Fructose transport in Bacillus subtilis.

The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP-dependent fructokinase (fruC), the fructose 1-phosphate kinase (fruB) the phosphofructokinase (pfk), the enzyme I of the phosphoenolpyruvate phosphotransferase system (the thermosensitive mutation ptsI1), and a transport activity (fruA). Combinations of these mutations indicated that the transport of fructose in Bacillus subtilis is tightly coupled to its phosphorylation either in fructose 1-phosphate, identified in vivo and in vitro or in fructose 6-phosphate identified by indirect lines of evidence. These steps of fructose metabolism were shown to depend on the activity of the enzyme I of the phosphoenolpyruvate phosphotransferase systems. The fruA mutations affect the transport of fructose when the bacteria are submitted to catabolite repression. The mutations were localized on the chromosome of Bacillus subtilis in a cluster including the fruB gene. When grown in a medium supplemented by a mixture of potassium glutamate and succinate the fruA mutants are able to carry on the two vectorial metabolisms generating fructose 6-phosphate as well as fructose 1-phosphate. A negative search of strictly negative transport mutants in fruA strains indicated that more than two structural genes are involved in the transport of fructose.     

Fructose catabolism in Xanthomonas campestris pv. campestris. Sequence of the PTS operon, characterization of the fructose-specific enzymes.

In Xanthomonas campestris pv. campestris, fructose is transported and phosphorylated into fructose 1-phosphate through a phosphoenolpyruvate-dependent phosphotransferase system. The nucleotide sequence of the fruA gene encoding the phosphotransferase system permease specific of fructose (EIIFru) was determined. The fructose 1-phosphate produced by the phosphotransferase system is phosphorylated into fructose 1,6-bisphosphate by a 1-phosphofructokinase. This enzyme was characterized and the corresponding gene (fruK) was sequenced. Sequence comparisons revealed that FruK is a member of a new family of ATP-binding proteins composed of sugar (or sugar-phosphate) kinases. In phosphotransferase system-deficient strains, fructose can still be transported by an unidentified permease. The intracellular fructose is then phosphorylated by a multimeric fructokinase of 135 kDa specific for fructose and inhibited by fructose, fructose 1,6-bisphosphate, and mannose. Several other enzymes of fructose metabolism were assayed and a potential pathway for fructose catabolism is presented.     

Characterization of two operons that encode components of fructose-specific enzyme II of the sugar:phosphotransferase system of Streptococcus mutans

Three genes, designated as fruC, fruD and fruI, were predicted to encode polypeptides homologous to fructose-specific enzyme II (II(Fru)) of the phosphoenolpyruvate-dependent sugar:phosphotransferase system, and were cloned from Streptococcus mutans, the primary etiological agent of human dental caries. The fruC and fruD genes encoded domains BC and domain A of II(Fru), respectively. The fruI gene encoded IICBA(Fru). Northern hybridization and slot blot analysis showed that expression of fruI was inducible by sucrose and fructose, while fruCD were expressed constitutively and at much lower levels. Inactivation of either fruI or fruCD alone, or of both fruCD and fruI, had no major impact on growth on fructose at a concentration of 0.5% (w/v). However, when the strains were grown with 0.2% fructose as the sole carbohydrate source, a significant decrease in the growth rate was seen with the fruCD/fruI double mutants. Assays of sugar:phosphotransferase activity showed that the fruCD/fruI double mutants had roughly 30% of the capacity of the wild-type strain to transport fructose via the phosphoenolpyruvate-dependent sugar:phosphotransferase system. Xylitol toxicity assays indicated that the inducible fructose permease was responsible for xylitol transport.     

Pyrophosphate: fructose 6-phosphate phosphotransferase in germinating castor bean seedlings

The distribution of enzymes interconverting fructose 6-phosphate and fructose 1,6-bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PP_i:fructose 6-phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6-bisphosphatase. PP_i:fructose 6-phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.     

Regulation of fructose uptake and catabolism by succinate in Azospirillum brasilense.

Fructose uptake and catabolism in Azospirillum brasilense is dependent on three fructose-inducible enzymes (fru-enzymes): (i) enzyme I and (ii) enzyme II of the phosphoenolpyruvate:fructose phosphotransferase system and (iii) 1-phosphofructokinase. In minimal medium containing 3.7 mM succinate and 22 mM fructose as sources of carbon, growth of A. brasilense was diauxic, succinate being utilized in the first phase of growth and fructose in the second phase with a lag period between the two growth phases. None of the fru-enzymes could be detected in cells grown with succinate as the sole source of carbon, but they were detectable toward the end of the first phase of diauxie. All the fru-enzymes were coinduced by fructose and coordinately repressed by succinate. Studies on the effect of succinate on differential rates of syntheses of the fru-enzymes revealed that their induced syntheses in fructose minimal medium were subject to transient as well as permanent (catabolite) repression by succinate. Succinate also caused a similar pattern of transient and permanent repression of the fructose transport system in A. brasilense. However, no inducer (fructose) exclusionlike effect was observed as there was no inhibition of fructose uptake in the presence of succinate with fructose-grown cells even when they were fully induced for succinate uptake activity.     

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.     

Inducible phosphoenolpyruvate-dependent hexose phosphotransferase activities in Escherichia coli

1. A method is described for measuring the rate of phosphoenolpyruvate-dependent phosphotransferase activity for a variety of hexoses in toluene-treated suspensions of Escherichia coli. 2. The specific activities of the phosphotransferases that catalyse the phosphorylation of hexoses are greatly affected by the carbon source for growth. 3. In all strains of E. coli tested, fructose phosphotransferase activity is induced by growth on fructose. 4. Strains of E. coli differ greatly in the rate at which they phosphorylate glucose, but all strains possess at least a low glucose phosphotransferase activity under any tested condition of growth. Glucose phosphotransferase activity is further induced by growth on glucose; this does not occur in a mutant that lacks the ability to take up methyl alpha-d-[(14)C]glucopyranoside and hence grows poorly on glucose. 5. When growing on fructose, two strains of E. coli synthesize the inducible glucose phosphotransferase system gratuitously, and to specific activities higher than observed during growth on glucose. A phosphotransferase catalysing the phosphorylation of mannose is similarly induced.     

菠萝叶片依赖焦磷酸的磷酸果糖激酶的纯化及其特性

经硫酸铵分部,DEAE—纤维素、羟基磷灰石、Sephadex G—200及磷酸纤维素柱层析,从菠萝叶片分离得到电泳均一的依赖焦磷酸的磷酸果糖激酶(PFP)。SDS电泳图谱表明有一条分子量为62kD的主带和一条57 kD的弱带。Fru—2,6—P_2对酶的正反应活性有促进作用。动力学研究表明,Fru—2,6—P_2增加V_(max)及酶对底物Fru—6—P和Mg~(2+)的亲和性。     

Catabolism of Fructose and Mannitol in Clostridium thermocellum: Presence of Phosphoenolpyruvate: Fructose Phosphotransferase, Fructose l-Phosphate Kinase, Phosphoenolpyruvate: Mannitol Phosphotransferase, and Mannitol l-Phosphate Dehydrogenase in Cell Extracts

Fructose and mannitol are fermented by Clostridium thermocellum in a medium containing salts and 0.5% yeast extract. The initial reaction in the catabolism of fructose was found to be the formation of fructose l-phosphate by phosphoenolpyruvate (PEP):fructose phosphotransferase which resembles the Kundig-Roseman phosphotransferase system. The phosphorylation of fructose l-phosphate to form fructose-1, 6-diphosphate is catalyzed by fructose l-phosphate kinase. Fructose-1, 6-diphosphate can be further metabolized by the Embden-Meyerhof pathway. The formation of both PEP:fructose phosphotransferase and fructose l-phosphate kinase is induced by growth in fructose medium. Mannitol catabolism was found to proceed by the phosphorylation of mannitol by PEP:mannitol phosphotransferase to form mannitol l-phosphate. Mannitol l-phosphate is converted to fructose 6-phosphate by a nicotinamide adenine dinucleotide-specific mannitol l-phosphate dehydrogenase. The fructose 6-phosphate formed in the reaction can enter the glycolytic scheme. The formation of both PEP:mannitol phosphotransferase and mannitol l-phosphate dehydrogenase is induced by growth in mannitol medium. Evidence is presented for the induction by mannitol of PEP:mannitol phosphotransferase and mannitol l-phosphate dehydrogenase in suspensions of fructose-grown cells.     

Separation of four components of the phosphoenolpyruvate: glucose phosphotransferase system in Vibrio parahaemolyticus.

Four classes of Vibrio parahaemolyticus mutants defective in the phosphoenolpyruvate: glucose phosphotransferase system (PTS) are described. They were phenotypically different, and were defective in different PTS components. The components designated tentatively as II, I, III, and H were separated by gel filtration of a wild-type extract. Component II, which was specific for glucose and found in the particulate fraction, is probably membrane-bound, glucose-specific enzyme II. Both components I and H were soluble proteins, and the latter was relatively heat-stable. Component I was required for phosphorylation of glucose, trehalose, fructose, mannose, and mannitol. Component H was also required for phosphorylating all the above sugars except fructose. These and some additional findings strongly suggest that components I and H correspond to enzyme I and HPr, respectively. Component III, a soluble heat-stable protein, may be equivalent to the sugar-specific factor III found in other organisms, although it seems to participate in phosphorylating two sugars, glucose and trehalose. There were evidences that mutants defective in components I and III were deficient in cyclic adenosine 3',5'-monophosphate synthesis under certain conditions.     

Existence of two alternative pathways for fructose and sorbitol metabolism in Bacillus subtilis Marburg.

Strains of Bacillus subtilis mutated for fructose phosphotransferase system (fruA), fructose-1-phosphate kinase (fruB), fructokinase (frucC) have been tested for their catabolism of sorbitol and fructose. It is shown that the previously known pathways of sorbitol and fructose degradation in B. subtilis, e.g.: (see article) may metabolize intracellular fructose produced either by sorbitol oxidation or by fructose-1-phosphate dephosphorylation. The intracellular fructore degradation via fructose-1-phosphate kinase has been found to require the fructose phosphotransferase system which ensures a vectorial transport of fructose.     

Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.     

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.     

Xylitol and D-arabitol toxicities due to derepressed fructose, galactitol, and sorbitol phosphotransferases of Escherichia coli.

d-Arabitol was observed to be toxic to many laboratory strains of Escherichia coli K-12, and xylitol was found to be toxic to an existing E. coli C mutant strain. Fructose-specific components of the phosphoenolpyruvate:sugar phosphotransferase system are required for xylitol toxicity. Selection for xylitol resistance results in Fru(-) strains blocked in fructose phosphotransferase. Introduction of the ptsF or ptsI mutation into a xylitol-sensitive strain eliminates sensitivity. [(14)C]fructose uptake experiments imply that the mutation to xylitol sensitivity, which is co-transducible with ara and leu, results in derepression of normally inducible fructose phosphotransferase. Wild-type strains also become xylitol sensitive if induced by (and then removed from) fructose. Xylitol toxicity is prevented by fructose in both wild-type and mutant strains. Circumstances causing xylitol, a new food additive, to become toxic to an otherwise insensitive wild-type organism have not been reported previously. The d-arabitol-sensitive laboratory strains are galactitol (dulcitol) utilizers, although most other strains are not. Selection for d-arabitol resistance results in Gat(-) strains blocked in a constitutive galactitol-specific component of the phosphotransferase system. A mutation causing d-arabitol sensitivity occurred many years ago in AB284, the parent of AB311, AB312, AB313, and many other strains. d-Arabitol sensitivity also occurs in sorbitol-constitutive strains and is shown, like the previous two instances of pentitol toxicities, to result from a constitutive phosphotransferase, which is blocked in mutants selected for resistance.     

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida characterization of a new mannitol-producing metabolic pathway.

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell-free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden-Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden-Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by alpha glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.     

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: Effect on PPi:fructose 6-phosphate phosphotransferase

Fructose 6-phosphate from several commercial sources was shown to be contaminated with fructose 2,6-bisphosphate. This contaminant was identified by its activation of PP_i:fructose 6-phosphate phosphotransferase, extreme acid lability and behaviour on ion-exchange chromatography. The apparent kinetic properties of PP_i:fructose 6-phosphate phosphotransferase from castor bean endosperm were considerably altered when contaminated fructose 6-phosphate was used as a substrate. Varying levels of fructose 2,6-bisphosphate in the substrate may account for differences that have been observed in the properties of the above enzyme from several plant sources.     

Adenosine triphosphate:creatine phosphotransferase of rat cerebral cortex: evidence for a bimodal subcellular localization

—(1) ATP: creatine phosphotransferase of rat cerebral cortex is soluble to the extent of 57 per cent when the tissue is homogenized in 0.25 M-sucrose and 80 per cent when distilled water is used for tissue dispersion. Among particulate fractions, the crude mitochondria] fraction contains the highest percentage of enzyme activity. (2) Discontinuous sucrose gradient fractionation of the crude mitochondrial fraction yields about 55 per cent of the particulate activity in the nerve ending fractions and 24 per cent in the mitochondrial pellet. (3) Rupturing of the nerve-ending particles by a moderate osmotic shock designed to spare the mitochondria results in about 60 per cent of the ATP:creatine phosphotransferase becoming soluble, the remainder preserving the association with heavy particles, presumably mitochondria. (4) Subfractionation of the microsomal fraction on a discontinuous sucrose gradient reveals that this particulate component of the enzyme is an adsorption artifact. (5) The overall evidence points to at least two distinct subcellular localizations of the enzyme in rat brain cortex, a major soluble component and a particulate component. It has not been unequivocally shown whether the latter, in turn, reflects the presence of a single, mitochondrial component or whether the soluble matrix of the nerve ending particles represents a third locale for the enzyme.     

Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon

The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.     

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.