Relationship of the oxidative pentose shunt pathway to lipid synthesis in Drosophila melanogaster

The tissue activities of the oxidative pentose shunt enzymes, glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and 6-phosphogluconate dehydrogenase (E.C. 1.1.1.44), in the larvae of Drosophila melanogaster are not dependent on the amount of flux through the oxidative pentose shunt pathway. An oxidative pentose shunt deficiency effects about a 40% reduction in the NADPH concentration in early third instar larvae, resulting in a six-fold difference in the NADPH/NADP+ ratio between wild-type and pentose-shunt-deficient larvae. The capacity of pentose-shunt-deficient larvae to synthesize triglyceride in response to a high concentration of dietary sucrose is only 73% of the wild-type level. Environmental temperature influences on the fatty acid composition of larvae are not altered by an oxidative pentose shunt deficiency.     

Dynamic simulation of red blood cell metabolism and its application to the analysis of a pathological condition

Background  

Cell simulation, which aims to predict the complex and dynamic behavior of living cells, is becoming a valuable tool. In silico models of human red blood cell (RBC) metabolism have been developed by several laboratories. An RBC model using the E-Cell simulation system has been developed. This prototype model consists of three major metabolic pathways, namely, the glycolytic pathway, the pentose phosphate pathway and the nucleotide metabolic pathway. Like the previous model by Joshi and Palsson, it also models physical effects such as osmotic balance. This model was used here to reconstruct the pathology arising from hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is the most common deficiency in human RBC.     

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.     

Interrelations between glycolysis and the hexose monophosphate shunt in erythrocytes as studied on the basis of a mathematical model

A mathematical model is presented which comprises the reactions of glycolysis, the hexose monophosphate shunt (HMS) and the glutathione system in erythrocytes. The model is used to calculate stationary and time-dependent metabolic states of the cell in vitro and in vivo. The model properly accounts for the following metabolic features observed in vitro: (a) stimulation of the oxidative pentose pathway after addition of pyruvate due to a NADP-dependent lactate dehydrogenase as coupling enzyme between glycolysis and the oxidative pentose pathway, (b) relative share of the oxidative pentose pathway in the total consumption of glucose amounting to approximately 10% in the normal case and to approximately 90% under conditions of oxidative stress excreted by methylene blue. From the application of the model to in vivo conditions it is predicted that (c) under normal conditions glycolysis and the HMS are independently regulated by the energetic and oxidative load, respectively, (d) under conditions of enhanced energetic or oxidative load both glycolysis and the HMS are mainly controlled by the hexokinase; in this situation the highest possible values of the energetic and oxidative load which are compatible with cell integrity are strongly coupled and considerably restricted in comparison with the normal case, (e) the stationary states possess bifurcation points at high and low values of the energetic load.     

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等。葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中。本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用。     

The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.     

Microsomal hexose-6-phosphate and 6-phosphogluconate dehydrogenases in extrahepatic tissues: human placenta and pig kidney cortex

An oxidative pathway of glucose-6-phosphate was found in the microsomal fraction of two extra-hepatic tissues: human placenta and pig kidney cortex. Oxidation activity in microsomes, measured by the formation of 14CO2 from [1-14C] glucose-6-phosphate, was observed only after Triton X-100 treatment and in the presence of methylene blue and NADP. Hexose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were present in a latent form and required treatment with detergent for full activation. Our results suggest that these enzymes are located in the luminal space of placental and kidney microsomes, and that, as in the liver, they generate NADPH on the inner side of the endoplasmic reticulum when G6P and NADP are available.     

Light modulation of the activity of carbon metabolism enzymes in the crassulacean Acid metabolism plant kalanchoë

When intact Kalanchoë plants are illuminated NADP-linked malic dehydrogenase and three enzymes of the reductive pentose phosphate pathway, ribulose-5-phosphate kinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose-1,7-diphosphate phosphatase, are activated. In crude extracts these enzymes are activated by dithiothreitol treatment. Light or dithiothreitol treatment does not inactivate the oxidative pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. Likewise, neither light, in vivo, nor dithiothreitol, in vitro, affects fructose-1,6-diphosphate phosphatase. Apparently the potential for modulation of enzyme activity by the reductively activated light effect mediator system exists in Crassulacean acid metabolism plants, but some enzymes which are light-dark-modulated in the pea plant are not in Kalanchoë.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

Glucose-6-phosphate metabolism in Plasmodium falciparum

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.     

Pentose synthesis in glucose-grown cells of Lactobacillus casei

The pathway of pentose synthesis in glucose-grown cells of Lactobacillus casei was ascertained. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were present in glucose-grown cells, while transaldolase and transketolase were present only in traces. This suggested that only the oxidative arm of this pathway was operative in glucose-grown cells. On the other hand, in ribose-grown cells, transaldolase was induced with a concomitant suppression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These results were confirmed by the detection of labelled CO2 produced by L. casei grown on [1-14C]glucose. The activities of the enzymes of the oxidative pentose phosphate pathway as also the rate of CO2 formation were higher in the exponential phase of growth as compared to the stationary phase, when the requirement of the cells for pentoses for the formation of DNA and RNA was higher.     

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.     

Metabolic flux analysis of postburn hepatic hypermetabolism

The hepatic response to severe injury is characterized by a marked upregulation of glucose, fatty acid, and amino acid turnover, which, if persistent, predisposes the patient to progressive organ dysfunction. To study the effect of injury on liver intermediary metabolism, metabolic flux analysis was applied to isolated perfused livers of burned and sham-burned rats. Intracellular fluxes were calculated using metabolite measurements and a stoichiometric balance model. Significant flux increases were found for multiple pathways, including mitochondrial electron transport, the TCA and urea cycles, gluconeogenesis, and pentose phosphate pathway (PPP). The burn-induced increase in gluconeogenesis did not significantly increase glucose output. Instead, glucose-6-phosphate was diverted into the PPP. These changes were paralleled by increases in glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activities. Given that G6PDH and GR are the most significant NADPH producers and consumers in the liver, respectively, and that GR is responsible for recycling the free radical scavenger glutathione, these data are consistent with the notion that hepatic metabolic changes are in part due to the induction of liver antioxidant defenses.     

In vivo measurements of control coefficients for hexokinase and glucose-6-phosphate dehydrogenase in Xenopus laevis oocytes

Hexokinase and glucose-6-phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO(2) (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1-(14)C]glucose and measurements of the radioactivity in glycogen and CO(2). Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=-0.01). Glucose-6-phosphate dehydrogenase injections did not affect the production of 14CO(2) by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.     

Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo.

The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell-Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P2), D-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 microM for both enzymes. Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases. NADP+ and NADPH levels were between 130 microM and 290 microM, which is very much higher than the respective Km and Ki values. The Glc6P concentration was around 500 microM in both strains. The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP+ concentrations and the specific enzyme activities of both dehydrogenases.     

Increased neuronal glucose-6-phosphate dehydrogenase and sulfhydryl levels indicate reductive compensation to oxidative stress in Alzheimer disease.

We analyzed glucose-6-phosphate dehydrogenase, the rate-controlling enzyme of the pentose phosphate pathway and free sulfhydryls, to study redox balance in Alzheimer disease. Glucose-6-phosphate dehydrogenase plays a pivotal role in homeostatic redox control by providing reducing equivalents to glutathione, the major nonenzymatic cellular antioxidant. There is a multitude of evidence that marks oxidative stress proximally in the natural history of Alzheimer disease. Consistent with a role for glutathione in defense against increased reactive oxygen, we found an upregulation of glucose-6-phosphate dehydrogenase together with increased sulfhydryls in Alzheimer disease. These data indicate that reductive compensation may play an important role in combating oxidative stress in Alzheimer disease.     

Changes in enzyme activity of white yam tubers after prolonged storage

There is a substantial increase in the activities of phosphorylase, hexokinase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase in white yam tubers as they age. The high glucose-6-phosphate dehydrogenase activities suggest that the pentose phosphate pathway is important in yam tuber tissue.     

Pathway analysis and metabolic engineering in Corynebacterium glutamicum

The gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of L-glutamate and L-lysine. During the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. In order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [13C]-labelling technique was combined with metabolite balancing to achieve a unifying comprehensive pathway analysis. These methods can determine the flux distribution at the branch point between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and 6-phosphogluconate dehydrogenases determined in vitro were in full accordance with the fluxes measured by the [13C]-labelling technique. These data indicate that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/NADP concentrations and the specific activity of glucose-6-phosphate dehydrogenase. The carbon flux via the oxidative pentose phosphate pathway correlated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylase fulfills a main anaplerotic function in C. glutamicum, we recently detected that a biotin-dependent pyruvate carboxylase exists as a further anaplerotic enzyme in this bacterium. In addition to the activities of these two carboxylases three enzymes catalysing the decarboxylation of the C4 metabolites oxaloacetate or malate are also present in this bacterium. The individual flux rates at this complex anaplerotic node were investigated by using [13C]-labelled substrates. The results indicate that both carboxylation and decarboxylation occur simultaneously in C. glutamicum so that a high cyclic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways exist for the synthesis of DL-diaminopimelate and L-lysine. As shown by NMR spectroscopy the relative use of both pathways in vivo is dependent on the ammonium concentration in the culture medium. Mutants defective in one pathway are still able to synthesise enough L-lysine for growth, but the L-lysine yields with overproducers were reduced. The luxury of having these two pathways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL-diaminopimelate as a building block for the synthesis of the murein sacculus.     

Coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.