Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Correlation between the rate of protein and nucleic acid synthesis and the intensity of various glucose metabolic pathways in rat liver cells

A sharp and strong suppression of protein synthesis by cycloheximide in liver cells of starving rats is paralleled with activation of RNA synthesis and glucose-6-phosphate dehydrogenase production. Subsequent reconstitution and stimulation of protein synthesis (6-12 hrs after cycloheximide injection) result in activation of hexokinase. Upon stimulation of DNA synthesis (48-60 hrs after cycloheximide injection) the activity of both enzymes is very low. Since glucose-6-phosphate dehydrogenase appears to be the limiting step of glucose decay via the pentose phosphate pathway, and hexokinase is the limiting step of glycolysis, it was assumed that RNA synthesis predominantly occurs via the pentose phosphate pathway, while that of proteins via glycolysis.     

The impact of HPV infection on human glycogen and lipid metabolism – a review

Reinterpretation of the Wartburg effect leads to understanding aerobic glycolysis as a process that provides considerable amount of molecular precursors for the production of lipids, nucleotides and amino acids that are necessary for continuous growth and rapid proliferation characteristic for cancer cells.Human papilloma virus (HPV) is a number one cause of cervical carcinoma with 99% of the cervical cancer patients being HPV positive. This tight link between HPV and cancer raises the question if and how HPV impact cells to reprogram their metabolism? Focusing on early phase proteins E1, E2, E5, E6 and E7 we demonstrate that HPV activates plethora of metabolic pathways and directly influences enzymes of the glycolysis pathway to promote the Warburg effect by increasing glucose uptake, activating glycolysis and pentose phosphate pathway, increasing the level of lactate dehydrogenase A synthesis and inhibiting β-oxidation. Our considerations lead to conclusion that HPV is substantially involved in metabolic cell reprogramming toward neoplastic phenotype and its metabolic activity is the fundamental reason of its oncogenicity.     

The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells

1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The K(m) values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and P(i) found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [(14)C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with phenazine methosulphate. 8. The pentose phosphate formed by way of the direct oxidative route and estimated from the (14)CO(2) yields represented 20% of the total accumulated pentose phosphate, the other 80% being formed by the non-oxidative reactions of the pentose phosphate pathway. 9. The pentose phosphate pathway appears to function as two separate pathways, both operating towards pentose phosphate formation. Control of the two pathways is discussed.     

Light and dark assimilation of nitrate in plants

Abstract. Heterotrophic assimilation of nitrate in roots and leaves in darkness is closely linked with the oxidative pentose phosphate pathway. The supply of glucose-6-phosphate to roots and chloroplasts in leaves in darkness is essential for assimilation of nitrite into amino acids. When green leaves are exposed to light, the key enzyme, glucoses-phosphate dehydrogenase, is inhibited by reduction with thioredoxin. Hence the dark nitrate assimilatory pathway is inhibited under photoautotrophic conditions and replaced by regulatory reactions functioning in light. On account of direct photo-synthetic reduction of nitrite in chloroplasts and availability of excess NADH for nitrate reduclase, the rate of nitrate assimilation is extremely rapid in light. Under dark anaerobic conditions also nitrate is equally rapidly reduced to nitrite on account of abolition of competition for NADH between nitrate reductase and mitochondrial oxidation.     

Intracellular interactions between the pathways of carbohydrate oxidation and nitrate assimilation in plant roots

Using density gradient techniques we have shown that in addition to a location within the cytoplasm all the enzymes of the pentose phosphate pathway are also present within the plastids of apical cells of pea roots. The data are discussed in relation to the hypothesis that the pentose phosphate pathway provides the NADPH for nitrite assimilation, the enzymes of which pathway have previously been shown to be located within the plastids of apical cells of pea roots.     

Coordination of Chloroplastic Metabolism in N-Limited Chlamydomonas reinhardtii by Redox Modulation (II. Redox Modulation Activates the Oxidative Pentose Phosphate Pathway during Photosynthetic Nitrate Assimilation)

The onset of photosynthetic NO3- assimilation in N-limited Chlamydomonas reinhardtii increased the initial extractable activity of the glucose-6-phosphate dehydrogenase (G6PDH), the key regulatory step of the oxidative pentose phosphate pathway. The total activated enzyme activity did not change upon NO3- resupply. The higher activity, therefore, represents activation of existing enzyme. No activation occurred during NH4+ assimilation. Incubation of extracts with DTT reversed the NO3- stimulation of G6PDH activity, indicating that the activation involved redox modulation of G6PDH. Phosphoribulosekinase, an enzyme activated by thioredoxin reduction, was inhibited at the onset of NO3- assimilation. A 2-fold stimulation of O2 evolution and a 70% decrease in the rate of photosynthetic CO2 assimilation accompanied the enzyme activity changes. There was an immediate drop in the NADPH and an increase in NADP upon addition of NO3-, whereas NH4+ caused only minor fluctuations in these pools. The response of C. reinhardtii to NO3- indicates that the oxidative pentose phosphate pathway was activated to oxidize carbon upon the onset of NO3- assimilation, whereas reduction of carbon via the reductive pentose phosphate pathway was inhibited. This demonstrates a possible role for the Fd-thioredoxin system in coordinating enzyme activity in response to the metabolic demands for reducing power and carbon during NO3- assimilation.     

Analyzing the effect of decreasing cytosolic triosephosphate isomerase on Solanum tuberosum hairy root cells using a kinetic–metabolic model

A kinetic–metabolic model of Solanum tuberosum hairy roots is presented in the interest of understanding the effect on the plant cell metabolism of a 90% decrease in cytosolic triosephosphate isomerase (cTPI, EC 5.3.1.1) expression by antisense RNA. The model considers major metabolic pathways including glycolysis, pentose phosphate pathway, and TCA cycle, as well as anabolic reactions leading to lipids, nucleic acids, amino acids, and structural hexoses synthesis. Measurements were taken from shake flask cultures for six extracellular nutrients (sucrose, fructose, glucose, ammonia, nitrate, and inorganic phosphate) and 15 intracellular compounds including sugar phosphates (G6P, F6P, R5P, E4P) and organic acids (PYR, aKG, SUCC, FUM, MAL) and the six nutrients. From model simulations and experimental data it can be noted that plant cell metabolism redistributes metabolic fluxes to compensate for the cTPI decrease, leading to modifications in metabolites levels. Antisense roots showed increased exchanges between the pentose phosphate pathway and the glycolysis, an increased oxygen uptake and growth rate. Biotechnol. Bioeng. 2013; 110: 924–935. © 2012 Wiley Periodicals, Inc.     

Simultaneous operation of three catabolic pathways in the metabolism of glucose by Thiobacillus A2

Enzymes essential to the operation of the Embden-Meyerhof glycolytic pathway, the Entner-Doudoroff pathway and oxidative pentose phosphate pathway were present in Thiobacillus A2 grown on glucose and other sugars. Radiorespirometry under various conditions with Thiobacillus A2 oxidising glucose specifically labelled with ¹⁴C in carbon atoms 1, 2, 3, 3+4, 6 or universally labelled demonstrated the simultaneous operation of the Embden-Meyerhof (48%), Entner-Doudoroff (28%), and pentose phosphate (24%) pathways in release of carbon dioxide from glucose. Growth on succinate, or autotrophically on formate or thiosulphate resulted in repression of most enzymes of the pathways, but high aldolase levels were retained indicating its role in gluconeogenesis and the Calvin cycle. Different fructose diphosphatase activities were found in succinate- and thiosulphate-grown organisms. The results indicate that all three major catabolic pathways for glucose function in Thiobacillus A2 grown on sugars. Thiobacillus acidophilus showed a different radiorespirometric pattern and apparently used the Entner-Doudoroff (64.5%) and pentose phosphate (35.5%) pathways, but showed unusually high release of carbon atom 6, as was also found for T. ferrooxidans.Abbreviations EM Embden-Meyerhof - ED Entner-Doudoroff - EDTA ethylene diamine tetra-acetic acid, disodium salt - FDP fructose 1,6-diphosphate - KDPG 2-keto-3-deoxy-6-phosphogluconate - 6-PG 6-phosphogluconate - Pa Pascal (10⁵ Pa=1 bar) - PP pentose phosphate - POPOP 1,4-di[2-(5-phenyloxazolyl)] benzene - PPO 2,5-diphenyloxazole     

Mutants of the pentose phosphate pathway in Aspergillus nidulans

Mutants of the pentose phosphate pathway have been isolated in Aspergillus nidulans. These fail to grow on a variety of carbohydrates that are catabolized through the pentose phosphate pathway. They also grow poorly on nitrate and nitrite as sole nitrogen sources. The pentose phosphate pathway mutations have been assigned to two unlinked genes. Mutants with lesions in the pppB locus have reduced activities of four enzymes of the pentose phosphate pathway, of glucose-phosphate isomerase, and of mannitol-1-phosphate dehydrogenase. pppA(-) mutants have elevated activities of these same enzymes except for transaldolase, for which they have much reduced activity. Both classes of mutants accumulate sedoheptulose-7-phosphate to an extent that is increased considerably when nitrate is present in the medium. Nitrate does not cause an increase in accumulation of sedoheptulose-7-phosphate in double mutants which, in addition to the pppA1 mutation, carry a mutation that leads to the lack of nitrate reductase activity. These last results suggest that nitrate stimulates the flux through the oxidative pentose phosphate pathway, but that this stimulation depends upon the metabolism of nitrate.     

Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation

A 14.5 kDa protein with antigenic components in common with pea leaf ferredoxin was detected on transblots of the soluble proteins of pea root plastids. The amount of this protein was found to increase during the induction of nitrate assimilation in pea roots, reaching a maximal level at 8–12 h. Concurrent with this, a fourfold increase in NADPH-dependent ferredoxin-NADP⁺ oxidoreductase (FNR) activity was observed corresponding to an increase in the amount of this protein detected immunologically on transblots using a leaf FNR antibody. These changes were not observed in plastids from roots of plants grown on ammonia or depleted of nitrogen. It is suggested that in addition to the already well reported induction by nitrate of nitrate reductase and nitrite reductase, there is a co-induction of a plastid located ferredoxin and FNR. Both these proteins are necessary for the transfer of reductant generated by the oxidative pentose phosphate pathway to nitrite reductase.     

A comparative developmental pattern of enzymes of carbon metabolism and pentose phosphate pathway in mungbean and lentil nodules

The activities of enzymes of pentose phosphate pathway (PPP) viz. glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carbon metabolism viz. phosphoenol pyruvate carboxylase, NADP- isocitrate dehydrogenase and NADP-malic enzyme were measured in the plant and bacteroid fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules along with the developing roots for comparison. The enzymes of pentose phosphate pathway in legume cytosol had higher activities at a stage of maximum nitrogenase activity and higher sucrose metabolism. However, bacteroids had only limited capacity for this pathway. The specific activities of these enzymes were greater in ureide than in amide exporter. CO₂ fixation via higher activity of phosphoenolpyruvate carboxylase in the plant part of the nodules in lentil might have been due to the greater synthesis of four carbon amino acids for amide export. The peak of NADP-isocitrate dehydrogenase in both legumes coincided with the pentose phosphate pathway enzymes at the time of high rates of sucrose metabolism and nitrogen fixation. Higher activities of NADP-malic enzyme were obtained in mungbean than in the lentil nodules. These findings are consistent with the role of these enzymes in providing reductant (NADPH) and substrates for energy yielding metabolism of bacteroids and carbon skeletons for ammonia assimilation.     

Interaction of cytosolic and plastidic nitrogen metabolism in plants

In angiosperms, the assimilation of ammonia resulting from nitrate reduction and from photorespiration depends on the operation of the plastidic GS/GOGAT cycle. The precursor for ammonia assimilation, 2-oxoglutarate, is synthesized in the mitochondria and in the cytosol. It is imported into the plastid by a 2-oxoglutarate/malate translocator (DiT1). In turn, the product of ammonia assimilation, glutamate, is exported from the plastids by a glutamate/malate translocator (DiT2). These transport processes link plastidic and cytosolic nitrogen metabolism and are essential for plant metabolism. DiT1 was purified to homogeneity from spinach chloroplast envelope membranes and identified as a protein with an apparent molecular mass of 45 kDa. Peptide sequences were obtained from the protein and the corresponding cDNA was cloned. The function of the DiT1 protein and its substrate specificity were confirmed by expression of the cDNA in yeast cells and functional reconstitution of the recombinant protein into liposomes. Recent advances in the molecular cloning of DiT2 and in the analysis of the in vivo function of DiT1 by antisense repression in transgenic tobacco plants will be discussed. In non-green tissues, the reducing equivalents required for glutamate formation by NADH-GOGAT are supplied by the oxidative pentose phosphate pathway. Glucose 6-phosphate, the immediate precursor of the oxidative pentose phosphate pathway is generated in the cytosol and imported into the plastids by the plastidic glucose 6-phosphate/phosphate translocator.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

Formation of a pentose phosphate cycle metabolite, erythrose-4-phosphate, from initial compounds of glycolysis by transketolase from the rat liver

Using ion-exchange chromatography of sucrose phosphates on Dowex-1, it was demonstrated that the highly purified rat liver transketolase (specific activity 1.7 mumol/min.mg protein) is capable of catalyzing the synthesis of erythrose-4-phosphate, a metabolite of the pentose phosphate pathway non-oxidizing step, from the initial participants of glycolysis, i. e., glucose-6-phosphate and fructose-6-phosphate. As can be evidenced from the reaction course, the second product of this synthesis is octulose-8-phosphate. The reaction was assayed by accumulation of erythrose-4-phosphate. The soluble fraction from rat liver catalyzes under identical conditions the synthesis of heptulose-7-phosphate (but not erythrose-4-phosphate), which points to the utilization of the erythrose-4-phosphate formed in the course of the transketolase reaction by transaldolase which is also present in the soluble fraction. The role of the transketolase reaction reversal from the synthesis of pentose phosphate derivatives to glycolytic products is discussed. The transketolase reaction provides for the relationship between glycolysis and the anaerobic step of the pentose phosphate pathway which share common metabolites, i. e. glucose-6-phosphate and fructose-6-phosphate.     

昆明小鼠体内发育早期胚胎葡萄糖代谢关键酶转录产物的分析

首次报道了昆明小鼠体内发育的早期胚胎1-细胞至桑椹期阶段葡萄糖代谢的3种关键酶-6-磷酸葡萄糖脱氢酶（G6PDH）、6-磷酸果糖激酶（PFK）和磷酸葡萄糖变位酶（PGM）的基因转录情况,其分别体现了磷酸戊糖、糖酵解、糖原的合成和分解等途径,根据G6PDH、PFK、PGM的cDNA序列分别设计和合成3套共6对内、外引物,采用巢式RT-PCR方法对其进行检测。结果表明：早期胚胎1-8细胞阶段均有G6PDH基因的转录,叠椹期胚胎不存在该基因的转录,说明早期胚胎1-8细胞阶段可能存在磷酸戊糖,而桑椹期则不存在;1-细胞至桑椹期均存在PFK基因的转录,说明该阶段的胚胎可能存在糖酵解代谢途径;1-细胞至桑椹期均不存在PGM基因的转录,说明该阶段的胚胎可能不存在糖原的合成与分解代谢途径。     

The crystal structure of glucose-6-phosphate isomerase from Leishmania mexicana reveals novel active site features.

Glucose-6-phosphate isomerase catalyzes the reversible aldose-ketose isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate in glycolysis and gluconeogenesis, and in the recycling of hexose-6-phosphate in the pentose phosphate pathway. The unicellular protozoans, Trypanosoma brucei, T. cruzi and Leishmania spp., of the order Kinetoplastida are important human parasites responsible for African sleeping sickness, Chagas' disease and leishmaniases, respectively. In these parasites, glycolysis is an important (and in some cases the only) metabolic pathway for ATP supply. The first seven of the 10 enzymes that participate in glycolysis, as well as an important fraction of the enzymes of the pentose phosphate pathway, are compartmentalized in peroxisome-like organelles called glycosomes. The dependence of the parasites on glycolysis, the importance of the pentose phosphate pathway in defense against oxidative stress, and the unique compartmentalization of these pathways, point to the enzymes contained in the glycosome as potential targets for drug design. The present report describes the first crystallographic structure of a parasite (Leishmania mexicana) glucose-6-phosphate isomerase. A comparison of the atomic structure of L. mexicana, human and other mammalian PGIs, which highlights unique features of the parasite's enzyme, is presented.     

几种化学药剂对‘NJ72’油桃休眠解除的影响

在进行果树温室栽培时,经常遇到萌芽率低、萌芽开花延迟、花器官发育差、座果率低的问题。本试验以‘NJ72’油桃为试材,观察了3种药剂对解除芽休眠的影响。结果表明,2%(NH2)2CS能提早花期,但存在药害现象。6% KNO3不能提早花期,并且花期不整齐,5% NH4NO3效果与6%KNO3类似。同时化学药剂处理促进花芽内H2O2的积累,抑制了过氧化氢酶(CAT)活性但促进了过氧化物酶(POD)活性,超氧物岐化酶(SOD)活性变化较小。化学药剂处理使花芽的呼吸速率增加,其中磷酸戊糖途径(PPP)代谢增加,糖酵解(EMP)降低,而三羧酸循环(TCA)代谢波动较小。葡萄糖_6_磷酸脱氢酶(G6PDH)活性在化学药剂处理时也增加。     

Nonoxidative Pentose Phosphate Pathway in Veillonella alcalescens

Crude cell-free extracts of Veillonella alcalescens C1, an anaerobe unable to ferment glucose, were assayed for individual enzymes of the pentose phosphate pathway. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were not detectable. Constituent enzymes of the nonoxidative limb of the pentose phosphate pathway were demonstrable. The presence of transaldolase, transketolase, phosphoribose isomerase, and phosphoribulose epimerase in this organism suggests a primarily biosynthetic role for these enzymes. It is postulated that ribose is synthesized from lactate in V. alcalescens C1 via a modified reversal of glycolysis and the nonoxidative limb of the pentose phosphate pathway.