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.     

Activities of the pentose phosphate pathway and enzymes of proline metabolism in legume root nodules

Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N₂ fixation, and production of NADP⁺ to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N₂ is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N₂-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N₂ fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities.     

Development and intracellular distribution of enzymes of the oxidative pentose phosphate cycle in radish cotyledons

Developmental and compartmentation studies were used to evaluate the relative roles of the oxidative pentose phosphate cycle, the Calvin cycle, and the glycolysis in cotyledons of radish (Raphanus sativus L.).     

Reductant for glutamate synthase in generated by the oxidative pentose phosphate pathway in non-photosynthetic root plastids

Purified pea root plastids were supplied with glutamine, 2-oxoglutarate and phosphorylated sugars. Formation of glutamate was linear for 75 min and dependent upon the intactness of the organelle. Glucose-6-phosphate and ribose-5-phosphate were the most effective substrates in supporting glutamate synthesis. Flux through the oxidative pentose phosphate pathway during glutamate synthesis in purified plastids was followed by monitoring the release of ¹⁴CO₂ from [1-¹⁴C]glucose-6-phosphate. ¹⁴CO₂ evolution from C-1 was dependent upon the presence of both glutamine and 2-oxoglutarate and could be inhibited by the application of azaserine. The data are discussed in view of the role of the oxidative pentose phosphate pathway in non-photosynthetic plastids.     

The oxidative pentose phosphate pathway: structure and organisation

The oxidative pentose phosphate pathway is a major source of reducing power and metabolic intermediates for biosynthetic processes. Some, if not all, of the enzymes of the pathway are found in both the cytosol and plastids, although the precise distribution of their activities varies. The apparent absence of sections of the pathway from the cytosol potentially complicates metabolism. These complications are partly offset, however, by exchange of intermediates between the cytosol and the plastids through the activities of a family of plastid phosphate translocators. Molecular analysis is confirming the widespread presence of multiple genes encoding each of the enzymes of the oxidative pentose phosphate pathway. Differential expression of these isozymes may ensure that the kinetic properties of the activity that catalyses a specific reaction match the metabolic requirements of a particular tissue. This hypothesis can be tested thanks to recent developments in the application of 13C-steady-state labelling strategies. These strategies make it possible to quantify flux through metabolic networks and to discriminate between pathways of carbohydrate oxidation in the cytosol and plastids.     

Recycling of carbon in the oxidative pentose phosphate pathway in non-photosynthetic plastids

Recycling of carbon in the oxidative pentose phosphate pathway (OPPP) of intact pea root plastids has been studied. The synthesis of dihydroxyacetone phosphate (DHAP) and evolution of CO₂ was followed in relation to nitrite reduction. A close coupling was observed between all three measured fluxes which were linear for up to 60 min and dependent upon the integrity of the plastids. However, the quantitative relationship between 1-¹⁴CO₂ evolution from glucose 6-phosphate and nitrite reduction varied with available hexose phosphate concentration. When 10 mM glucose 6-phosphate was supplied to intact plastids a stoichiometry of 1.35 was observed between ¹⁴CO₂ evolution and nitrite reduction. As exogenous glucose 6-phosphate was decreased this value fell, becoming 0.47 in the presence of 0.2 mM glucose 6-phosphate, indicative of considerable recycling of carbon. This conclusion was reinforced when using [2-¹⁴C]glucose-6-phosphate. The measured release of 2-¹⁴CO₂ was consistent with the data for 1-¹⁴CO₂, suggesting complete recycling of carbon in the OPPP. Ribose 5-phosphate was also able to support nitrite reduction and DHAP production. A stoichiometry of 2 NO ₂ ^? reduced: 1 DHAP synthesised was observed at concentrations of 1 mM ribose 5-phosphate or less. At concentrations of ribose 5-phosphate greater than 1 mM this stoichiometry was lost as a result of enhanced DHAP synthesis without further increase in nitrite reduction. It is suggested that this decoupling from nitrite reduction is a function of excess substrate entering directly into the non-oxidative reactions of the OPPP, and may be useful when the demand for OPPP products is not linked to the demand for reductant. The significance of recycling in the OPPP is discussed in relation to the coordination of nitrate assimilation with carbohydrate oxidation in roots and with the utilisation of carbohydrate by other pathways within plastids.     

Activity of pentose phosphate pathway enzymes in alkane-oxidizing yeast cells]

The activity of the key enzymes of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transketolase) was determined in cell-free homogenates of Candida lipolytica 695 and Candida tropicalis 303 growing on different carbon sources. The activity of these enzymes remained almost the same in the course of growth of both cultures. The activity of the enzymes differed only slightly in the cells metabolizing hexadecane and glucose. The activity of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the cell-free homogenates of C. tropicalis 303 was twice as high as in the cells of C. lipolytica 695. The activity of transketolase was the same in both cultures. The main role of the pentose phosphate pathway is presumed to consist not in catabolism of the carbon source, but in biosynthesis of pentoses and other important intermediates.     

Estimation of the activity of the oxidative pentose phosphate pathway in pea chloroplasts

The fraction of glucose 6-phosphate metabolism in isolated intact chloroplasts of Pisum sativum in the dark that occurs via the oxidative pentose phosphate pathway has been estimated from the distribution of ¹⁴C from specifically labelled glucose-[¹⁴C] supplied to the chloroplasts.     

Capacities of pea chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis

The aim of this work was to measure the capacities of pea (Pisum sativum) shoot chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis. Of the total activities in the unfractionated homogenates, appreciable proportions of those of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphofructokinase, and smaller but significant proportions of those of phosphopyruvate hydratase and pyruvate kinase were recovered in crude preparations of chloroplasts, and co-purified with intact chloroplasts on sucrose gradients. The activities in the chloroplasts showed considerable latency that was closely correlated with chloroplast integrity. Phosphoglyceromutase activity in the above preparations of chloroplasts did not exceed that expected from cytoplasmic contamination. The mass-action ratio for phosphoglyceromutase in illuminated isolated chloroplasts differed markedly from the enzyme's equilibrium constant. Isolated chloroplasts converted 2-phosphoglycerate to pyruvate. The enzyme activities of the chloroplasts were compared with the rates of respiration and starch breakdown in pea leaves in the dark. It is concluded that in the dark chloroplasts could metabolize all the products of starch breakdown and catalyse much of the respiration of pea shoots via the oxidative pentose phosphate pathway and/or glycolysis as far as 3-phosphoglycerate. It is suggested that pea shoot chloroplasts lack phosphoglyceromutase but contain some phosphopyruvate hydratase and pyruvate kinase.     

Levels of pentose phosphate pathway enzymes fromCandida shehatae grown in continuous culture

Summary Candida shehatae exhibits different fermentative capacities when grown under different aeration conditions. These studies investigated the titers of xylose reductase, xylitol dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase in crude extracts ofCandida shehatae grown in continuous culture with various specific aeration rates. Carbon source, aeration rate, dilution rate and temperature were examined as variables. Xylose reductase and xylitol dehydrogenase were induced by xylose and were largely absent in glucose-grown cells. Alcohol dehydrogenae levels were higher in glucose-grown cells than in xylose-grown cells. The levels of this enzyme also correlated with the fermentative character of metabolism, having a low value under fully aerobic conditions, a high value under anaerobic conditions, and intermediate levels under various semi-aerobic conditions. Temperature had no effect on any enzyme level over the range of 20–30°C.Maintained in cooperation with the University of Wisconsin-Madison     

Requirement for the plastidial oxidative pentose phosphate pathway for nitrate assimilation in Arabidopsis

Sugar metabolism and the oxidative pentose phosphate pathway (OPPP) are strongly implicated in N assimilation, although the relationship between them and the roles of the plastidial and cytosolic OPPP have not been established genetically. We studied a knock‐down mutant of the plastid‐localized OPPP enzyme 6‐phosphogluconolactonase 3 (PGL3). pgl3‐1 plants exhibited relatively greater resource allocation to roots but were smaller than the wild type. They had a lower content of amino acids and free in leaves than the wild type, despite exhibiting comparable photosynthetic rates and efficiency, and normal levels of many other primary metabolites. When N‐deprived plants were fed via the roots with , pgl3‐1 exhibited normal induction of OPPP and nitrate assimilation genes in roots, and amino acids in roots and shoots were labeled with ¹⁵N at least as rapidly as in the wild type. However, when N‐replete plants were fed via the roots with sucrose, expression of specific OPPP and N assimilation genes in roots increased in the wild type but not in pgl3‐1. Thus, sugar‐dependent expression of N assimilation genes requires OPPP activity and the specificity of the effect of the pgl3‐1 mutation on N assimilation genes establishes that it is not the result of general energy deficiency or accumulation of toxic intermediates. We conclude that expression of specific nitrate assimilation genes in the nucleus of root cells is positively regulated by a signal emanating from OPPP activity in the plastid.     

Involvement of the oxidative pentose phosphate pathway in thiamine biosynthesis in Salmonella typhimurium.

purF mutants of Salmonella typhimurium are known to require a source of both purine and thiamine; however, exogenous pantothenate may be substituted for the thiamine requirement. We show here that the effect of pantothenate is prevented by blocks in the oxidative pentose phosphate pathway, gnd (encoding gluconate 6-phosphate [6-P] dehydrogenase) or zwf (encoding glucose 6-P dehydrogenase). We further show that the defects caused by these mutations can be overcome by increasing ribose 5-P, suggesting that ribose 5-P may play a role in the ability of pantothenate to substitute for thiamine.     

Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway

RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltransferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltransferase (UPRT) were sub-cloned into pET15b vectors. All four constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21(AI) strain for protein over-expression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni-NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113 U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most 2 days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.     

Regulation of the pentose phosphate pathway in cancer

Energy metabolism is significantly reprogrammed in many human cancers, and these alterations confer many advantages to cancer cells, including the promotion of biosynthesis, ATP generation, detoxification and support of rapid proliferation. The pentose phosphate pathway (PPP) is a major pathway for glucose catabolism. The PPP directs glucose flux to its oxidative branch and produces a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), an essential reductant in anabolic processes. It has become clear that the PPP plays a critical role in regulating cancer cell growth by supplying cells with not only ribose-5-phosphate but also NADPH for detoxification of intracellular reactive oxygen species, reductive biosynthesis and ribose biogenesis. Thus, alteration of the PPP contributes directly to cell proliferation, survival and senescence. Furthermore, recent studies have shown that the PPP is regulated oncogenically and/or metabolically by numerous factors, including tumor suppressors, oncoproteins and intracellular metabolites. Dysregulation of PPP flux dramatically impacts cancer growth and survival. Therefore, a better understanding of how the PPP is reprogrammed and the mechanism underlying the balance between glycolysis and PPP flux in cancer will be valuable in developing therapeutic strategies targeting this pathway.     

Activity of some glycolytic and pentose phosphate pathway enzymes during the development of adventitious roots

Activities of phosphofructokinase (PFK, EC 2.7.1.11), glyceraldehyde 3-phosphate (NAD) dehydrogenase [G-3-PD(NAD), EC 1.2.1.12], glucose 6-phosphate dehydrogenase (G-6-PD, EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (6-PGD, EC 1.1.1.44) were determined in bean cuttings (Phaseolus vulgaris L. cv. Top Crop) over 4 days, encompassing adventitious root primordium initiation and development. Effects of applied auxin and “endogenous root-forming stimulus”(ERS) on enzyme activities, concentrations of reducing sugars, and primordium development were also determined during the first 4 days of propagation. Effects of auxin were determined through use of applied indole-3-acetic acid (IAA) or 2,3,5-triiodobenzoic acid. Effects of ERS were evaluated by means of decapitation of cuttings. Increased basipetal transport and increased metabolism of reducing sugars occurred in leafy cuttings in response to applied IAA and to ERS. Primordium development and activities of the four enzymes increased in leafy cuttings under conditions that simultaneously increased basipetal transport and metabolism of reducing sugars. Three types of enzyme activity response were found: (i) activity increased over time by ERS and by applied IAA [G-3-PD(NAD)], (ii) activity increased over time by ERS but not by applied IAA (PFK, G-6-PD), (iii) activity increased over time but not by ERS or applied IAA (6-PGD). Increases in G-3-PD(NAD), G-6-PD, and PFK activity in leafy cuttings were positively related to primordium development. 6-PGD activity increased in leafy cuttings during primordium development and may have supported it. However, equal increases occurred in decapitated cuttings, in which the long-term development of primordia was supressed. Results for G-3-PD(NAD) that were obtained in an experiment with jack pine (Pinus banksiana Lamb.) seedling cuttings were similar to results for the same enzyme in bean cuttings. G-3-PD(NAD) activity in naphthaleneacetic acid-treated jack pine cuttings increased with time, in comparison with untreated cuttings, before root emergence.