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PubMed1986

Inhibition of E. coli adenylate cyclase activity by inorganic orthophosphate is dependent on IIIglc of the phosphoenolpyruvate:glycose phosphotransferase system.

The relationship of adenylate cyclase, inorganic orthophosphate and the proteins of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was studied. A strain deleted for the genes for Enzyme I and IIIglc of the PTS was transformed with plasmids expressing either Enzyme I and HPr, IIIglc or all three proteins. The fully reconstituted strain showed a Pi-dependent stimulation of adenylate cyclase activity; in contrast, the strain expressing only IIIglc showed a Pi-dependent inhibition of adenylate cyclase activity.

Adenylyl Cyclase Inhibitors↗

PubMed2003

Why the phosphotransferase system of Escherichia coli escapes diffusion limitation.

We calculated the implications of diffusion for the phosphoenolpyruvate:glucose phosphotransferase system (glucose-PTS) of Escherichia coli in silicon cells of various magnitudes. For a cell of bacterial size, diffusion limitation of glucose influx was negligible. Nevertheless, a significant concentration gradient for one of the enzyme species, nonphosphorylated IIA(Glc), was found. This should have consequences because the phosphorylation state of IIA(Glc) is an important intracellular signal. For mammalian cell sizes we found significant diffusion limitation, as well as strong concentration gradients in many PTS components, and strong effects on glucose and energy signaling. We calculated that the PTS may sense both extracellular glucose and the intracellular free-energy state. We discuss i), that the effects of diffusion on cell function should prevent this highly effective bacterial system from functioning in eukaryotic cells, ii), that in the larger eukaryotic cell any similar chain of mobile group-transfer proteins can neither sustain the same volumetric flux as in bacteria nor transmit a signal far into the cell, and iii), that systems such as these may exhibit spatial differentiation in their sensitivity to different signals.

Cell Size↗

PubMed2003

Metabolic switching in the sugar phosphotransferase system of Escherichia coli.

Bacteria grown in a mixture of multiple sugars will first metabolize a preferred sugar until it is nearly depleted, only then turning to other carbon sources in the medium. This sharp switching of metabolic preference is characteristic of systems that optimize fitness. Here we consider the mechanism by which switching can occur in the Escherichia coli phosphotransferase system (PTS), which regulates the uptake and metabolism of several sugars. Using a model combining the description of fast biochemical processes and slower genetic regulation, we derive metabolic phase diagrams for the uptake of two PTS sugars, indicating regions of distinct sugar preference as a function of external sugar concentrations. We then propose a classification of bacterial phenotypes based on the topology of the metabolic phase diagram, and enumerate the possible topologically distinct phenotypes that can be achieved through mutations of the PTS. This procedure reveals that there is only one nontrivial switching phenotype that is insensitive to large changes in biochemical parameters. This phenotype exhibits diauxic growth, a manifestation of the winner-take-all dynamics enforced by PTS architecture. Winner-take-all behavior is implemented by the induction of sugar-specific operons, combined with competition between sugars for limited phosphoryl flux. We propose that flux-limited competition could be a common mechanism for introducing repressive interactions in cellular networks, and we argue that switching behavior similar to that described here should occur generically in systems that implement such a mechanism.

Adaptation, Physiological↗

PubMed1985

Synthesis of methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D-mannopyranoside and use of this compound for the determination of N-acetylglucosamine-1-phosphotransferase.

Methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D-mannopyranoside was synthesized and identified by 1H-n.m.r. and 13C-n.m.r. data, acid hydrolysis, and elemental analysis. It was utilized for the determination of UDP-N-acetylglucosamine-1-phosphotransferase in an assay procedure that employed methyl alpha-D-mannopyranoside as an acceptor. The assay product was identified and characterized by thin-layer chromatography with the title reference compound. The present technique does not require [32P]UDP-N-acetylglucosamine, but effectively uses commercially available UDP-[14C]GlcNAc.

Acetylglucosamine↗

PubMed1999

Evidence that fructose 1,6-bisphosphate specifically protects the alpha-subunit of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase against proteolytic degradation.

Pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase (PFP) consists of alpha (regulatory) and beta (catalytic) subunits. The alpha-subunit was previously reported to be much more susceptible to tryptic digestion than the beta-subunit. In this study, ligand-induced protection of PFP subunits against proteolysis by subtilisin was investigated in vitro and the data obtained demonstrated that fructose 1,6-bisphosphate (Fru-1,6-P(2)), while exerting negligible effect on the beta-subunit, remarkably protected the alpha-subunit against proteolytic degradation. Western blot analysis revealed a good correlation between the Fru-1,6-P(2) concentration and the degree of corresponding protection on the alpha-subunit against proteolysis. In contrast, none of other examined ligands including fructose 2,6-bisphosphate, fructose 6-phosphate and pyrophosphate had such protection on the alpha-subunit. This finding (1) indicates that the stability of the alpha-subunit can be selectively increased by Fru-1,6-P(2), and (2) suggests that Fru-1,6-P(2) is likely a special effector of the alpha-subunit.

Electrophoresis, Polyacrylamide Gel↗

PubMed1985

The bacterial phosphoenolpyruvate-dependent phosphotransferase system. Isolation of active site peptides by reversed-phase high-performance liquid chromatography and determination of their primary structure.

Using reversed-phase high-performance liquid chromatography (HPLC) it was possible to isolate 32P-labelled active-site regions of various proteins from the bacterial phosphoenolpyruvate-dependent phosphotransferase system. The purified peptides obtained by proteolytic cleavage with Lys-C protease and trypsin were sequenced by the gas phase method. The fragments derived from enzyme I (MW 70 000) of two streptococcal species show 100% homology. The analogous peptide of Staphylococcus aureus Enzyme I differs in the N-terminal region. A labelled peptide from the glucose-specific enzyme III protein of Escherichia coli obtained by cleavage with alkaline protease was isolated and sequenced. It could be fitted into the primary structure of this protein, which was derived from DNA sequence data. The active-site histidine residue of this protein is therefore localized at position 91. The HPLC separation method described is suitable for the isolation of peptides derived from active sites containing labile amino acid derivatives such as phosphohistidines.

Amino Acid Sequence↗

PubMed1997

Dependence of divalent metal ions on phosphotransferase activity of osseous plate alkaline phosphatase.

Kinetic evidence for the role of divalent metal ions in the phosphotransferase activity of polidocanol-solubilized alkaline phosphatase from osseous plate is reported. Ethylenediamine tetreacetate, 1,10-phenanthrolin, and Chelex-100 were used to prepare metal-depleted alkaline phosphatase. Except for Chelex-100, either irreversible inactivation of the enzyme or incomplete removal of metal ions occurred. After Chelex-100 treatment, full hydrolase activity of alkaline phosphatase was recovered upon addition of metal ions. On the other hand, only 20% of transferase activity was restored with 0.1 microM ZnCl2, in the presence of 1.0 M diethanolamine as phosphate acceptor. In the presence of 0.1 mM MgCl2, the recovery of transferase activity increased to 63%. Independently of the phosphate acceptor used, the transferase activity of the metal-depleted alkaline phosphatase was fully restored by 8 microM ZnCl2 plus 5 mM MgCl2. In the presence of diethanolamine as phosphate acceptor, manganese, cobalt, and calcium ions did not stimulate the transferase activity. However, manganese and cobalt-enzyme catalyzed the transfer of phosphate to glycerol and glucose.

Alkaline Phosphatase↗

PubMed1976

The phosphoenolpyruvate : methyl-alpha-D-glucoside phosphotransferase system in Bacillus subtilis Marburg 168 : purification and identification of the phosphocarrier protein (HPr).

The phosphocarrier protein (HPr) of the phosphoenol pyruvate : alpha-methyl-D-glucoside-phosphotransferase system (PTS) has been purified from Bacillus subtilis Marburg 168. The molecular weight is about 8300. HPr contains 1 histidine residue. Phophoenzyme I appears to be an intermediate in the initial phosphoryl transfer from phosphoenolpyruvate (PEP) to HPr. Phospho-HPr is isolated and characterized as a component of the complete system.

Bacillus subtilis↗

PubMed1978

The phosphoenolpyruvate : methyl-alpha-d-glucoside phosphotransferase system in Bacillus subtilis Marburg : kinetic studies of enzyme ii and evidence for a phosphoryl enzyme ii intermediate.

The Enzyme II complex catalyzing the phosphoryl transfer from P-HPr to sugar in the inducible methyl-alpha-D-glucoside : phosphotransferase system in Bacillus subtilis acts according to a ping-pong mechanism, implying a phosphorylated Enzyme II intermediate. This result is supported by the demonstration of a specific transphosphorylation between [14C] alphaMG and glucose-6-phosphate in the presence of an induced Enzyme II preparation.

Bacillus subtilis↗

PubMed2004

The role of histidine residues conserved in the putative ATP-binding region of macrolide 2'-phosphotransferase II.

Macrolide 2'-phosphotransferase (MPH(2')) catalyzes the transfer of the gamma-phosphate of ATP to the 2'-hydroxyl group of macrolide antibiotics. In this study, H198 and H205, conserved in the ATP-binding region motif 1 in the putative amino acid sequence of MPH(2')II, were replaced by Ala to investigate their role. H205 was also subsequently replaced by Asn. H198A and H205N mutant enzymes retained more than 50% of the specific activity of the original enzyme to substrate oleandomycin. On the other hand, the specific activity of the H205A mutant enzyme was reduced to less than 1% of that of the wild enzyme. The results suggested that H205 is crucial for maintaining the catalytic activity of MPH(2')II, and Asn can substitute for His at this position.

Adenosine Triphosphate↗

PubMedDate not supplied

Structural relationship between the genes encoding 3'-aminoglycoside phosphotransferases in Campylobacter and in gram-positive cocci.

Campylobacter coli strain BM2509 resistant to ampicillin, chloramphenicol, erythromycin, kanamycin, spectinomycin, streptomycin and tetracycline was isolated from a patient with hospital-acquired diarrhoea. Resistance to kanamycin has not been thus far described in Campylobacter. Phosphocellulose paper-binding assay indicated that resistance to kanamycin and structurally related antibiotics in strain BM2509 was due to synthesis of a 3'-aminoglycoside phosphotransferase of type III (APH(3')-III), an enzyme so far confined to Gram-positive cocci. The kanamycin and tetracycline resistances were transferable en bloc by conjugation to C. fetus but not to Escherichia coli. Analysis by agarose gel electrophoresis of crude bacterial lysates revealed the presence, in BM2509 and in the transconjugants, of a plasmid, pIP1433, with a size of 47.2 kilobases (Kb). Strain BM2509 also harboured a 4.5-Kb cryptic plasmid. DNA annealing studies indicated a close structural relationship between the kanamycin resistance gene of C. coli BM2509 and that representative of this type of resistance determinant in Gram-positive cocci. These results indicate that emergence of resistance to kanamycin in Campylobacter is due to acquisition in vivo of a gene or a plasmid from Gram-positive bacteria.

Aminoglycosides↗

PubMed2002

Dependency of sugar transport and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system on membranous phosphatidyl glycerol in Escherichia coli: studies with a pgsA mutant lacking phosphatidyl glycerophosphate synthase.

It has been reported that phosphatidyl glycerol (PG) is specifically required for the in vitro activities of the hexose-phosphorylating Enzymes II of the Escherichia coli phosphoenolpyruvate-dependent sugar transporting phosphotransferase system (PTS). We have examined this possibility by measuring the properties of a null pgsA mutant that lacks detectable PG. The mutant showed lower in vitro phosphorylation activities towards several sugars when both PEP-dependent and sugar-phosphate-dependent [14C]sugar phosphorylation reactions were measured. The order of dependency on PG for the different enzymes II was: IIMannose > IIGlucose > IIFructose > IIMannitol. Nonsedimentable (40000 rpm for 2 h) Enzymes II exhibited a greater dependency on PG than pelletable Enzymes II. Western blot analyses showed that the glucose Enzyme II is present in normal amounts. Transport and fermentation measurements revealed diminished activities for all Enzymes II. Thermal stability of all of these enzymes except the mannitol-specific Enzyme II was significantly decreased by the pgsA mutation, and sensitivity to detergent treatments was enhanced. Sugar transport proved to be the most sensitive indicator of proper Enzyme II-phospholipid association. Our results show that PG stimulates but is not required for Enzyme II function in E. coli.

Biological Transport↗

PubMed1998

Transport of glucose by a phosphoenolpyruvate:mannose phosphotransferase system in Pasteurella multocida.

Pasteurella multocida was examined for glucose and mannose transport. P. multocida was shown to possess a phosphoenolpyruvate (PEP):mannose phosphotransferase system (PTS) that transports glucose as well as mannose and was functionally similar to the Escherichia coli mannose PTS. Phosphorylated proteins with molecular masses similar to those of E. coli mannose PTS proteins were visualized when incubated with 32P-PEP. The presence of an enzyme IIAGlc which could play an important role in regulation, as described in other Gram-negative bacteria, was detected. The enzymes of the pentose-phosphate pathway were present in P. multocida growth on glucose. The activity of 6-phosphofructokinase (the key enzyme of the Embden-Meyerhof pathway (EMP)), was very low in cell extracts, suggesting that EMP is not the major pathway for glucose catabolism.

Biological Transport↗

PubMed2000

High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin.

This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to > 70% homogeneity. The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed.

Amikacin↗

PubMed1996

The first step in sugar transport: crystal structure of the amino terminal domain of enzyme I of the E. coli PEP: sugar phosphotransferase system and a model of the phosphotransfer complex with HPr.

BACKGROUND: The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) transports exogenous hexose sugars through the membrane and tightly couples transport with phosphoryl transfer from PEP to the sugar via several phosphoprotein intermediates. The phosphate group is first transferred to enzyme I, second to the histidine-containing phosphocarrier protein HPr, and then to one of a number of sugar-specific enzymes II. The structures of several HPrs and enzymes IIA are known. Here we report the structure of the N-terminal half of enzyme I from Escherichia coli (EIN). RESULTS: The crystal structure of EIN (MW approximately 30 kDa) has been determined and refined at 2.5 A resolution. It has two distinct structural subdomains; one contains four alpha helices arranged as two hairpins in a claw-like conformation. The other consists of a beta sandwich containing a three-stranded antiparallel beta sheet and a four-stranded parallel beta sheet, together with three short alpha helices. Plausible models of complexes between EIN and HPr can be made without assuming major structural changes in either protein. CONCLUSIONS: The alpha/beta subdomain of EIN is topologically similar to the phosphohistidine domain of the enzyme pyruvate phosphate dikinase, which is phosphorylated by PEP on a histidyl residue but does not interact with HPr. It is therefore likely that features of this subdomain are important in the autophosphorylation of enzyme I. The helical subdomain of EIN is not found in pyruvate phosphate dikinase; this subdomain is therefore more likely to be involved in phosphoryl transfer to HPr.

Amino Acid Sequence↗

PubMed1996

Mechanism of aminoglycoside 3'-phosphotransferase type IIIa: His188 is not a phosphate-accepting residue.

BACKGROUND: The enzyme aminoglycoside 3'-phosphotransferase Type IIIa (APH(3')-IIIa), confers resistance to many aminoglycoside antibiotics by regiospecific phosphorylation of their hydroxyl groups. The chemical mechanism of phosphoryl transfer is unknown. Based on sequence homology, it has been suggested that a conserved His residue, His188, could be phosphorylated by ATP, and this phospho-His would transfer the phosphate to the incoming aminoglycoside. We have used chemical modification, site-directed mutagenesis and positional isotope exchange methods to probe the mechanism of phosphoryl transfer by APH(3')-IIIa. RESULTS: Chemical modification by diethylpyrocarbonate implicated His in aminoglycoside phosphorylation by APH(3')-IIIa. We prepared His --> Ala mutants of all four His residues in APH(3')-IIIa and found minimal effects of the mutations on the steady-state phosphorylation of several aminoglycosides. One of these mutants, His188Ala, was largely insoluble when compared to the wild-type enzyme. Positional isotope exchange experiments using gamma-[18O]-ATP did not support a double-displacement mechanism. CONCLUSIONS: His residues are not required for aminoglycoside phosphorylation by APH(3')-IIIa. The conserved His 188 is thus not a phosphate accepting residue but does seem to be important for proper enzyme folding. Positional isotope exchange experiments are consistent with direct attack of the aminoglycoside hydroxyl group on the gamma-phosphate of ATP.

Adenosine Triphosphate↗

PubMed1998

Formation of a novel four-helix bundle and molecular recognition sites by dimerization of a response regulator phosphotransferase.

A basis for understanding specificity of molecular recognition between phosphorelay proteins has been deduced from the 2.6 A structure of the Spo0B phosphotransferase of the phosphorelay regulating sporulation initiation. Spo0B consists of two domains: an N-terminal alpha-helical hairpin domain and a C-terminal alpha/beta domain. Two subunits of Spo0B dimerize by a parallel association of helical hairpins to form a novel four-helix bundle from which the active histidine protrudes. Docking studies show that both the monomers interact with a Spo0F molecule at the region surrounding the active site aspartate to position it for phosphotransfer. It is apparent that different surfaces of response regulators may be involved in recognition of the protein partners to which they are paired.

Amino Acid Sequence↗

PubMed2001

Variations in the cytomegalovirus DNA polymerase and phosphotransferase genes in relation to foscarnet and ganciclovir sensitivity.

BACKGROUND: Identification of human cytomegalovirus (CMV) genome variation is important for understanding mutations associated with drug resistance. OBJECTIVES: To investigate the CMV resistance to foscarnet (PFA) and ganciclovir (GCV) in patients treated with antiviral drugs and to identify the DNA polymerase (UL54) and phosphotransferase (UL97) gene mutations inducing resistance. STUDY DESIGN: Antiviral susceptibility of CMV strains/isolates for PFA and GCV was compared by plaque reduction assay and in situ ELISA. UL54 and UL97 gene mutations were identified by sequencing. Growth phenotype of two CMV recombinants with mutations in UL54 was studied. RESULTS: Six of seven GCV resistant strains had alterations within the UL97. Five of them also had alterations in the UL54 (F412C, L802M or K513E), previously shown to induce GCV resistance. Seven isolates had no or reduced susceptibility to PFA, which had alterations in the UL54 (D588N, E756K, V781I or L802M). By in vitro mutagenesis, it was shown that a mutation at codon D588N of UL54 conferred 9-fold reduced susceptibility to PFA, while a mutation at codon V781I induced 4-fold reduced susceptibility to PFA and GCV. Both recombinants showed the same kinetics of protein expression (IE, E, and L antigen) and virus yields as the CMV Towne strain. CONCLUSIONS: The recombinants containing alterations within the UL54 (D588N and V781I) showed a reduced susceptibility to antiviral drugs but no change in the replication rate compared to the CMV Towne.

Antiviral Agents↗