SEARCH · PubMed Health

Results for “PHOSPHOTRANSFERASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources

PubMed2003

Bioinformatic analyses of the bacterial L-ascorbate phosphotransferase system permease family.

The tripartite L-ascorbate permease of Escherichia coli is the first functionally characterized member of a large family of enzyme II complexes (SgaTBA, encoding enzymes IIC, IIB and IIA) of the bacterial phosphotransferase system (PTS). We here report bioinformatic analyses of these proteins. Forty-five homologous systems from a wide variety of bacteria were identified, but no homologues were found in archaea or eukaryotes. These systems fell into five structural types: (1) IIC, IIB and IIA are encoded by distinct genes; (2) IIC and IIB are encoded by distinct genes, but the IIA-encoding gene is absent; (3) IIC and IIB are encoded by a fused gene, but IIA is a distinct gene product; (4) IIA and IIB are fused, but IIC is encoded by a distinct gene, and (5) IIC and IIB are encoded by distinct genes, but IIA is fused to a transcriptional regulator. Phylogenetic analyses revealed that gene fusion/splicing events have occurred repeatedly during the evolutionary divergence of family members, although no evidence for shuffling of constituents between systems was obtained. The SgaTBA family proved to be distantly related to the GatCBA family of PTS permeases, and this family was also analyzed. In contrast to the SgaTBA family, no gene splicing/fusion has occurred during the evolutionary divergence of GatCBA family members as each domain is always encoded by a distinct gene. However, GatC homologues were identified in organisms that lack other PTS proteins, suggesting a transport mechanism not coupled to substrate phosphorylation. Topological analyses suggest that in contrast to all other PTS permeases, IIC proteins of the Sga and Gat families exhibit 12 transmembrane alpha-helical segments and are distantly related to secondary carriers. Like many secondary carriers, GatC (IIC) homologues could be shown to have arisen by an ancient intragenic duplication event. These results suggest that the Sga and Gat families of PTS permeases comprise a small superfamily in which the transmembrane IIC domains evolved independently of all other known PTS permeases.

Amino Acid Transport Systems↗

PubMed2004

Role of pyruvate oxidase in Escherichia coli strains lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system.

We report a study to determine the role of pyruvate oxidase among Escherichia coli isogenic strains with active and inactive phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Strain PB11, displaying a specific growth rate (mu) in glucose minimal medium of 0.1 h(-1) is a ptsHI, crr operon deletion derivative of wild-type JM101 (displaying a mu of 0.70 h(-1)). Strain PB12 is a spontaneous mutant obtained from PB11 after selection for its capacity to grow on glucose with a mu of 0.40 h(-1). In minimal medium cultures supplemented with glucose plus acetate, strain JM101 displayed preferential consumption of glucose, whereas strains PB11 and PB12 did not display glucose catabolic repression of acetate consumption. Inactivation of poxB caused a severe reduction in growth rate in strain PB11 when grown in the fermentor with medium containing glucose or glucose plus acetate, whereas under the same conditions poxB(-)derivative strains of JM101 and PB12 were not affected. Relative transcript levels for 29 genes related to poxB transcriptional regulation and central metabolism were determined using RT-PCR. This analysis revealed 2-fold lower transcript levels for genes encoding subunits of the pyruvate dehydrogenase complex (Pdh) in strain PB11 and 4- to 6-fold higher transcript levels for poxB in strains PB11 and PB12, when compared to JM101. In addition, in the PTS(-) strains, upregulation of the poxB transcription factors rpoS, soxS and marA, was detected. The results presented here strongly suggest that AcCoA is mainly synthesized from acetate produced by pyruvate oxidase in strain PB11, whereas in strains JM101 and PB12, AcCoA is synthesized preferentially from pyruvate by Pdh.

Acetic Acid↗

PubMed2005

Nutrient-scavenging stress response in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system, as explored by gene expression profile analysis.

The physiological role of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been studied in Escherichia coli. It has been shown that it directly or indirectly regulates the activity of most catabolic genes involved in carbohydrate transport. Accordingly, strains lacking PTS have pleiotropic phenotypes and are impaired in their capacity to grow on glucose and other PTS sugars. We have previously reported the characterization of a mutant harboring a pts operon deletion (PB11) which, as expected, showed a severe reduction of its growth capacity when incubated on glucose as carbon source, as compared to that of the isogenic wild-type strain. These observations corroborate that PTS is the main determinant of the capacity to grow on glucose and confirm the existence of other systems that allow glucose utilization although at a reduced level. To explore the physiological state and the metabolic pathways involved in glucose utilization in a pts(-) background, we analyzed the global transcriptional response of the PB11 mutant when growing in minimal medium with glucose as carbon source. Genome-wide transcriptional analysis using microarrays revealed that, under this condition, expression of several genes related to carbon transport and metabolism was upregulated, as well as that of genes encoding transporters for certain nucleotides, nitrogen, phosphorus and sulfur sources. In addition, upregulation of rpoS and several genes transcribed by this sigma subunit was detected. These results indicate that the reduced capacity of glucose utilization present in the PB11 strain induces a general nutrient-scavenging response and this behavior is not dependent on a functional PTS. This condition is responsible of the utilization of secondary carbon sources in the presence of glucose.

Biological Transport↗

PubMed2006

Engineering transport protein function: theoretical and technical considerations using the sugar-transporting phosphotransferase system of Escherichia coli as a model system.

Potential experimental approaches for developing and applying protein-engineering protocols to transmembrane transport systems are described. We specifically consider procedures designed to alter protein function. These procedures are designed for the specific purposes of (1) changing protein interaction specificities and (2) changing a protein's catalytic function. We use sugar-transporting bacterial phosphotransferase systems as model systems to illustrate the proposed approaches. These and other similar procedures are likely to prove to be of utility for biotechnological manipulation of proteins as well as for elucidating potential evolutionary pathways taken for the appearance of novel functions within a protein family.

Bacterial Proteins↗

PubMed2006

Topological predictions for integral membrane permeases of the phosphoenolpyruvate:sugar phosphotransferase system.

We report bioinformatic analyses of the largest superfamily of integral membrane permeases of the bacterial phosphotransferase system (PTS), the Enzyme IIC constituents of the Glc superfamily. Phylogenetic analyses reveal that this superfamily consists of five equally distant families, the Glucose (Glc), beta-Glucoside (Bgl), Fructose (Fru), Mannitol (Mtl) and Lactose (Lac) families. Average hydropathy, amphipathicity and similarity plots were generated for these five families as well as for the entire superfamily. Charged residue distribution was analyzed, and the most conserved sequence motif, common to all five families, was identified. The results show that the members of all five families exhibit similar average hydropathy plots with regions of average amphipathicity and relative conservation also being similar. Evidence is presented suggesting that the Glucitol (Gut) family of Enzyme IIC constituents is a distant member of the Glc superfamily. Based on our analyses we offer a topological model that resembles, but differs in detail from the two previously proposed models.

Amino Acid Sequence↗

PubMed2007

The phosphotransferase system of Lactobacillus casei: regulation of carbon metabolism and connection to cold shock response.

Genome sequencing of two different Lactobacillus casei strains (ATCC334 and BL23) is presently going on and preliminary data revealed that this lactic acid bacterium possesses numerous carbohydrate transport systems probably reflecting its capacity to proliferate under varying environmental conditions. Many carbohydrate transporters belong to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but all different kinds of non-PTS transporters are present as well and their substrates are known in a few cases. In L. casei regulation of carbohydrate transport and carbon metabolism is mainly achieved by PTS proteins. Carbon catabolite repression (CCR) is mediated via several mechanisms, including the major P-Ser-HPr/catabolite control protein A (CcpA)-dependent mechanism. Catabolite response elements, the target sites for the P-Ser-HPr/CcpA complex, precede numerous genes and operons. PTS regulation domain-containing antiterminators and transcription activators are also present in both L. casei strains. Their activity is usually controlled by two PTS-mediated phosphorylation reactions exerting antagonistic effects on the transcription regulators: P~EIIB-dependent phosphorylation regulates induction of the corresponding genes and P~His-HPr-mediated phosphorylation plays a role in CCR. Carbohydrate transport of L. casei is also regulated via inducer exclusion and inducer expulsion. The presence of glucose, fructose, etc. leads to inhibition of the transport or metabolism of less favorable carbon sources (inducer exclusion) or to the export of accumulated non-metabolizable carbon sources (inducer expulsion). While P-Ser-HPr is essential for inducer exclusion of maltose, it is not necessary for the expulsion of accumulated thio-methyl-beta-D-galactopyranoside. Surprisingly, recent evidence suggests that the PTS of L. casei also plays a role in cold shock response.

Biological Transport↗

PubMed1985

Lysosomal enzyme phosphorylation in human fibroblasts. Kinetic parameters offer a biochemical rationale for two distinct defects in the uridine diphospho-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase.

The primary genetic defect in the lysosomal storage disease mucolipidosis III (ML III) is in the enzyme uridine diphospho-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. This enzyme has two well-defined functions: specific recognition of lysosomal enzymes (recognition function) and phosphorylation of their oligosaccharides (catalytic function). Using fibroblasts from patients with ML III as the source of enzyme, and alpha-methylmannoside and two lysosomal enzymes as the substrates, we have identified defects in both of these functions. In one group of fibroblasts, the catalytic activity of the N-acetylglucosaminylphosphotransferase is decreased while the ability to recognize lysosomal enzymes as specific substrates remains intact. In the second group of fibroblasts, the ability to recognize lysosomal enzymes is impaired while the catalytic activity of the enzyme is normal. These data provide a biochemical rationale for the previously described genetic heterogeneity among patients with ML III (Honey, N. K., O. T. Mueller, L. E. Little, A. L. Miller, and T. B. Shows, 1982, Proc. Natl. Acad. Sci. USA., 79:7420-7424).

Acid Phosphatase↗

PubMed1978

Assessment of third trimester choline phosphotransferase activity in uncomplicated human pregnancies.

CDP--choline:1,2-diglyceride choline phosphotransferase (CPT; EC 2.7.8.2) undergoes a marked surge in activity in human amniotic fluid when assayed from 30 weeks of gestation to term. The activity of this enxyme, plotted against gestational age, follows a highly significant regression correlation from which an equation can be obtained for the prediction of gestational age.

Amniotic Fluid↗

PubMed1998

Expression in Pseudomonas aeruginosa of an erythromycin-resistance determinant that encodes the mphA gene for macrolide 2'-phosphotransferase I from Escherichia coli.

We studied the expression in Pseudomonas aeruginosa of an erythromycin-resistance (EMr) determinant that included the mphA gene for macrolide 2'-phosphotransferase I and originated in Escherichia coli. A recombinant plasmid, pTZ3609, that consisted of the EMr determinant and a broad-host-range vector RSF1010, endowed P. aeruginosa with high-level resistance to erythromycin. Furthermore, the EMr determinant on a self-transferable plasmid, RP1, was transferred from E. coli to P. aeruginosa by conjugation.

Anti-Bacterial Agents↗

PubMed1999

Nucleotide sequence of the gene cluster containing the mphB gene for macrolide 2'-phosphotransferase II.

Escherichia coli BM2506 produced macrolide 2'-phosphotransferase II [MPH (2') II]. A gene for MPH (2') II, designated mphB, is located on plasmids pTZ3721 and pTZ3723 in E. coli BM2506. In the present study, we determined the nucleotide sequence of the 6.5-kb EcoRI-PstI fragment containing mphB on pTZ3721. The DNA region of 6.5-kb EcoRI-PstI fragment contained five open reading frames (ORFs). ORF4 corresponded to mphB. Respective products deduced from ORF1, ORF2, ORF3, and ORF5 were similar to the penicillin-binding protein 4 of Streptomyces lactamduras, the repressor protein AcrR of the acrAB operon, the enzyme RdmC involved in the biosynthesis of the antibiotic aklavinone, and IS801 transposase-like protein from Pseudomonas pseudoalcakigenes, respectively. Among these genes, ORF2, ORF3, and mphB formed a gene cluster with ORF2 in the lead sequence. Our results suggest that mphB may originate from an operon related to antibiotic biosynthesis.

Base Sequence↗

PubMed2005

Structure-function analysis of the yeast NAD+-dependent tRNA 2'-phosphotransferase Tpt1.

Tpt1 is an essential 230-amino-acid enzyme that catalyzes the final step in yeast tRNA splicing: the transfer of the 2'-PO4 from the splice junction to NAD+ to form ADP-ribose 1''-2''cyclic phosphate and nicotinamide. To understand the structural requirements for Saccharomyces cerevisiae Tpt1 activity, we performed an alanine-scanning mutational analysis of 14 amino acids that are conserved in homologous proteins from fungi, metazoa, protozoa, bacteria, and archaea. We thereby identified four residues-Arg23, His24, Arg71, and Arg138-as essential for Tpt1 function in vivo. Structure-activity relationships at these positions were clarified by introducing conservative substitutions. The activity of the Escherichia coli ortholog KptA in complementing tpt1Delta was abolished by alanine substitutions at the equivalent side chains, Arg21, His22, Arg69, and Arg125. Deletion analysis of Tpt1 shows that the C-terminal 20 amino acids, which are not conserved, are not essential for activity in vivo at 30 degrees C. These findings attest to the structural and functional conservation of Tpt1-like 2'-phosphotransferases and identify likely constituents of the active site.

Amino Acid Motifs↗

PubMed2005

In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus.

An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55 degrees C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58 degrees C and then 61 degrees C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67 degrees C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65 degrees C.

Base Sequence↗

PubMed1998

Xylose transport insensitivity to catabolite inhibition by phosphoenolpyruvate:sugar phosphotransferase system in Tetragenococcus halophila.

Tetragenococcus halophila accumulates glucose and 2-deoxyglucose (dGlc) via the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and pentoses, maltose, and glycerol via non-PTS carriers. Based on the discovery that xylose metabolism in T. halophila was subject to catabolite repression but not to catabolite inhibition, we designed a selection protocol for thermosensitive mutants pleiotropically unable to use sugars. One such mutant was a ptsI mutant with a thermosensitive enzyme I (EI) of the PTS (leaky at 30 degrees C). Using this ptsI mutant, catabolite inhibitions was studied. dGlc was more strongly inhibitory of glycerol uptake in the mutant than in the parent because of the leaky ptsI mutation. Thermoinactivation of EI at 42 degrees C resulted in the total loss of uptake of PTS sugars and in the virtual abolishment of glycerol uptake. However, xylose uptake of the ptsI mutant was scarcely inhibited by dGlc even after thermoinactivation of EI. These results suggest that sensitivities of non-PTS uptakes to PTS-mediated inhibition vary among non-PTS sugars.

Biological Transport↗

PubMed1998

Selective fermentation of xylose by a mutant of Tetragenococcus halophila defective in phosphoenolpyruvate:mannose phosphotransferase, phosphofructokinase, and glucokinase.

Tetragenococcus halophila is a Gram-positive halophilic lactic acid bacterium used for soy sauce fermentation. We isolated a mutant, T. halophila 3E4, triply defective in phosphoenolpyruvate:mannose phosphotransferase, phosphofructokinase, and glucokinase. 3E4 selectively metabolized pentoses such as xylose and arabinose in the presence of hexoses such as glucose and galactose. We present here an example of the metabolic engineering of catabolite control.

Fermentation↗

PubMed1998

Identification, separation and characterisation of two forms of cytosolic 5'-nucleotidase/nucleoside phosphotransferase in calf thymus.

Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, endowed with phosphotransferase activity, is a widespread enzyme responsible for the regulation of intracellular IMP and GMP concentrations and the phosphorylation of purine nucleoside pro-drugs. The enzyme activity is stimulated by ATP, ADP and 2,3-bisphosphoglycerate (BPG), and is inhibited by phosphate. Calf thymus possesses two active proteins with a different electrophoretic mobility. In this report we show that the two forms can be separated by ADP-agarose affinity chromatography. Whereas form A binds weakly to the column, form B is tightly bound and is released by the addition of ADP into the elution buffer. The two enzyme forms differ in terms of electrophoretic, chromatographic behaviour and regulatory characteristics. Form B, as already described for the enzyme purified from the same source (Pesi et al., 1996, Biochim Biophys Acta 294, 191-194), exhibits three different sites for the three activators with a synergistic effect between ADP and BPG. Form A has a high affinity regulatory site for BPG, while ADP and ATP appear to share the same low affinity site and no synergistic effect is observed.

5'-Nucleotidase↗

PubMed1986

Nucleoside phosphotransferase from malt sprouts. I. Isolation, characterization and specificity of the enzyme.

The nucleoside phosphotransferase of barley was isolated in four steps from dried malt sprouts and was obtained homogeneous according to several criteria. The enzyme transfers the phospho group from the 3'- or 5'-position of a nucleotide to the 5'-OH group of another nucleoside. Activated phosphomonoesters (e.g. 4-nitrophenyl phosphate) can be used as donors as well. In the absence of nucleosides the enzyme shows pronounced phosphatase activity towards 3'- and 5'-nucleotides as well as towards activated phosphomonoesters. 2'-Nucleotides are not hydrolysed. Studies of the phosphatase activity with a number of modified nucleotides demonstrate that the nucleotide substrates are bound by the phosphate group and by the base. The base can be extensively modified compared to naturally occurring nucleobases. Obviously, only a hydrophobic interaction with the enzyme is required; base-specific hydrogen bonds are of no importance. In the transferase reaction the acceptor nucleoside interacts only with the base which has a much more restricted specificity. Nucleotides which are donors do not need to be acceptors when they are nucleosides, while all acceptor nucleosides are efficient donors. Due to the pre-orientation of the base, the phosphate groups of the 3'- and 5'-nucleotides are in different positions relative to the groups of the enzyme that are involved in the catalysis. This is reflected in the kinetic parameters which are interpreted on the basis of a mechanism with a phospho-intermediate as postulated for the enzyme of Ives et al. [(1979) J. Biol. Chem. 254, 4339; (1982) J. Biol. Chem. 257, 4931]. Evidence is obtained that Km represents the ratio of the rate constants for the decay and the formation of the intermediate and that the kcat/Km-value can be used as indicator for the formation step.

Cyanogen Bromide↗

PubMed1986

Nucleoside phosphotransferase from malt sprouts. II. Studies on the active site and the phospho-intermediate.

The phospho-intermediate formed in the reaction of the nucleoside phosphotransferase is an acyl phosphate, the phosphorus bound to the gamma-carboxylate group of a glutamic acid. Reduction of this intermediate with sodium cyanoborotritiide yields labeled 2-amino-5-hydroxyvaleric acid after hydrolysis of the protein. Nucleophilic trapping of the intermediate with hydroxylamine during the reaction with substrates leads to N-phosphohydroxylamine, which is the only reaction product at a higher concentration of hydroxylamine. Evidence is obtained from modification experiments that in addition to the carboxylate group a histidine is involved in the reaction. The pKa-value for the histidine derived from the photoinactivation of the enzyme is 7.6, indicating that this group forms a salt bridge to a carboxylate group, probably that group attacking the phosphorus. The acceptor nucleosides are bound only by hydrophobic interactions of the base, a conclusion obtained from fluorescence studies and quenching experiments. The hydrophobic interaction obviously does not involve pi-interactions to tyrosine and tryptophan residues, since their fluorescence is not affected by addition of nucleotide inhibitors. Modification of these residues leads only to unspecific inactivation. From the Scatchard plot of the titration of the enzyme with 1,N6-ethenoadenosine 5'-phosphoramidate, an efficient inhibitor (Kd = 1.2 X 10(-5) M), it can be concluded that there is only one binding site on the dimeric enzyme.

Binding Sites↗

PubMed1986

Nucleoside phosphotransferase from malt sprouts. III. Studies on metal ion requirement, solvent effects, kinetics and reaction mechanism.

The nucleoside phosphotransferase from malt sprouts contains one Mg2 per dimeric enzyme molecule. This cation can be removed by EDTA, while other chelating agents are less efficient. The metal-free apoenzyme is inactive. Activity can be restored to its initial value by Mg2 or Co2 and to a minor extent by Mn2, Zn2, Cu2, Ni2 and Fe2. Thermal stability is reduced when the metal is removed but can be restored by addition of Mg2. Adenosine 3',5'-phosphate (cAMP) and arsenate, strong competitive inhibitors, reduce the rate of inactivation by EDTA considerably but do not reduce the rate for the reconstitution with Mg2. We therefore conclude that the phosphate group interacts electrostatically with Mg2 and that the inhibitor is not bound to the apoenzyme. The Sp-isomer of adenosine 3',5'-thionophosphate is a hundred times stronger inhibitor than the Rp-isomer and ten times stronger than cAMP; this allowed us to determine the relative position of the Mg2 in the active site. The imidazolium cation, previously detected as an essential part of the active site, obviously forms a salt bridge to the carboxylate group which attacks the phosphorus opposite to the leaving alcohol group. This conclusion is derived from the fact that organic solvents increase the rate of formation of the phospho-intermediate considerably, while higher concentrations of salt decrease it strongly. In addition, the imidazolium cation seems to polarize the P = O bond and to stabilize the negative charge at the phosphoryl oxygen in the pentacoordinate transition state; this is followed from the pH-dependence of the hydrolysis reaction. The kinetic results reveal that Km does not represent a binding equilibrium, but is the ratio of the rates for decay and formation of the covalent intermediate, while kcat/Km is an indicator for the formation step of the acyl phosphate. On the basis of all these informations it should be allowed to formulate a reaction mechanism, in which all steps of the transferase reaction have to be microscopically reversible.

Apoenzymes↗