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 667 records · Page 37Linked to original sources

PubMed1992

Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase.

N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+ or Mg2+, for its activity and exhibits an optimal pH at 7.2-7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparent Km of 24 microM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor alpha-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is approximately 1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).

Chromatography, Affinity↗

PubMed1995

Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance.

The UL97 phosphotransferase coding sequences of clinical cytomegalovirus (CMV) isolates, 10 resistant and 11 sensitive to ganciclovir, were compared to define mutations associated with drug resistance. In each ganciclovir-resistant isolate, a mutation was found that resulted in an amino acid substitution at codon 460 (4 isolates), codon 594 (2 isolates), or codon 595 (4 isolates). No sensitive isolate carried any of these mutations. Marker transfer studies showed that each mutation was capable of conferring ganciclovir resistance to the laboratory CMV strain AD169. Rapid diagnostic tests based on DNA amplification and restriction enzyme analysis were developed for these mutations. Specific mutant DNAs were detected when they constituted at least 10% of the population in the specimen. Several mutations in UL97 appear to be common markers for ganciclovir resistance, and their detection may be a rapid alternative to conventional cell culture susceptibility testing.

Amino Acid Sequence↗

PubMed1986

Aminoglycoside-phosphotransferases APH(3')-IV and APH(3") synthesized by a strain of Campylobacter coli.

Campylobacter coli strain 981 of animal origin was resistant to erythromycin, tetracycline, streptomycin, kanamycin, ribostamycin, neomycin, paromomycin, lividomycin, and butirosin. Resistance to aminoglycosides of strain 981 was mediated by phosphotransferases APH (3') type-IV and APH (3"). C. coli 981 harboured three plasmids of 24, 34, and 40 Megadaltons respectively. None of these plasmids were transferable to Escherichia coli K-12 by conjugation.

Aminoglycosides↗

PubMed1986

Nucleotide sequence of the hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

The nucleotide sequence of a 1467 bp fragment of Streptomyces hygroscopicus DNA containing the gene (hyg) encoding a hygromycin B phosphotransferase (HPH) has been determined. The N-terminal amino acid sequence of HPH determined by automated Edman degradation has allowed the coding sequence of the hyg gene to be identified. The translation initiation triplet is GTG and 5 bp preceding it there is a sequence complementary to the 3'-end of 16S rRNA from S. lividans. The transcriptional start and termination sites have been determined; the presumptive promoter region has only partial homology to that of the Streptomyces vinaceus vph gene and is different to the promoter sequences of other Streptomyces genes.

Amino Acid Sequence↗

PubMed1978

Transient repression of beta-galactosidase synthesis by glucose-6-phosphate in a mutant of Escherichia coli lacking enzyme II specific for glucose in the phosphoenolpyruvate-sugar phosphotransferase system.

The effects of glucose and glucose-6-phosphate in initiating the repression of beta-galactosidase synthesis were studied using a mutant of Escherichia coli K12 which lacks glucose-specific enzyme II of the phosphoenolpyruvate-sugar phosphotransferase system. It was found that glucose-6-phosphate causes transient repression of beta-galactosidase synthesis but glucose does not cause transient repression in this mutant. Evidence was obtained that both the presence of an active transport system for glucose-6-phosphate in the cells and glucose-6-phosphate in the medium are necessary for the initiation of transient repression. No metabolism of glucose-6-phosphate is required. Upon depletion of glucose-6-phosphate in the medium the transient repression was reversed. After the reversal the rate of enzyme synthesis was high in the cells which had been exposed to a high concentration of glucose-6-phosphate. It was concluded that the translocation of glucose-6-phosphate across the membranes is the primary event which affects both the initiation of and the recovery from the transient repression. During the transient repression the cellular content of cyclic adenosine 3',5'-monophosphate decreased significantly.

Biological Transport, Active↗

PubMed1991

Enzyme IIIlac of the staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis of histidine residues, biochemical characterization and 1H-NMR studies.

The lactose-specific phosphocarrier protein enzyme III of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacF gene in order to replace the histidine residues 78 and 82 of the amino acid sequence with a serine residue. Wild-type and both mutant genes were overexpressed in Escherichia coli and the gene products were purified to homogeneity. The conformation of wild-type and mutant proteins were monitored by 1H-NMR spectroscopy. In vitro phosphorylation studies on mutant lactose-specific enzyme III, as well as evidence from NMR spectroscopy, lead to the conclusion that His78 is the active-site for phosphorylation of lactose-specific enzyme III by phospho-HPr (histidine-containing protein). The role of His82 probably is the enhancement of velocity and efficiency of the phosphotransfer from lactose-specific enzyme III to lactose-specific enzyme II. This result refutes the conclusion of former work based on data by protelytic cleavage and sequencing of the 32P-labeled peptide of lactose-specific enzyme III that His82 is the active-site for phosphorylation.

Bacterial Proteins↗

PubMed1991

Staphylococcal lactose phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis on the lacE gene gives evidence that a cysteine residue is responsible for phosphorylation.

The lactose-specific phosphocarrier protein enzyme II of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacE gene in order to replace the histidine residues 245, 274 and 510 and the cysteine residue 476 of the amino acid sequence with a serine residue. The wild-type and mutant genes were expressed in Escherichia coli and the gene products were characterized in different in vitro test systems. In vitro phosphorylation studies on mutant derivatives of the lactose-specific enzyme II led to the conclusion that cysteine residue 476 is the active-site for phosphorylation of this enzyme II by a phospho-enzyme III of the same sugar specificity. A cysteine residue phosphorylated intermediate was first postulated for the mannitol-specific enzyme II of E. coli and studies performed independently concerning the lactose-specific enzyme II of Lactobacillus casei are in agreement with the above results.

Amino Acid Sequence↗

PubMed1992

Role of the phosphoenolpyruvate-dependent fructose phosphotransferase system in the utilization of mannose by Escherichia coli.

Mutants of Escherichia coli devoid of the membrane-spanning proteins PtsG and PtsMP, which are components of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and which normally effect the transport into the cells of glucose and mannose, do not grow upon or take up either sugar. Pseudorevertants are described that take up, and grow upon, mannose at rates strongly dependent on the mannose concentration in the medium (apparent Km > 5 mM); such mutants do not grow upon glucose but are derepressed for the components of the fructose operon. Evidence is presented that mannose is now taken up via the fructose-PTS to form mannose 6-phosphate, which is further utilized for growth via fructose 6-phosphate and fructose 1,6-bisphosphate.

Biological Transport, Active↗

PubMed1982

A specialized transducing phage, lambda psrlA, for the sorbitol phosphotransferase of Escherichia coli K12.

A specialized transducing phage for the srlA gene, specifying the sorbitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system, was constructed and its DNA was analysed by restriction endonuclease digestion. Phage construction involved four steps: (1) integration of lambda into the srlA gene; (2) selection of phage carrying (a) the left and (b) the right end of the srlA gene by means independent of the function of the new DNA acquired; (3) reconstitution of the srlA gene in a dilysogen of these two phage; and (4) the excision, using the heteroimmune lambdoid phage 21, of a plaque-forming srlA+ phage from the dilysogenic chromosome. Comparison of the DNA restriction digests of the transducing phage with those of its parents and of wild-type lambda revealed fragments consisting partly of lambda and partly of Escherichia coli DNA. The junction points in the intermediate phage define a site that must lie within the reconstituted gene of the final phage. This technique should be of general application in relating genes, cloned by our method, to DNA sequences.

Bacteriophage lambda↗

PubMed1983

Phosphotransferase-mediated regulation of carbohydrate utilization in Escherichia coli K12: the nature of the iex (crr) and gsr (tgs) mutations.

Mutants of Escherichia coli K12 defective in the gene iex (crr) no longer utilize glucose or N-acetylglucosamine in preference to lactose, but competition between either of these sugars and another that also enters by a phosphotransferase (PT) mechanism is not affected. In this they differ from gsr (tgs) mutants. In gsr mutants, glucose does not exclude any other sugar, though N-acetylglucosamine still does so. In gsr mutants that are also ptsM the phosphoenolpyruvate-dependent phosphorylation of glucose or methyl alpha-glucoside is reduced by 90%: N-acetylglucosamine phosphorylation is not affected. The iex mutation does not affect the phosphorylation of either of these compounds. The wild-type alleles iex+ and gsr+ are dominant in lambda heterozygotes. Glucose inhibits the lactose permease of wild-type cells, but only when the permease is present in low amounts. The inhibition is also relieved (1) by induction of another transport system that is subject to regulation by the iex system or (2) by an iex mutation. We suggest that the iex gene specifies a protein that, in cells transporting certain sugars by a PT mechanism, acts to inhibit active transport systems. The protein is present in limiting concentration in the cell, sufficient only to inhibit the basal, uninduced, level of the active transport systems. In consequence the inducer (or its precursor) may be excluded from the cell and induction thus prevented.

Acetylglucosamine↗

PubMed1984

An inducible phosphoenolpyruvate: dihydroxyacetone phosphotransferase system in Escherichia coli.

A phosphoenolpyruvate: dihydroxyacetone phosphotransferase was induced in Escherichia coli grown on dihydroxyacetone as sole carbon source or in its presence. This is the first example of a triose which can be acted upon by the membrane complex to provide a central intermediate in glycolysis. The presence of this system explains the ability of a mutant, in which the ATP-dependent glycerol kinase is genetically replaced by a glycerol: NAD 2-oxidoreductase, to grow on glycerol.

Dihydroxyacetone↗

PubMed1987

The roles of osmotic stress and water activity in the inhibition of the growth, glycolysis and glucose phosphotransferase system of Clostridium pasteurianum.

Growth of Clostridium pasteurianum was inhibited in media of high solute content. At equal osmolarities, 'permeant' solutes (glycerol and acetamide) were much less growth-inhibitory than 'non-permeant' solutes (KC1 and xylitol). Glycolysis by washed cell suspensions was inhibited by these solutes in parallel with growth. However, in their inhibition of glucose 6-phosphate dissimilation by permeabilized cells the distinction between 'permeant' and 'impermeant' solutes was significantly less marked. The glucose phosphotransferase system (PTS) of intact cells was much more strongly inhibited by 'non-permeant' than by 'permeant' solutes. It was concluded that the predominant inhibitory effects on this organism of media of high solute content are due not to the low water activity of such media per se, but to the creation of an osmotic pressure across the bacterial cytoplasmic membrane, which acts to inhibit the glucose PTS by which the organism effects glucose uptake. Parallel measurements of the effects of xylitol on both glycolysis and the activity of the glucose PTS suggested that despite this correlation between the osmotic inhibition of growth, glycolysis and the PTS, the flux-control coefficient of the PTS on glycolysis did not exceed 0.2 under the conditions used.

Acetamides↗

PubMed1987

Nucleotide sequence of bglC, the gene specifying enzymeIIbgl of the PEP:sugar phosphotransferase system in Escherichia coli K12, and overexpression of the gene product.

The EnzymeIIbgl of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system catalyses the uptake and concomitant phosphorylation of beta-glucosides by Escherichia coli; it is specified by the gene bglC. The nucleotide sequence of a 3.6 kb HindIII restriction fragment spanning bglC, cloned on a plasmid, was determined. DNA analysis strongly suggests that the published order of this and other genes involved in beta-glucoside utilization, bgl C, S, B, is incorrect, and that the regulatory gene bglS may be located upstream of the structural genes bglC and bglB. From the deduced amino acid sequence it is predicted that the membrane protein specified by bglC consists of 625 amino acid residues (66.48 kDa). The protein has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.62). Comparisons between the amino acid sequences deduced for the EnzymeIIbgl and for the mannitol-specific EnzymeIImtl show that these proteins are related, and a little direct homology is apparent. A 2.3 kb AluI fragment spanning bglC was subcloned into an expression vector which carries the lambda PL promoter and then transformed into a host strain which produces thermolabile cI857 repressor and the anti-terminator N; thermoinduction resulted in the overproduction of a membrane protein and the appearance of Bgl activity.

Amino Acid Sequence↗

PubMed1987

The nucleotide sequence of a streptomycin streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from a streptomycin producer.

The nucleotide sequence of the DNA fragment containing the streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of fruA, the gene specifying enzyme IIfru of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Escherichia coli K12.

The Enzyme IIfru of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system (PTS), which catalyses the uptake of fructose and its concomitant phosphorylation to fructose 1-phosphate by Escherichia coli, is specified by a gene designated fruA. The nucleotide sequence of a 2.5 kb PvuII restriction fragment spanning fruA+, cloned on a plasmid, was determined. This fragment contained three open reading frames (ORFs) but only one complete ORF, 1689 base pairs long, which was preceded by a well-defined Shine-Dalgarno sequence and ended with a rho-independent transcription terminator. The amino acid sequence deduced from this DNA corresponds to that of a protein of 563 amino acids (57.5 kDa), which has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.40) and which is characterized by a number of well-marked hydrophobic loops that may correspond to membrane-spanning regions. There is relatively little overall homology between this protein and those of other Enzymes II of the PTS but there is considerable correspondence between the region surrounding one of the six histidine residues (His381) of Enzyme IIfru and those surrounding the particular histidines of other Enzymes II, and of HPr, known to be involved in phosphorylation. A plasmid carrying the complete fruA+ nucleotide sequence, but not that of any other functional protein, fully restored the ability of fruA mutants to grow on fructose and of extracts of fruA mutants to phosphorylate fructose, which confirms that the nucleotide sequence determined species Enzyme IIfru.

Amino Acid Sequence↗

PubMed1992

Expression of an inducible enzyme II fructose and activation of a cryptic enzyme II glucose in glucose-grown cells of spontaneous mutants of Streptococcus salivarius lacking the low-molecular-mass form of IIIman, a component of the phosphoenolpyruvate:mannose phosphotransferase system.

We have reported previously that the phosphoenolpyruvate:mannose phosphotransferase system (mannose PTS) of Streptococcus salivarius, consisting of an Enzyme II mannose (EIIman) and two forms of Enzyme III mannose (IIIman) with Mr values of 38,900 and 35,200, respectively, concomitantly transports and phosphorylates mannose, as well as glucose and fructose. In this paper, we report the presence, in S. salivarius, of alternative specific fructose and glucose PTSs encoded by inducible and cryptic genes, respectively. Protein phosphorylation experiments conducted with [32P]phosphoenolpyruvate have allowed us to identify by SDS-PAGE and autoradiography the EII fructose (EIIfru) (Mr 57,500) and the EII glucose (EIIglc) (Mr 58,700). No proteins corresponding to IIIfru or IIIglc could be detected. EIIfru phosphorylated fructose on the C-1 position rather than, as with the constitutive mannose PTS, on the C-6 position. Growth on fructose resulted in the induction of EIIfru as well as an increase of 1-phosphofructokinase activity. Nevertheless, the genes encoding these proteins were independently regulated. Studies carried out with spontaneous mutants lacking the low-molecular-mass form of IIIman (mutants A37, G29 and B31) showed that EIIfru was expressed in glucose-grown cells of strains G29 and B31, but not in strain A37, whereas the cryptic gene encoding EIIglc was activated in all three mutant strains. The results obtained with the mutants suggest that the three spontaneous mutants were not all mutated on the gene encoding IIIman although all of them lacked IIIman.

Enzyme Induction↗

PubMed1995

Identification and characterization of phosphoenolpyruvate:fructose phosphotransferase systems in three Streptomyces species.

Streptomyces lividans, S. coelicolor and S. griseofuscus were examined for the presence of the enzymes of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). All three species were shown to possess Enzyme I, HPr and fructose-specific Enzyme II (IIFru) activities. In S. lividans and S. coelicolor, all three PTS enzymes were fructose-inducible, but in S. griseofuscus the system was expressed constitutively. These organisms apparently lack the HPr(Ser) kinase and HPr(Ser-P) phosphatase that characterize low-GC Gram-positive bacteria.

Bacillus subtilis↗

PubMed1996

Surface location of HPr, a phosphocarrier of the phosphoenolpyruvate: sugar phosphotransferase system in Streptococcus suis.

HPr is a low-molecular-mass phosphocarrier protein of the bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) found in the cytoplasm or associated with the inner surface of the cytoplasmic membrane. Treatment of Streptococcus suis cells with a Sorvall Omnimixer, a technique used to extract cell surface components, resulted in the extraction of a major protein with a molecular mass of 9 kDa. Several lines of evidence suggested that this protein was HPr: (i) the S. suis protein showed homology over the first 35 N-terminal amino acid residues with the HPrs of Streptococcus salivarius and Streptococcus mutans, including the signature sequence for the site of PEP-dependent phosphorylation; (ii) it cross-reacted with the S. salivarius anti-HPr antibody preparation; (iii) it could be phosphorylated by enzyme I at the expense of PEP, and by a membrane-associated kinase at the expense of ATP; and (iv) it possessed phosphocarrier activity when used as a source of HPr in an in vitro PTS assay. The data suggested that a portion of the cellular HPr is associated with the external cell surface in S. suis, a result that was confirmed by immunogold electron microscopy. The cellular HPr of S. suis consisted of two forms that could be distinguished by the presence or the absence of the N-terminal methionine. Amino acid sequence analysis indicated that the cell-surface-associated HPr of S. suis lacked the N-terminal methionine residue.

Amino Acid Sequence↗