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At least 433 records · Page 24Linked to original sources

PubMed2007

Genomic analysis of the phosphotransferase system in Clostridium botulinum.

Clostridium botulinum is capable of fermenting carbohydrates, but there have been no detailed studies of the uptake of sugars and related substrates. In bacteria, a common and often predominant system of carbohydrate uptake is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). This multi-protein complex catalyses a group translocation involving both uptake and phosphorylation of carbohydrates, and is also known to play an important role in environmental sensing and metabolic regulation. The genome of C. botulinum encodes 15 PTSs which have a similar domain structure to the PTS in other bacteria. Based on phylogenetic relationships and analysis of gene clusters, the C. botulinum PTS appears to be involved in the uptake of hexoses, hexose derivatives and disaccharides. C. botulinum also contains the components of PTS-associated regulatory mechanisms which have been characterised in other bacteria. It therefore seems likely that the PTS plays a significant, and previously unrecognised, role in the physiology of this bacterium.

Biological Transport↗

PubMed2006

Biophysical characterization of the enzyme I of the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system.

The first protein in the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system is the homodimeric 60-kDa enzyme I (EI), which autophosphorylates in the presence of PEP and Mg2+. The conformational stability and structure of the EI from Streptomyces coelicolor, EI(sc), were explored in the absence and in the presence of its effectors by using several biophysical probes (namely, fluorescence, far-ultraviolet circular dichroism, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry) and computational approaches. The structure of EI(sc) was obtained by homology modeling of the isolated N- and C-terminal domains of other EI proteins. The experimental results indicate that at physiological pH, the dimeric EI(sc) had a well-folded structure; however, at low pH, EI(sc) showed a partially unfolded state with the features of a molten globule, as suggested by fluorescence, far-ultraviolet circular dichroism, FTIR, and 8-anilino-1-naphthalene-sulfonic acid binding. The thermal stability of EI(sc), in the absence of PEP and Mg2+, was maximal at pH 7. The presence of PEP and Mg2+ did not change substantially the secondary structure of the protein, as indicated by FTIR measurements. However, quenching experiments and proteolysis patterns suggest conformational changes in the presence of PEP; furthermore, the thermal stability of EI(sc) was modified depending on the effector added. Our approach suggests that thermodynamical analysis might reveal subtle conformational changes.

Enzyme Activation↗

PubMed1995

Nucleotide sequence of the region between crr and cysM in Salmonella typhimurium: five novel ORFs including one encoding a putative transcriptional regulator of the phosphotransferase system.

A 4471 bp region between crr and cysM on the Salmonella typhimurium chromosome (49.5 min) has been sequenced. Five ORFs were found within this region, one of which is likely to be the putative regulatory gene, ptsJ, that corresponds in map position to a gene which when mutated allows expression of a cryptic Enzyme I of the phosphotransferase system. The deduced amino acid sequence of the encoded protein is similar to those of several open reading frames (ORFs) including ORFT2 of Rhodobacter spheroides with which it is 28% identical throughout most of its length (comparison score of 21 S.D.). PtsJ exhibits a putative, N-terminal, helix-turn-helix, DNA binding domain that is similar in sequence to those in members of the GntR family of transcriptional regulators. Analyses of the sequences of the ORFs encoded within this region are presented.

Amino Acid Sequence↗

PubMed1996

Acarbose 7-phosphotransferase from Actinoplanes sp.: purification, properties, and possible physiological function.

A phosphotransferase which modifies the alpha-glucosidase inhibitor acarbose by phosphorylation at its 7-position was isolated from the acarbose producer Actinoplanes sp. and purified to homogeneity. The sequence of the first 20 amino acids of the enzyme was determined. The enzyme is an ATP-dependent kinase and shows high specificity for acarbose and some related compounds containing the pseudodisaccharide moiety (acarviosin). The product formed by the enzyme, acarbose-7-phosphate, shows a significant lower inhibitory activity towards disaccharidases than acarbose itself. The acarbose producing organism contains a maltase which is inhibited by acarbose, but to a much lesser extent by acarbose-7-phosphate. The possible role of acarbose 7-phospho-transferase as part of a self-defense mechanism against acarbose in the producing organism is discussed.

Actinomycetales↗

PubMed1999

Novel phosphotransferase systems revealed by bacterial genome analysis: the complete repertoire of pts genes in Pseudomonas aeruginosa.

We herein describe all genes encoding constituents of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in the 6Mbp genome of the opportunistic human pathogen, Pseudomonas aeruginosa. Only four gene clusters were found to encode identifiable PTS homologues. These genes clusters encode novel multidomain proteins, two complete sugar-specific PTS phosphoryl transfer chains for the metabolism of fructose and N-acetylglucosamine, and a complex regulatory system that may function to coordinate carbon and nitrogen metabolism. No previously characterized organism has been shown to exhibit such a novel and restricted complement of PTS proteins.

Genome, Bacterial↗

PubMed1999

A reporter gene assay for inhibitors of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) plays a key role in sugar uptake and metabolic regulation in bacteria. PTS proteins form a divergent phosphorylation cascade. Enzyme I (EI) is at the top of the cascade and mediates phosphoryl-transfer from phosphoenolpyruvate to the phosphoryl-carrier protein HPr, which then distributes the phosphoryl-groups to the different carbohydrate transporters. In addition, some PTS proteins have a regulatory function in catabolite repression, inducer exclusion and chemotaxis which is modulated by their degree of phosphorylation in response to the availability of substrates. Using as a reporter the IacZ gene under control of the bgl t2 transcriptional terminator and as an effector the transcriptional antiterminator LicT from B. subtilis, a two-plasmid reporter gene system was constructed in order to monitor PTS activity. LicT, when present at low concentration in E. coli, is inactivated by EI/HPr-dependent phosphorylation and conversely is active in a ptsl- mutant lacking El. Active LicT allows for transcriptional readthrough at bgl t2, resulting in a full-length lacZ transcript. Beta-galactosidase activities are increased 4-8-fold in a ptsl+ strain growing on PTS substrates relative to growth on non-PTS substrates and approximately 30-fold in a ptsl- mutant. This gain-of-function in response to dephosphorylation of El or lack of active El can be used to monitor changes of El activity caused by mutations and environmental factors and for screening and validation of inhibitors of the PTS as potentially novel antibacterial compounds.

Bacterial Proteins↗

PubMed1975

Inhibition by 6-O-tosyl galactosides of beta-galactoside phosphorylation and transport by the lactose phosphotransferase system of Staphylococcus aureus.

The effect of various galactose derivatives, substituted at C-6, on the phosphoenolpyruvate:beta-galactoside phosphotransferase system of Staphylococcus aureus was studied. Cells were grown by an improved procedure, which resulted in a 5- to 10-fold increase in cell yield. The four protein components of the system were separated. A membrane fraction containing negligible levels of the soluble components was prepared by alternate cycles of sonic treatment and differential centrifugation. The in vitro system reconstituted from these fractions was used to test the ability of the galactose derivatives to inhibit the phosphorylation of lactose analogs, under conditions where the membrane-bound component, Enzyme IIlac, was rate limiting. Derivaites in which the hydroxyl group of C-6 was missing, or replaced by a fluoro, O-methyl, or carboxyl group had no affinity for Enzyme IIlac, as judged by their inability to inhibit phosphorylation. Surprisingly, derivatives containing arylsulfonyl groups at C-6 were potent inhibitors; the O-tosyl compound has an apparent affinity five times that of galactose. The arylsulfonyl substitution in an absolute requirement; neither O-benzyl or O-methanesulfonyl derivatives were inhibitory. The specificity of the inhibition by tosyl derivatives parallels that of unsubstituted substrates; tosyl galactosides of the beta configuration were inhibitory, but those of the alpha configuration were not. The tosyl derivatives also strongly inhibited the uptake of lactose analogs into whole cells; the requirement for the arylsulfonyl moiety was again observed. The chemical analogy between the tosyl galactosides and possible intermediates in the transport-phosphorylation step catalyzed by Enzyme IIlac provides a possible explanation for the unexpected properties of these derivatives.

Bacterial Proteins↗

PubMed2002

[Isolation and genetic study of Erwinia mutants devoid of common components of the phosphoenolpyruvate-dependent phosphotransferase system].

Mutants of bacteria belonging the genus Erwinia (Erwinia chrysanthemi and Erwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis. Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively. The ptsI+ allele in both Erwinia species was cloned in vivo. Mapping of obtained mutations indicated that the ptsI and ptsH genes of E. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas the ptsH gene is located at 175 min. Sequencing of a portion of the E. chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I.

Bacterial Proteins↗

PubMed1990

Sugar transport by the bacterial phosphotransferase system. Characterization of the sulfhydryl groups and site-specific labeling of enzyme I.

Enzyme I is the first protein of the phospho transfer sequence in the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This protein exhibits a temperature-dependent monomer/dimer equilibrium. The nucleotide sequence of Escherichia coli ptsI indicates four -SH residues per subunit (Saffen, D. W., Presper, K. A., Doering, T. L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253). In the present experiments, the sulfhydryl groups of the E. coli enzyme were studied with various -SH-specific reagents. Titration of Enzyme I with 5,5'-dithiobis-2-nitrobenzoic acid also revealed four reacting -SH groups. The kinetics of the 5,5'-dithiobis-2-nitrobenzoic acid reaction with Enzyme I exhibit biphasic character, with pseudo-first order rate constants of 2.3 x 10(-2)/s and 2.3 x 10(-3)/s at pH 7.5, at room temperature. Fractional amplitudes associated with the rate constants were 25 +/- 5% for the fast and 75 +/- 5% for the slow rate. The "slow" rate was influenced by ligands that react with Enzyme I (the protein HPr, Mg2+, Mg2+ plus P-enolpyruvate), and also by temperature (at the temperature range where the monomer/dimer association occurs). The fractional ratio of the two rates remained at 1:3 under these conditions. Thus, under all conditions tested, two classes of -SH groups were detected, one reacting more rapidly than the other three -SH groups. Modification of the "fast" -SH group results in an active enzyme capable of forming dimer, whereas modification of the slow -SH groups results in inactive and monomeric Enzyme I. The enzyme was labeled with pyrene maleimide under conditions where only the more reactive sulfhydryl group was derivatized. Hydrolysis by trypsin followed by reverse-phase high performance liquid chromatography analysis of the peptide mixture resulted in only one fluorescent peak. This peak was not observed when the more reactive sulfhydryl residue was protected prior to pyrene maleimide labeling. Amino acid sequencing of the fluorescent peak indicated that the more reactive residue is the C-terminal amino acid residue, cysteine 575. The results provide a means for selectively labeling Enzyme I with a fluorophore at a single site while retaining full catalytic activity.

Amino Acid Sequence↗

PubMed1990

Sugar transport by the bacterial phosphotransferase system. Fluorescence studies of subunit interactions of enzyme I.

Enzyme I of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) exhibits a temperature-dependent monomer/dimer equilibrium. The accompanying paper (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995) shows that the C-terminal -SH residue (Cys-575) can be modified specifically with fluorescent probes such as pyrene maleimide. The derivative retains full enzyme activity, and is capable of forming dimers at room temperature. In the present studies, Enzyme I labeled in this way is found to exhibit a temperature-, concentration-, and pH-dependent monomer/dimer association. The kinetics of dimer formation of Enzyme I is measured in the following way. A derivatized Enzyme I sample is prepared with a pyrene moiety irreversibly attached to the C-terminal -SH residue and 5,5'-dithiobis-2-nitrobenzoic acid reversibly attached to the other 3 -SH residues. This modified enzyme does not form dimers at room temperature. Addition of dithiothreitol results in total release of the thionitrobenzoate anion within 2 min. After the three -SH groups are unblocked, steady-state and nanosecond time-resolved emission anisotropy measurements indicate the dimer is formed over a period of 30 min. In a similar experiment, little dimer formation is observed at 3 degrees C, at temperature at which the native enzyme also does not form dimers. Tryptophan fluorescence is also examined during the release of the thionitrobenzoate. After the completion of thionitrobenzoate release, additional slow steady-state tryptophan fluorescence changes are observed. These results suggest that dimer formation may be preceded by a conformational change following thionitrobenzoate release.

Dithionitrobenzoic Acid↗

PubMed1985

Existence in animal tissues of adenosine triphosphate thiamin diphosphate phosphotransferase [EC 2.7.4.15].

An enzyme which catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate (TDP), thiamindiphosphate kinase (ATP:thiamin diphosphate phosphotransferase) [EC 2.7.4.15], was detected in animal tissues. The enzyme was partially purified (150-fold) from the cytosol fraction of guinea pig brain. The enzyme reaction required free (not protein-bound) TDP, ATP, Mg2+, and a cofactor, which is a low molecular weight and heat-stable compound. The enzyme activity was optimal at pH 11 and at 25 degrees C. A stoichiometric transfer of 32P from [gamma-32P]ATP to TDP was demonstrated. Km values for TDP and ATP were calculated to be 1.1 mM and 10 microM, respectively, and Vmax was 868 nmol/mg of protein/hr. The enzyme was found solely in the cytosol fraction of guinea pig brain and was also detectable in the skeletal muscle and heart. These results provide strong evidence for the existence of TDP kinase in animal tissues.

Animals↗

PubMed1980

Affinity chromatographic purification and properties of flavokinase (ATP:riboflavin 5'-phosphotransferase) from rat liver.

Flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26) has been purified to apparent homogeneity from rat liver by affinity chromatography using flavinyl agarose beads (agarose-OCH2CONH(CH2)2NHCO(CH2)/N10-7,8-dimethylisoalloxazine). The specific activity of the pure enzyme is 9,900 units (nmol of FMN formed/h at 37 degrees C)/mg of protein, and reflects a one-step, 7000-fold purification. Flavokinase thus obtained, unlike previous preparations from mammalian sources, is free from contaminating phosphatase and FAD synthase. The purified enzyme rapidly loses activity upon storage but is stabilized by riboflavin and thiol-protecting reagents. The apparent molecular weight, estimated by gel filtration on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 28,000 +/- 1,000. Flavokinase phosphorylates and/or is inhibited by a large number of riboflavin analogs; however, the physiologically important 8 alpha-(amino acid)riboflavins are poorly accommodated. The strongly preferred phosphate donors are ATP and dATP. Both Zn2+ and Mg2+, as well as several other divalent cations, activate flavokinase, but Zn2+ yields greatest activity (1.8 times that with Mg2+). The pH optimum for activity with either Zn2+ or Mg2+ is approximately 9.3; at pH 7.0, the activity is 40% of that at the pH optimum.

Animals↗

PubMed1982

Sugar transport by the bacterial phosphotransferase system. Studies on the molecular weight and association of enzyme I.

Studies were conducted on the physical properties of Enzyme I, the first protein in the Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Since values lower than those previously reported for the monomer molecular weight were obtained, experiments were performed to determine whether Enzyme I had been partially degraded during isolation of homogeneous protein. Crude extracts and partially purified and homogeneous protein preparations exhibited identical behavior in crossed immunoelectrophoresis analyses, indicating that the isolated protein represented native, intact Enzyme I. The monomeric subunit of Enzyme I is globular, with a frictional ratio of about 1. Sedimentation equilibrium experiments provided a monomer molecular weight of 57,700 +/- 3,400, and gel filtration studies under denaturing conditions gave a comparable value of 57,000. The values previously obtained from polyacrylamide gel electrophoresis analyses in the presence of sodium dodecyl sulfate varied with the conditions used, but under one set of conditions agreed with those given above. The sedimentation equilibrium studies were conducted at 8 degrees C, in the absence of substrates and cofactor (phosphoenolpyruvate, pyruvate, Mg2+). Under these conditions Enzyme I self-associates, but the association is weak, favoring primarily monomer. Because of solubility limitations, the sedimentation experiments were performed with Enzyme I at an initial concentration of 0.5 mg/ml, providing a concentration distribution of 0.1 to 2 mg/ml. Computer analysis of the results showed that within this concentration range it was not possible to distinguish between two modes of self-association, monomer-dimer and isodesmic. The physiological significance of the results is discussed.

Biological Transport↗

PubMed1997

Purification and preliminary crystallographic studies on the sporulation response regulatory phosphotransferase protein, Spo0B, from Bacillus subtilis.

The phosphotransferase protein Spo0B, a component of the sporulation signal transduction system in Bacillus subtilis was expressed from the Escherichia coli strain BL21DE3. It was purified, crystallized, and 2.25 A data measured using the synchrotron source at the Stanford Linear Accelerator Center. The search for heavy atom derivatives is in progress.

Bacillus subtilis↗

PubMed2001

Determination of 3-deoxy-D-arabino-heptulosonate 7-phosphate productivity and yield from glucose in Escherichia coli devoid of the glucose phosphotransferase transport system.

The effect of inactivation of the glucose phosphotransferase transport system (PTS) on 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) productivity and yield from glucose in Escherichia coli is reported. Strains used in this study were the PTS(+) PB103 and its PTS(-) glucose(+) derivative NF9. Their aroB(-) derivatives PB103B and NF9B were constructed to allow accurate measurement of total carbon flow into the aromatic pathway. The measured specific rates of DAHP synthesis were 0.55 and 0.94 mmol/g-dcw. h and the DAHP molar yields from glucose were 0.43 and 0.71 mol/mol for the PTS(+) aroB(-)and the PTS(-) glucose(+) aroB(-)strains, respectively. For the latter strain, this value represents 83% of the maximum theoretical yield for DAHP synthesis from glucose.

Biological Transport↗

PubMed2005

Synthetic neomycin-kanamycin phosphotransferase, type II coding sequence for gene targeting in mammalian cells.

The bacterial neomycin-kanamycin phosphotransferase, type II enzyme is encoded by the neo gene and confers resistance to aminoglycoside drugs such as neomycin and kanamycin-bacterial selection and G418-eukaryotic cell selection. Although widely used in gene targeting in mouse embryonic stem cells, the neo coding sequence contains numerous cryptic splice sites and has a high CpG content. At least the former can cause unwanted effects in cis at the targeted locus. We describe a synthetic sequence, sneo, which encodes the same protein as that encoded by neo. This synthetic sequence has no predicted splice sites in either strand, low CpG content, and increased mammalian codon usage. In mouse embryonic stem cells sneo expressability is similar to neo. The use of sneo in gene targeting experiments should substantially reduce the probability of unwanted effects in cis due to splicing, and perhaps CpG methylation, within the coding sequence of the selectable marker.

Animals↗

PubMed1984

Molecular cloning, sequencing, and expression of the crr gene: the structural gene for IIIGlc of the bacterial PEP:glucose phosphotransferase system.

The phosphoenolpyruvate:glucose phosphotransferase system (PTS) of Salmonella typhimurium is involved both in glucose transport and in the regulation and synthesis of adenylate cyclase and several transport systems. The crr gene has been implicated in this regulating mechanism. A 9.6-kb segment of the S. typhimurium chromosome containing the crr gene was cloned in pAT153. The cloned fragment also complemented cysA mutations but did not contain a functional pts operon which is closely linked to the crr gene and codes for two enzymes of the PTS. Although cysA and crr have been reported to be located on opposite sides of ptsHI, our results suggest that the correct gene order is cysK-ptsHI-crr-cysA. Expression of crr plasmids in a maxicell system yielded two proteins which reacted with specific anti-serum against IIIGlc. The apparent mol. wts. in SDS-polyacrylamide gels were 20 000 and 21 000, the former corresponding to the major band of purified IIIGlc. Both forms were also observed in bacterial extracts and purified IIIGlc. The crr gene was localized on a 1-kb EcoRI-EcoRV fragment of the 9.6-kb insert and sequenced. It codes for a single protein (18 556 D) containing 169 amino acid residues and identified as IIIGlc.

Base Sequence↗

PubMed1989

Mechanistic and physiological consequences of HPr(ser) phosphorylation on the activities of the phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: studies with site-specific mutants of HPr.

The bacterial phosphotransferase system (PTS) catalyzes the transport and phosphorylation of its sugar substrates. The protein-kinase-catalyzed phosphorylation of serine 46 in the phosphocarrier protein, HPr, inhibits PTS activity, but neither the mechanism of this inhibition nor its physiological significance is known. Site-specific HPr mutants were constructed in which serine 46 was replaced by alanine (S46A), threonine (S46T), tyrosine (S46Y) or aspartate (S46D). The purified S46D protein exhibited markedly lower Vmax and higher Km values than the wild-type, S46T or S46A protein for the phosphoryl transfer reactions involving HPr(His approximately P). Interactions of HPr with the enzymes catalyzing phosphoryl transfer to and from HPr regulated the kinase-catalyzed reaction. These results establish the inhibitory effect of a negative charge at position 46 on PTS-mediated phosphoryl transfer and suggest that HPr is phosphorylated on both histidyl and seryl residues by enzymes that recognize its tertiary rather than its primary structure. In vivo studies showed that a negative charge on residue 46 of HPr strongly inhibits PTS-mediated sugar uptake, but that competition of two PTS permeases for HPr(His approximately P) is quantitatively more important to the regulation of PTS function than serine 46 phosphorylation.

Amino Acids↗