is presented.

Cell Membrane↗

PubMed1992

NAD+ and NADH regulate an ATP-dependent kinase that phosphorylates enzyme I of the Escherichia coli phosphotransferase system.

Crude extracts of Escherichia coli contain a protein kinase, EI-K, that phosphorylates enzyme I (EI) of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Phosphorylation occurs at the active site histidine residue. The activity of EI-K was lost during purification. However, kinase activity was restored by adding NAD+ or NADP+.NADH reversed NAD+ activation of the kinase, and the level of EI-K activity was dependent on the NAD+/NADH ratio. Although crude preparations of EI-K showed no NAD+ requirement, they were completely inhibited by NADH, either in the assay mixture or when the enzyme was pretreated and the NADH was removed prior to the assay. NAD+ restored full activity to the NADH-pretreated inactive fractions. The results suggest that EI-K contains a bound cofactor that is lost during purification and that may be analogous to NAD+. EI-K activity may serve to link some of the diverse functions of the PTS, such as sugar transport, to the metabolic state of the cell.

Adenosine Triphosphate↗

PubMed1999

Deduction of consensus binding sequences on proteins that bind IIAGlc of the phosphoenolpyruvate:sugar phosphotransferase system by cysteine scanning mutagenesis of Escherichia coli lactose permease.

Mediated by the protein IIAGlc, the phosphoenolpyruvate:sugar phosphotransferase system plays a role in the regulation of activity of other sugar transport systems in Escherichia coli. By using a direct binding assay, a collection of single-Cys replacement mutants in cytoplasmic loops of lactose permease were evaluated for their capacity to bind IIAGlc. Selected Cys replacements in loops IV/V or VI/VII result in loss of binding activity. Analysis of the mutagenesis results together with multiple sequence alignments of a family of proteins that interacts with IIAGlc provides the basis for developing two regions of consensus sequence in those partner proteins necessary for binding to IIAGlc. The requirement for two interaction regions is interpreted in the regulatory framework of a substrate-dependent conformational change that brings those two regions into an orientation optimal for binding IIAGlc.

Amino Acid Sequence↗

PubMed2000

Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli.

From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (K(m) for growth approximately 8 mM but with V(max) for generation time approximately 1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme II(Glc) of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG(+) from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (V(max) approximately 7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme II(Man) (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl alpha-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

Cell Division↗

PubMed1996

Active site phosphorylation of enzyme I of the bacterial phosphotransferase system by an ATP-dependent kinase.

Enzyme I (EI) of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) is autocatalytically phosphorylated by P-enolpyruvate. We report here an ATP-dependent kinase (EI-K) from Escherichia coli that reversibly phosphorylates EI at its active site histidine; ATP and EI-K can therefore replace P-enolpyruvate. EI-K contains a bound cofactor that is lost during purification with concomitant loss of activity. NAD+ and NADP+ substitute for the cofactor and restore activity to the apoenzyme, whereas their analogues are inactive. The pyridine nucleotides do not activate EI-K by covalent modification (e.g. ADP-ribosylation), but must be present during the kinase reaction. NADH and NADPH are potent inhibitors of EI-K at all stages of purity, and enzyme activity in a mixture of NAD+ and NADH depends on the ratio of the two pyridine nucleotides. Inhibition is observed with reduced beta-NMN and alpha-NADH, but neither is as effective as beta-NADH. The reverse reaction, the transfer of the phosphoryl moiety from phospho-EI to ADP, also requires NAD+ or NADP+. In the absence of NAD+ or NADH, [32P]phospho-EI is hydrolyzed to 32Pi, suggesting that EI-K can act as a phospho-EI phosphatase. EI kinase may serve as a link between PTS-driven sugar transport and the electron transport chain.

Binding Sites↗