Localization of Phosphoglucose Isomerase in Escherichia coli and Its Relation to the Induction of the Hexose Phosphate Transport System

The localization of phosphoglucose isomerase (PGI) was studied in relation to the induction of hexose phosphate uptake in Escherichia coli. The uptake system is induced only by extracellular glucose-6-phosphate (G6P); there is no induction by intracellular G6P. Fructose-6-phosphate (F6P) is an indirect inducer, and isomerization of F6P to G6P must occur before induction. PGI has been considered to be an internal enzyme; therefore, uptake of F6P by noninduced cells and leakage of the G6P formed would be required for induction. In this study, it was concluded that part of the PGI activity is located in the cell surface because: (i) uninduced, intact cells are able to convert F6P to G6P, whereas the activity of G6P dehydrogenase is not detectable; (ii) when cells are subjected to osmotic shock, about 10% of the PGI activity is found in the shock fluid; and (iii) sorbitol-6-phosphate (S6P) inhibits both PGI activity of whole cells and the induction of hexose phosphate transport system by F6P. S6P was not taken by intact cells. The data indicate that the isomerization of F6P to G6P can take place on the cell surface, and this explains the indirect induction of hexose phosphate transport by F6P.     

Intramolecular deuterium distributions reveal disequilibrium of chloroplast phosphoglucose isomerase

D, deuterium
δ D(NMR), chemical shift axis in a deuterium NMR spectrum
F6P, fructose-6-phosphate
G6P, glucose-6-phosphate
IRMS, isotope ratio mass spectrometry
NMR, nuclear magnetic resonance
PGI, phosphoglucose isomerase

Intramolecular deuterium distributions of the carbon-bound hydrogens of glucose were measured using deuterium nuclear magnetic resonance. Glucose isolated from leaf starch of common bean ( Phaseolus vulgaris cv. Linden) or spinach ( Spinacia oleracea cv. Giant nobel) was depleted in deuterium in the C(2) position, compared with glucose isolated from leaf sucrose or bean endosperm starch. In beans, the depletion of C(2) was independent of the light intensity during growth (150 or 700 μ mol photons s^–1 m^–2). The ratio of glucose-6-phosphate to fructose-6-phosphate ([G6P]/[F6P]) in bean chloroplasts was 0·9 in high light, indicating that the phosphoglucose isomerase reaction was not in equilibrium ([G6P]/[F6P]) ≈ 3). This implies that the kinetic isotope effect of phosphoglucose isomerase depleted deuterium in the C(2) position of G6P. Because the depletion was the same, the chloroplastic ([G6P]/[F6P]) ratio was in disequilibrium irrespective of the light intensity. If the ([G6P]/[F6P]) ratio was in equilibrium, a large chloroplastic pool of G6P would be unavailable for regeneration of ribulose-1,5-bisphospate. We argue that chloroplast phosphoglucose isomerase activity is regulated to avoid this. The deuterium depletion of C(2) explains the known low overall deuterium abundance of leaf starch. This example shows that measurements of intramolecular deuterium distributions can be essential to understand overall deuterium abundances of plant material.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis

The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in ²H₂O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.     

Kinetics of Exogenous Induction of the Hexose-6-Phosphate Transport System of Escherichia coli

The kinetics of the exogenous induction of the hexose-phosphate transport system by glucose-6-phosphate (G6P) was investigated. The induction of this system by extracellular but not intracellular G6P was confirmed. The differential rate of synthesis was linear, a function of the extracellular concentration of G6P and independent of the previous induction history of the culture. Neither maintenance nor autocatalysis, phenomena described in the induction of the lac operon, were observed in the exogenous induction of hexose-phosphate transport. Fructose-6-phosphate, a potent competitive inhibitor of G6P influx, had no effect on the induction of the system by G6P, indicating that the transport of inducer was not involved in the induction process.     

Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts

The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.     

The crystal structure of phosphoglucose isomerase/autocrine motility factor/neuroleukin complexed with its carbohydrate phosphate inhibitors suggests its substrate/receptor recognition

Phosphoglucose isomerase catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. In addition, phosphoglucose isomerase has been shown to have functions equivalent to neuroleukin, autocrine motility factor, and maturation factor. Here we present the crystal structures of phosphoglucose isomerase complexed with 5-phospho-D-arabinonate and N-bromoacetylethanolamine phosphate at 2.5- and 2.3-A resolution, respectively. The inhibitors bind to a region within the domains' interface and interact with a histidine residue (His(306)) from the other subunit. We also demonstrated that the inhibitors not only affect the enzymatic activity of phosphoglucose isomerase, but can also inhibit the autocrine motility factor-induced cell motility of CT-26 mouse colon tumor cells. These results indicate that the substrate and the receptor binding sites of phosphoglucose isomerase and autocrine motility factor are located within close proximity to each other. Based on these two complex structures, together with biological and biochemical results, we propose a possible isomerization mechanism for phosphoglucose isomerase.     

Enzymes related to fructose utilization in Pseudomonas cepacia

Growth of Pseudomonas cepacia on fructose, mannitol, or sorbitol depended on formation of an inducible fructokinase (forming fructose-6-phosphate) and the presence of enzymes of the Entner-Doudoroff pathway. Mutants deficient in any of these enzymes failed to utilize the aforementioned carbohydrates. Fructokinase deficiency did not affect growth of the bacteria on glucose. Fructose was accumulated intracellularly by active transport. Mutants blocked in transport of fructose grew normally on mannitol or sorbitol despite their inability to utilize fructose. Growth on either of these hexitols or on galactitol was accompanied by induction of two hexitol dehydrogenases, one active primarily with mannitol and the other active with sorbitol and galactitol. As expected, a mutant deficient in mannitol dehydrogenase failed to utilize mannitol as a carbon and energy source but grew normally on sorbitol and galactitol. Extracts of bacteria grown on fructose, mannitol, or sorbitol and higher levels of phosphoglucose isomerase than extracts of bacteria grown on alternate carbon sources such as citrate or phthalate. The higher levels were due to appearance of a second phosphoglucose isomerase species not present in cells with the lower activity. The results indicate that the initial steps in fructose utilization by P. cepacia differ from those of most other pseudomonads, which transport fructose by phosphoenolpyruvate-dependent translocation, forming fructose-1-phosphate, and suggest that degradation of fructose, mannitol, and sorbitol occurs primarily via the Entner-Doudoroff pathway.     

Alginic acid synthesis in Pseudomonas aeruginosa mutants defective in carbohydrate metabolism.

Mutant cells of mucoid Pseudomonas aeruginosa isolated from cystic fibrosis patients were examined for their ability to synthesize alginic acid in resting cell suspensions. Unlike the wild-type strain which synthesizes alginic acid from glycerol, fructose, mannitol, glucose, gluconate, glutamate, or succinate, mutants lacking specific enzymes of carbohydrate metabolism are uniquely impaired. A phosphoglucose isomerase mutant did not synthesize the polysaccharide from mannitol, nor did a glucose 6-phosphate dehydrogenase mutant synthesize the polysaccharide from mannitol or glucose. Mutants lacking the Entner-Doudoroff pathway dehydrase or aldolase failed to produce alginate from mannitol, glucose, or gluconate, as a 3-phosphoglycerate kinase or glyceraldehyde 3-phosphate dehydrogenase mutant failed to produce from glutamate or succinate. These results demonstrate the primary role of the Entner-Doudoroff pathway enzymes in the synthesis of alginate from glucose, mannitol, or gluconate and the role of glyceraldehyde 3-phosphate dehydrogenase reaction for the synthesis from gluconeogenic precursors such as glutamate. The virtual absence of any activity of phosphomannose isomerase in cell extracts of several independent mucoid bacteria and the impairment of alginate synthesis from mannitol in mutants lacking phosphoglucose isomerase or glucose 6-phosphate dehydrogenase rule out free mannose 6-phosphate as an intermediate in alginate biosynthesis.     

Crystal structures of mouse autocrine motility factor in complex with carbohydrate phosphate inhibitors provide insight into structure-activity relationship of the inhibitors

Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is identical to the extracellular cytokines neuroleukin and maturation factor and, interestingly, to the intracellular enzyme phosphoglucose isomerase. The cytokine activity of AMF is inhibited by carbohydrate phosphate compounds as they compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven transmembrane helix protein. Here, we report the first comprehensive high-resolution crystal structure analyses of the inhibitor-free form and the eight types of inhibitor (phosphate, erythrose 4-phosphate (E4P), arabinose 5-phosphate (A5P), sorbitol 6-phosphate (S6P), 6-phosphogluconic acid (6PGA), fructose 6-phosphate (F6P), glucose 6-phosphate (G6P), or mannose 6-phosphate (M6P)) complexes of mouse AMF (mAMF). We assayed the inhibitory activities of these inhibitors against the cytokine activity of mAMF. The inhibitory activities of the six-carbon sugars (G6P, F6P, M6P, and 6PGA) were found to be significantly higher than those of the four or five-carbon sugars (E4P or A5P). The inhibitory activities clearly depend on the length of the inhibitor molecules. A structural comparison revealed that a water-mediated hydrogen bond between one end of the inhibitor and a rigid portion of the protein surface in the shorter-chain inhibitor (E4P) complex is replaced by a direct hydrogen bond in the longer-chain inhibitor (6PGA) complex. Thus, to obtain a new compound with higher inhibitory activities against AMF, water molecules at the inhibitor binding site of AMF should be replaced by a functional group of inhibitors in order to introduce direct interactions with the protein surface. The present structure-activity relationship studies will be valuable not only for designing more effective AMF inhibitors but also for studying general protein-inhibitor interactions.     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Cell Lysis of Bacillus subtilis Caused by Intracellular Accumulation of Glucose-1-Phosphate

Mutants deficient in both glucose-6-phosphate dehydrogenase and phosphoglucose isomerase lysed 4 to 5 h after growth in nutrient medium containing glucose, or after prolonged incubation if the medium contained galactose. The lysis could be prevented by the addition of any other rapidly metabolizable carbon source such as fructose, glucosamine, or glycerol. The glucose-induced lysis was also abolished by introduction of a third mutation lacking phospho-glucose mutase activity but not by a third mutation lacking uridine diphosphate-glucose pyrophosphorylase or teichoic acid glucosyl transferase activity. Galactose-induced lysis was prevented only if the additional mutation abolished the uridine diphosphate-glucose pyrophosphorylase activity. The results showed that lysis was caused by the intracellular accumulation of glucose-1-phosphate, which in turn inhibited at least one of the two enzymes that convert glucosamine-6-phosphate to N-acetyl glucosamine-6-phosphate.     

Pathway and regulation of erythritol formation in Leuconostoc oenos.

It was recently observed that Leuconostoc oenos GM, a wine lactic acid bacterium, produced erythritol anaerobically from glucose but not from fructose or ribose and that this production was almost absent in the presence of O2. In this study, the pathway of formation of erythritol from glucose in L. oenos was shown to involve the isomerization of glucose 6-phosphate to fructose 6-phosphate by a phosphoglucose isomerase, the cleavage of fructose 6-phosphate by a phosphoketolase, the reduction of erythrose 4-phosphate by an erythritol 4-phosphate dehydrogenase and, finally, the hydrolysis of erythritol 4-phosphate to erythritol by a phosphatase. Fructose 6-phosphate phosphoketolase was copurified with xylulose 5-phosphate phosphoketolase, and the activity of the latter was competitively inhibited by fructose 6-phosphate, with a Ki of 26 mM, corresponding to the Km of fructose 6-phosphate phosphoketolase (22 mM). These results suggest that the two phosphoketolase activities are borne by a single enzyme. Extracts of L. oenos were also found to contain NAD(P)H oxidase, which must be largely responsible for the reoxidation of NADPH and NADH in cells incubated in the presence of O2. In cells incubated with glucose, the concentrations of glucose 6-phosphate and of fructose 6-phosphate were higher in the absence of O2 than in its presence, explaining the stimulation by anaerobiosis of erythritol production. The increase in the hexose 6-phosphate concentration is presumably the result of a functional inhibition of glucose 6-phosphate dehydrogenase because of a reduction in the availability of NADP.     

Competitive inhibition of Trypanosoma brucei phosphoglucose isomerase by D-arabinose-5-phosphate derivatives

We report four new strong high energy intermediate analog competitive inhibitors of fructose-6-phosphate isomerization catalyzed by purified Trypanosoma brucei phosphoglucose isomerase: D-arabinonhydroxamic acid-5-phosphate, D-arabinonate-5-phosphate, D-arabinonamide-5-phosphate and D-arabinonhydrazide-5-phosphate. For comparison, the inhibitory properties of the corresponding non-phosphorylated analogues D-arabinonhydroxamic acid, D-arabinonate, D-arabinonamide and D-arabinonhydrazide were also evaluated. D-Arabinonhydroxamic acid-5-phosphate appears as the most potent competitive inhibitor ever evaluated on a phosphoglucose isomerase with an inhibition constant value of 50 nM and a Michaelis constant over inhibition constant ratio of about 2000. Our results show that anionic high energy intermediate analogues, and more particularly D-arabinonhydroxamic acid-5-phosphate, display a weak but significant specificity for Trypanosoma brucei phosphoglucose isomerase versus yeast phosphoglucose isomerase, while neutral high energy intermediate analogues are not selective at all. This would indicate the presence of more positively charged residues in the active site for Trypanosoma brucei phosphoglucose isomerase as compared to that of yeast phosphoglucose isomerase.     

Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

On selectivity and sensitivity of synthetic multifunctional pores as enzyme sensors: discrimination between ATP and ADP and comparison with biological pores

This report delineates scope and limitation of the selectivity of synthetic multifunctional pores as enzyme sensors using glycolytic enzymes as example (G. Das, P. Talukdar, and S. Matile, Science, 2002, Vol. 298, pp. 1600-1602). Unproblematic detectability of hexokinase and phosphofructokinase demonstrates that the selectivity of synthetic multifunctional pore (SMPs) sensors suffices to sense ATP in mixed analytes containing ADP, whereas detection of the isomerization of glucose 6-phosphate into fructose 6-phosphate by phosphoglucose isomerase is not possible with confidence. The sensitivity of SMP sensors is sufficient for end-point detection of one picomole poly-L-glutamate hydrolyzed by papain in unoptimized assay format; the sensitivity of melittin as representative biological pore of similar charge and aggregation number to detect the same reaction is more than four orders of magnitude inferior.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.