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PubMed1994

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5'-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2',3'-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5'-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD50 = 0.32 microM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5'-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5'-nucleotidase is not essential for the activation of this nucleoside analog.

5'-Nucleotidase↗

PubMed1984

Phosphoproteins and the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIImannose, IIIfructose, IIIglucitol, and the phosphorylation of enzyme IImannitol and enzyme IIN-acetylglucosamine.

Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [32P]phosphoenolpyruvate or [gamma 32P]ATP have been resolved and detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Simple techniques were found such that distinctions could be made between phosphoproteins containing acid-labile or stable phosphoamino acids and between N1-P-histidine and N3-P-histidine. Phosphoproteins were found to be primarily formed from phosphoenolpyruvate, but because of an efficient phosphoexchange, ATP also led to the formation of the major phosphoenolpyruvate-dependent phosphoproteins. These proteins had the following apparent subunit molecular weights: 65,000, 65,000, 62,000, 48,000, 40,000, 33,000, 25,000, 20,000, 14,000, 13,000, 9,000, 8,000. Major ATP-dependent phosphoproteins were detected with apparent subunit molecular weights of 75,000, 46,000, 30,000, and 15,000. Other minor phosphoproteins were detected. The phosphorylation of the 48,000- and 25,000-MW proteins by phosphoenolpyruvate was independent of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme IIN-acetylglucosamine (membrane bound; MW = 65,000); enzyme IImannitol (membrane bound; MW = 62,000); IIIfructose (soluble; MW = 40,000); IIImannose (partially membrane associated; MW = 33,000); IIIglucose (soluble; MW = 20,000); IIIglucitol (soluble; MW = 13-14,000); HPr (soluble; MW = 9,000); FPr (fructose induced HPr-like protein (soluble; MW = 8,000). HPr and FPr are phosphorylated on the N-1 position of a histidyl residue while all the others appear to be phosphorylated on an N-3 position of a histidyl residue. These studies identify some previously unknown proteins of the PTS and show the phosphorylation of others, which although previously known, had not been shown to be phosphoproteins.

Acetylglucosamine↗

PubMed1993

The role of phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, in the regulation of carbon metabolism in gram-positive bacteria.

HPr of the Gram-positive bacterial phosphotransferase system (PTS) can be phosphorylated by an ATP-dependent protein kinase on a serine residue or by PEP-dependent Enzyme 1 on a histidyl residue. Both phosphorylation events appear to influence the metabolism of non-PTS carbon sources. Catabolite repression of the gluconate (gnt) operon of B. subtilis appears to be regulated by the former phosphorylation event, while glycerol kinase appears to be regulated by the latter phosphorylation reaction. The extent of our understanding of these processes will be described.

Adenosine Triphosphate↗

PubMed1993

Regulatory interactions involving the proteins of the phosphotransferase system in enteric bacteria.

Sugar uptake and cytoplasmic inducer generation as well as cyclic AMP synthesis are regulated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Gram-negative enteric bacteria. In these organisms, the free form of the glucose-specific Enzyme IIA (IIAglc) of the PTS, which can be phosphorylated on a histidyl residue by PEP and the PTS energy coupling proteins, inhibits the activities of non-PTS carbohydrate permeases and catabolic enzymes. By contrast, the phosphorylated form of IIAglc appears to activate adenylate cyclase, the cyclic AMP biosynthetic enzyme. What is known of the molecular details of these regulatory interactions will be summarized, and a novel regulatory mechanism involving the fructose repressor, FruR, which controls the transcription of genes encoding enzymes which catalyze reactions in central pathways of carbon metabolism, will be presented.

ATP-Binding Cassette Transporters↗

PubMed1993

Signal transduction in chemotaxis mediated by the bacterial phosphotransferase system.

Gram-negative bacteria are able to respond chemotactically to carbohydrates which are substrates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The mechanism of signal transduction in PTS-mediated chemotaxis is different from the well-studied mechanism involving methyl-accepting chemotaxis proteins (MCPs). In PTS-mediated chemotaxis, carbohydrate transport is required, and phosphorylation seems to be involved in both excitation and adaptation. In this review the roles of the components of the PTS in chemotactic signal transduction are discussed.

Bacterial Proteins↗

PubMed1993

Three-dimensional structures of the central regulatory proteins of the bacterial phosphotransferase system, HPr and enzyme IIAglc.

Enzyme IIA and HPr are central regulatory proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase (PTS) system. Three-dimensional structures of the glucose enzyme IIA domain (IIAglc) and HPr of Bacillus subtilis and Escherichia coli have been studied by both X-ray crystallography and Nuclear Magnetic Resonance (NMR) Spectroscopy. Phosphorylation of HPr of B. subtilis and IIAglc of E. coli have also been characterized by NMR spectroscopy. In addition, the binding interfaces of B. subtilis HPr and IIAglc have been identified from backbone chemical shift changes. This paper reviews these recent advances in the understanding of the three-dimensional structures of HPr and IIAglc and their interaction with each other.

Bacillus subtilis↗

PubMed1991

Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity.

The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte-macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2-4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV-1) LTR. All of the G418 resistant (neo(r)) U937 cell lines and 10 of 14 neo(r) NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV-1 LTR by phorbol 12-myristate 13-acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV-1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor alpha. The expression of other eukaryotic promoters including the HTLV-1 LTR, SV40 ori sequences, and the human beta-actin gene promoter was similarly affected. However, differentiation of the neo(r) U937 cells into macrophages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV-1 directed gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neo(r) U937 cells. G418 at concentrations too low to affect cell growth (12-150 micrograms/ml) inhibited PMA-induced transactivation responses in wild type cells but did not inhibit PKC-dependent protein phosphorylation in vitro. PKC activities in the wild type and neo(r) cells were similar in absolute amounts and in the cytosol-membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca(2+)-phospholipid-dependent form of PKC disappeared from the neo(r) cells within 30 min after PMA induction. The results suggested that, depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.

Blotting, Northern↗

PubMed1980

Catalytic activities associated with the enzymes II of the bacterial phosphotransferase system.

The phosphotransferase system (PTS) in Escherichia coli is a multifunctional, multicomponent enzyme system. Its primary functions deal with carbon source acquisition, while its secondary functions are concerned with the regulation of bacterial physiology. The primary functions of the system include 1) extracellular detection, 2) unidirectional and exchange transmembrane transport, and 3) phosphoenolpyruvate-dependent and sugar phosphate-dependent phosphorylation of the sugar substrates of the system. The secondary functions include 1) regulation of the activities of adenylate cyclase and various non-PTS permeases and 2) regulation of the induced synthesis of several PTS enzymes. Both the primary and secondary functions appear to be elicited by the binding of a sugar substrate to an Enzyme II complex. One of these integral transmembrane enzymes, the mannitol Enzyme II (IImtl), has been solubilized with detergent, purified to homogeneity, and reconstituted in an artificial membrane system. The molecular weight of this protein, IImtl, is 60,000 daltons. It possesses an extracellular sugar binding site and distinct intracellular combining sites for sugar phosphate and phospho-HPr. An essential sulfhydryl group and an antibody combining site are localized to the cytoplasmic surface of the enzyme, while a dextran combining site is localized to the external surface. Preliminary experiments suggest that the different functions of the Enzyme IImtl can be dissected by genetic and biochemical techniques. These studies emphasize the functional complexity of the PTS and its integral membrane protein constituents.

Biological Transport, Active↗

PubMed2001

Structural basis for chloramphenicol tolerance in Streptomyces venezuelae by chloramphenicol phosphotransferase activity.

Streptomyces venezuelae synthesizes chloramphenicol (Cm), an inhibitor of ribosomal peptidyl transferase activity, thereby inhibiting bacterial growth. The producer escapes autoinhibition by its own secondary metabolite through phosphorylation of Cm by chloramphenicol phosphotransferase (CPT). In addition to active site binding, CPT binds its product 3-phosphoryl-Cm, in an alternate product binding site. To address the mechanisms of Cm tolerance of the producer, the crystal structures of CPT were determined in complex with either the nonchlorinated Cm (2-N-Ac-Cm) at 3.1 A resolution or the antibiotic's immediate precursor, the p-amino analog p-NH(2)-Cm, at 2.9 A resolution. Surprisingly, p-NH(2)-Cm binds CPT in a novel fashion. Additionally, neither 2-N-Ac-Cm nor p-NH(2)-Cm binds to the secondary product binding site.

Bacterial Proteins↗

PubMed1993

Sequence analyses and evolutionary relationships among the energy-coupling proteins Enzyme I and HPr of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158-9169). We now report the sequencing of the ptsI gene of B. subtilis encoding Enzyme I (570 amino acids and 63,076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source-dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate: phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequence HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross-reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N-terminal and the C-terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Enzyme IIBcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy.

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.

Amino Acid Sequence↗

PubMed1994

Novel phosphotransferase system genes revealed by bacterial genome analysis: unique, putative fructose- and glucoside-specific systems.

Analyses of sequences made available through the Escherichia coli genome project in the 87.2-89.2-min and 81.5-84.5-min regions have revealed 2 putative operons encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The first putative operon, designated frv, includes 4 open reading frames (ORFs), ORFf147, ORFf485, ORFf356, and ORFf582, ORFf147 and ORFf485 comprise an Enzyme IIA-Enzyme IIBC pair of the PTS. The sequence similarity of ORFf485 to previously characterized fructose-specific Enzymes IIBC suggests that ORFf485 may be specific for fructose. ORFf147 encodes a protein with comparable degrees of sequence similarity to fructose and mannitol-specific Enzymes IIA as well as homologous proteins implicated in sigma 54-dependent transcriptional regulation. Unique features of this system include a detached IIA protein and the absence of a IIB domain duplication. ORFf356 and ORFf582 are functionally unidentified and nonhomologous to other ORFs in the current protein databases, but ORFf582 contains 2 N-terminal helix-turn-helix motifs, suggestive of a role in frv operon transcriptional regulation. The second putative operon, designated glv, includes 3 ORFs, ORFf455, ORFf161, and ORFf212. We suggest that ORFf455 was incorrectly assigned and should be designated ORFf368. ORFf368 and ORFf161 encode an Enzyme IIC and IIB pair of the PTS showing greatest sequence similarity to Enzymes II specific for sugars of the gluco configuration. ORFf212 encodes a protein with sequence similarity to a phospho-beta-glucosidase and an alpha-galactosidase. No putative transcriptional regulator of the glv operon was found. This operon is the first one encoding a putative PTS permease with detached Enzymes IIB and IIC and lacking an Enzyme IIA. It is suggested that both the frv and glv operons are cryptic in E. coli and that additional genes encoding novel PTS-related proteins will be revealed by bacterial genome sequence analyses.

Amino Acid Sequence↗

PubMed1997

The NMR side-chain assignments and solution structure of enzyme IIBcellobiose of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms. IIBcel consists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand. A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.

Amino Acid Sequence↗

PubMed1998

Tautomeric state and pKa of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy. The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Ndelta1-H tautomer, and its Nepsilon2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.

Bacterial Proteins↗

PubMed1987

Nucleotide phosphotransferase, nucleotide kinase and inorganic pyrophosphatase activities of killer virions of yeast.

The intracellular killer virions of yeast co-purify with an RNA polymerase activity which catalyzes the synthesis of full-length transcripts of the two viral genomic double-stranded RNA segments. This polymerase utilizes ribonucleoside diphosphates or triphosphates as substrates. The virions have other associated nucleotide-metabolizing enzyme activities, including nucleoside diphosphate kinase, adenosine monophosphate kinase, and nucleoside triphosphate phosphotransferase, an activity which catalyzes the exchange of gamma-phosphate from any ribonucleoside triphosphate with any ribonucleoside or deoxyribonucleoside triphosphate. The purified virions also contain an inorganic pyrophosphatase activity. These enzymes may allow the virus to utilize nucleotide pools distinct from those utilized in host cell transcription.

Adenosine Diphosphate↗

PubMed1998

Autophosphorylation of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system requires dimerization.

Enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system undergoes a slow monomer-dimer transition. In vitro autophosphorylation of Enzyme I by PEP was studied at limiting concentrations of the protein. Addition to incubation mixtures containing wild-type Enzyme I of inactive or low-activity mutant forms of Enzyme I resulted in stimulation of autophosphorylation activity. The kinetics of the activation fit well to a model in which the active form of Enzyme I is the dimer. These experiments provide support for the argument that only the dimeric form of Enzyme I can be autophosphorylated.

Dimerization↗

PubMed1994

Crystallization of enzyme IIB of the cellobiose-specific phosphotransferase system of Escherichia coli.

Crystals of enzyme IIB of the cellobiose-specific phosphotransferase system have been obtained from 15% polyethylene glycol 4000 using both streak-seeding and macroseeding techniques at 4 degrees. Crystals were grown with the hanging drop method of vapour diffusion. Addition of 2-propanol and benzamidine/HCl proved essential to obtain single crystals suitable for X-ray analysis. The crystals diffract to 1.8 A resolution and have the monoclinic space group P2(1), with cell dimensions a = 53.6 A, b = 31.7 A, c = 60.0 A and beta = 101.7 degrees. From a self-rotation function it seems likely that there are two molecules in the asymmetric unit related by a non-crystallographic 2-fold axis.

Cellobiose↗

PubMed2001

NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively. Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16.

Binding Sites↗