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PubMed1990

Induction of pyrophosphate:fructose 6-phosphate 1-phosphotransferase by anoxia in rice seedlings.

Rice (Oryza sativa) seeds were imbibed for 3 days and the seedlings were further incubated for 8 days in the presence of either air or nitrogen. In aerobiosis, the specific activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase and that of the ATP-dependent phosphofructokinase increased about fourfold. In anaerobiosis, the specific activity of ATP-dependent phosphofructokinase remained stable, whereas that of pyrophosphate:fructose 6-phosphate 1-phosphotransferase increased as much as in the presence of oxygen and there was also a fourfold increase in the concentration of fructose 2,6-bisphosphate, a potent stimulator of that enzyme. These data suggest a preferential involvement of pyrophosphate:fructose 6-phosphate 1-phosphotransferase rather than of ATP-dependent phosphofructokinase in glycolysis during anaerobiosis.

Journal Article↗

PubMed1991

Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje.

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2 degrees C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4 degrees C. During storage at 8 degrees C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8 degrees C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8 degrees C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4 degrees C but remained the same throughout storage at 8 degrees C. After exposure to 2 degrees C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4 degrees C but decreased slightly at 8 degrees C. All observed changes became annulled after transfer of cold stored tubers to 18 degrees C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.

Journal Article↗

PubMed1991

Regulation of pp90rsk phosphorylation and S6 phosphotransferase activity in Swiss 3T3 cells by growth factor-, phorbol ester-, and cyclic AMP-mediated signal transduction.

Somatic cell homologs to the Xenopus laevis S6 protein kinases (referred to collectively as pp90rsk) have recently been identified and partially characterized. Here we examine alterations in pp90rsk phosphorylation and S6 phosphotransferase activity in response to regulators of multiple signal transduction systems: purified growth factors, phorbol ester, changes in cyclic AMP (cAMP) levels, and sodium vanadate. All reagents tested increased pp90rsk serine and threonine phosphorylation, but only those agents that regulate cell proliferation and sodium vanadate activated its S6 kinase activity. In addition to the cAMP-stimulated phosphorylation of pp90rsk, a simple correlation between the extent of growth-regulated pp90rsk phosphorylation and S6 phosphotransferase activity was not observed. Quantitative phosphorylation of pp90rsk continued to increase after its S6 kinase activity began its return towards basal levels. However, a close correlation between the appearance and disappearance of a slow-mobility form of phosphorylated pp90rsk (by electrophoresis) and pp90rsk activity was observed. In addition, pp90rsk was regulated by both protein kinase C-independent and -dependent signaling mechanisms. The extent of protein kinase C participation, however, varied depending on which growth factor receptor was activated. Furthermore, growth factor-specific differences in the temporal regulation of pp90rsk S6 phosphotransferase activity were also observed. These results support the notion that the complex regulation of the rsk gene product constitutes one of the primary responses of animal cells to mitogenic signals.

Animals↗

PubMed1982

Phosphorylation of lysosomal enzymes in fibroblasts. Marked deficiency of N-acetylglucosamine-1-phosphotransferase in fibroblasts of patients with mucolipidosis III.

N-Acetylglucosamine-1-phosphotransferase activity was assayed in human skin fibroblasts using [beta-32P]UDP-N-acetylglucosamine as donor and dephosphorylated beta-N-acetyl-D-hexosaminidase as acceptor. An optimal transfer rate of N-acetylglucosamine 1-phosphate required CDP-choline and ADP in order to inhibit the breakdown of [beta-32P]UDP-N-acetylglucosamine and a combination of leupeptin and iodoacetamide to protect the transferase. The transferase required Mg2 or Mn2. Using doubly labelled UDP-N-acetylglucosamine, simultaneous transfer of N-acetyl-[6-3H]glucosamine and [32P]phosphate to endogenous acceptors was demonstrated. Membranes prepared from fibroblasts from patients with mucolipidosis III were defective in transfer of N-acetylglucosamine 1-phosphate. A residual transferase activity of less than 10% of controls was detectable in fibroblast membranes of eight patients with mucolipidosis III. In membranes from fibroblasts from patients with mucolipidosis II,N-acetylglucosamine-1-phosphotransferase activity was not detectable. Our results indicate that the primary defect in mucolipidoses II and III is a deficiency in N-acetylglucosamine-1-phosphotransferase, the residual activity being higher in mucolipidosis III than in mucolipidosis II.

Cell Membrane↗

PubMed1997

[Determination of the nucleotide sequence and characterization of the novel aminoglycoside phosphotransferase aphVIII gene from the Streptomyces rimosus strain].

The nucleotide sequence of a BglII-PstI DNA fragment that contains the cloned aphVIII gene from the Streptomyces rimosus P3 strain, the producer of oxytetracycline, was determined. It was established that the aph gene encodes neomycin phosphotransferase that differs by substrate specificity from neomycin phosphotransferases encoded by aph genes in producers of aminoglycoside antibiotics and clinical bacterial strains. The gene was shown to be 777 bp in length with the mean GC content equal to 67%. The amino acid sequence possesses all highly conserved regions typical for aminoglycoside phosphotransferases; however, this sequence contained several amino acid substitutions that have been detected for the first time, including those in the domain responsible for the association with antibiotics.

Amino Acid Sequence↗

PubMed1982

Evidence for the functional association of enzyme I and HPr of the phosphoenolpyruvate-sugar phosphotransferase system with the membrane in sealed vesicles of Escherichia coli.

Several independent assay procedures were used to estimate the activities of the enzyme constituents of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) in osmotically shocked bacterial membrane vesicles. The soluble enzymes of the system were found to be in association with the membrane by several criteria. Phosphoenolpyruvate-dependent sugar phosphorylation was catalyzed by this membrane-bound enzyme system far more efficiently than by a mixture of the individual enzymes at corresponding concentrations. By contrast, the rates of the phosphoryl exchange reactions catalyzed by enzyme I and the enzyme II complexes were essentially the same for the associated and dissociated forms of the system. Functional association of the PTS-enzyme complex was stabilized by Mg++ and phosphoenolypyruvate and could be destroyed by detergent treatment, sonication, or by passage of the vesicle preparation through a French pressure cell. These results lead to the possibility that in the intact bacterial cell the soluble enzymes of the phosphotransferase system exist, in part, as peripheral membrane constituents associated with the integral membrane enzyme II complexes.

Bacterial Proteins↗

PubMed1994

Identification of the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system produced by proteolytic digestion.

The phosphoenolpyruvate:sugar phosphotransferase system of bacteria plays an important role in the concomitant uptake and phosphorylation of numerous sugars. The first protein in the pathway of phosphotransfer of the phosphoenolpyruvate:sugar phosphotransferase system is Enzyme I. It has been shown that a stable N-terminal domain can be produced by treatment of the purified protein with various proteolytic enzymes. We show here that the region from glutamate-252 to leucine-264 is accessible to proteolysis resulting in N-terminal cores ranging from M(r) 27521 to 28799.

Amino Acid Sequence↗

PubMed1987

Characterisation of the hydroxystreptomycin phosphotransferase gene (sph) of Streptomyces glaucescens: nucleotide sequence and promoter analysis.

The nucleotide sequence of a 1384 bp fragment containing the coding and promoter sequences of the streptomycin phosphotransferase gene (sph) of the hydroxystreptomycin-producing Streptomyces glaucescens was determined. Evidence for an ATG as translation start codon for sph was derived from a comparison with the amino-terminal amino acid sequence of an aminoglycoside phosphotransferase (aphD gene product) of S. griseus, exhibiting a high degree of amino acid homology to the deduced amino acid sequence of the S. glaucescens sph gene product. Transcriptional start and termination sites for the sph gene were identified by primer extension and/or nuclease S1 mapping experiments. The promoter region of the sph gene appears to be complex since tandemly arranged promoters (orfIp1, orfIp2) initiating transcription of a likely coding region (ORFI) in the opposite direction overlap sph promoter sequences. The presumptive sphp and orfIp1 promoters show considerable sequence similarities in the -10 region to Escherichia coli consensus promoter sequences but no homology to E. coli or Streptomyces -35 regions.

Amino Acid Sequence↗

PubMed1984

Cloning and expression in Streptomyces lividans of a paromomycin phosphotransferase from Streptomyces rimosus Forma paromomycinus.

The paromomycin producing organism Streptomyces rimosus forma paromomycinus is resistant to this antibiotic and contains a phosphotransferase which inactivates paromomycin. The gene encoding this enzyme has been inserted in the Streptomyces vector pIJ702 and then cloned in Streptomyces lividans, selecting for paromomycin-resistance. Three plasmids have been isolated and one of them, pMJ1, contains a 2.2 kb insert with a single HindIII restriction site. Insertion of foreign DNA in this site blocks the expression of the phosphotransferase enzyme indicating that it is within the cloned gene. These findings provide a new dominant selective marker for Streptomyces cloning vectors with the versatility of insertional inactivation.

Cloning, Molecular↗

PubMed1992

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

Animals↗

PubMed1992

A gene (hur) from Streptomyces aureofaciens, conferring resistance to hydroxyurea, is related to genes encoding streptomycin phosphotransferase.

A novel gene (hur) conferring resistance to hydroxyurea (HU) in Escherichia coli has been identified in a Streptomyces aureofaciens genomic library. The expression of hur in E. coli was under the control of the external plasmid tet promoter. Sequence analysis of a minimal fragment revealed an open reading frame (ORF) encoding a protein of 340 amino acids with an M(r) of 36,049 and an average hydropathy index of 1.13. The predicted protein product was similar to streptomycin phosphotransferases from Streptomyces glaucescens and Streptomyces griseus (52.4% and 50.8% identity, respectively), but it did not confer resistance to streptomycin or to any of the other aminoglycoside antibiotics tested. It is inferred that hur encodes a phosphotransferase that inactivates HU by phosphorylation of the hydroxy group in the hydroxylamine moiety.

Amino Acid Sequence↗

PubMed2005

Transient state kinetics of Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system of Escherichia coli: equilibrium and second-order rate constants for the phosphotransfer reactions with phosphoenolpyruvate and HPr.

The first two reactions in the phosphotransfer sequence of bacterial phosphoenolpyruvate:glycose phosphotransferase systems are the autophosphorylation of Enzyme I by phosphoenolpyruvate followed by the transfer of the phospho group to the low-molecular weight protein, HPr. Transient state kinetic methods were used to estimate the second-order rate constants for both phosphotransfer reactions. These measurements support previous conclusions that only the dimer of Enzyme I, EI2, is autophosphorylated, and that the rate of formation of dimer is slow compared to the rate of its phosphorylation. The rate constants of the two autophosphorylation reactions of EI2 by PEP are 6.6 x 10(6) M(-1) s(-1), and differ from one another by a factor of less than 3. The rate constant for the transfer reaction between phospho-EI2 and HPr is unusually large for a covalent reaction between two proteins (220 x 10(6) M(-1) s(-1)), while the constant for the reverse reaction is 4.2 x 10(6) M(-1) s(-1). Using the previously reported equilibrium constant for the autophosphorylation reaction, 1.5, the overall equilibrium constant for phosphotransfer from PEP to HPr is 80, somewhat higher than that previously reported. The results also show that EI2 can phosphorylate multiple molecules of HPr without dissociating to a monomer (EI), and that EI can accept a phospho group from phospho-HPr. These results are directly applicable to predicting the rates of phosphoenolpyruvate phosphotransferase system sugar uptake in whole cells.

Bacterial Proteins↗

PubMed1996

Importance of the region around glycine-338 for the activity of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system from an Escherichia coli enzyme I mutant was cloned and sequenced. The mutation was shown to be a guanine to adenine transition resulting in an altered protein in which glycine-338 was replaced by aspartic acid. The enzyme I structural gene was mutated to change glycine-338 to a variety of other amino acid residues. Fermentation tests indicated that glycine-338 could be mutated to alanine with no gross loss in phosphotransferase activity, while mutation to valine, glutamic acid, aspartic acid, arginine, histidine, or asparagine led to significant loss of activity. An expression vector for enzyme I was mutated to change glycine-338 to a variety of other amino acid residues and highly purified mutant proteins were prepared. Analysis of phosphorylation of the proteins by PEP indicated that mutation of glycine-338 to alanine had little effect on phosphorylation, mutation to valine substantially decreased phosphorylation, change to histidine or arginine drastically diminished phosphorylation, and mutation to aspartic or glutamic acids abolished phosphorylation activity. Mutation at glycine-338 influences the autophosphorylation rather than the phosphoryl transfer activity of enzyme I.

Amino Acid Sequence↗

PubMed1981

Purification and characterization of N-acetylglutamate 5-phosphotransferase from pea (Pisum sativum) cotyledons.

N-Acetylglutamate 5-phosphotransferase (acetylglutamate kinase, EC 2.7.2.8) has been isolated from pea (Pisum sativum) cotyledons and purified 312-fold by using heat treatment, (NH4)2SO4 fractionation, affinity chromatography on ATP--Sepharose and ion-exchange chromatography on DEAE-cellulose. This preparation was shown on polyacrylamide-gel electrophoresis to yield one band staining with Coomassie Blue. The enzyme was shown by a variety of techniques to be composed of two different kinds of subunits, of mol.wts. 43000 and 53000 respectively. These subunits are arranged to give either a dimeric or tetrameric enzyme composed of equal numbers of each type of subunit. The dimeric and tetrameric enzyme forms are thought to be interconvertible, the equilibrium between these forms being influenced by the type of ligand bound to the subunits. Kinetic studies performed on the purified enzyme, indicated a random Bi Bi type of mechanism. The enzyme displayed apparent negative co-operativity with respect to one of its substrates, N-acetylglutamate; as a result, two Km values were found for this substrate, one at 1.9 X 10(-3) M and the other at 6.2 X 10(-3) M. A single Km value for ATP was found to be 1.7 X 10(-3) M. Allosteric regulation by arginine was also shown. A model, based on the Koshland, Némethy & Filmer [(1966) Biochemistry 5, 365-385] Sequential model, which adequately describes the kinetic and structural properties of N-acetylglutamate 5-phosphotransferase, is presented.

Arginine↗

PubMed2002

The phosphotransferase system of Streptomyces coelicolor.

We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion. As in E. coli, the crr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIACrr is expressed in vivo. The functionality of IIACrr was demonstrated by phosphoenolpyruvate-dependent phosphorylation via enzyme I and the histidine-containing phosphoryl carrier protein HPr. Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E. coli IIAGlucose, was mutated. The capacity of IIACrr to operate in sugar transport was shown by complementation of the E. coli glucose-PTS. The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIAGlucose-like protein may be involved in carbon metabolism in S. coelicolor.

Amino Acid Sequence↗

PubMed1998

Identification of peptides inhibiting enzyme I of the bacterial phosphotransferase system using combinatorial cellulose-bound peptide libraries.

The phosphoenolpyruvate(P-pyruvate)-dependent sugar phosphotransferase system (PTS) is a transport and signal-transduction system which is almost ubiquitous in bacteria but does not occur in eucaryotes. It catalyzes the uptake and phosphorylation of carbohydrates and is involved in signal transduction, e.g. catabolite repression, chemotaxis, and allosteric regulation of metabolic enzymes and transporters. EI (Enzyme I of the PTS) is the first and central component of the divergent PTS (P-pyruvate-dependent sugar phosphotransferase system) phosphorylation cascade. Using immobilized combinatorial peptide libraries and phosphorimaging, heptapeptides and octapeptides were identified which selectively inhibit EI in vitro. The IC50 of the best peptides is 30 microM which is close to the K(M) (6 microM) of EI for its natural substrate HPr (histidine containing phosphoryl carrier protein of the PTS). The affinity-selected peptides are better inhibitors than a peptide with the active-site sequence of HPr. The selected peptides contain several basic residues and one aromatic residue which do not occur in the active site of HPr. The large proportion of basic residues most likely reflects charge complementarity to the strongly acidic active-site pocket of EI. Guanidino groups might facilitate by complexation of the phosphoryl group the slow phosphorylation of the peptide.

Amino Acid Sequence↗

PubMed2006

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein.

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg(2+)-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg(2+) and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg(2+) and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189 approximately P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an alpha-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189 approximately P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.

Binding Sites↗

PubMed1998

In vivo and in vitro complementation of the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system by the cloned C-terminal domain.

Enzyme I (EI) is the first protein in the phosphoryl transfer sequence from phosphoenolpyruvate (PEP) to sugar in carbohydrate uptake via the bacterial PEP:glycose phosphotransferase system. The EI monomer/dimer transition may regulate the phosphotransferase system because only the EI dimer is autophosphorylated by PEP. We previously showed that the EI monomer comprises two major domains: (i) a compact, protease-resistant N-terminal domain (EI-N), containing the active site His, and (ii) a flexible, protease-sensitive C-terminal domain (EI-C), which is required for EI dimerization. EI-N interacts with the second protein, HPr, and phospho-HPr, but EI-N neither dimerizes nor is phosphorylated by PEP. We report here the molecular cloning and some properties of EI-C. EI-C is rapidly proteolyzed in vivo. Therefore, two different overexpression vectors encoding fusion proteins were constructed. Fusion Xa contains MalE (the maltose-binding protein), the four-amino acid sequence required by protease factor Xa, followed by EI-C. Fusion G contains His-Tyr between MalE and EI-C and is cleaved by the protease genenase. Homogenous EI-C was isolated from fusion G. [32P]PEP phosphorylated EI-N when supplemented with EI-C, fusion Xa, or fusion G. EI-C may act catalytically. Complementation was also demonstrated in vivo. An Escherichia coli ptsI deletion grew on mannitol as the sole source of carbon after it was transformed with two compatible vectors; one vector encoded EI-N and the other encoded fusion Xa or fusion G. The molecular details underlying important properties of EI can now be studied.

Cloning, Molecular↗