Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Metabolic engineering of Corynebacterium glutamicum for production of sunscreen shinorine

Ultraviolet-absorbing chemicals are useful in cosmetics and skin care to prevent UV-induced skin damage. We demonstrate here that heterologous production of shinorine, which shows broad absorption maxima in the UV-A and UV-B region. A shinorine producing Corynebacterium glutamicum strain was constructed by expressing four genes from Actinosynnema mirum DSM 43827, which are responsible for the biosynthesis of shinorine from sedoheptulose-7-phosphate in the pentose phosphate pathway. Deletion of transaldolase encoding gene improved shinorine production by 5.2-fold. Among the other genes in pentose phosphate pathway, overexpression of 6-phosphogluconate dehydrogenase encoding gene further increased shinorine production by 60% (19.1 mg/L). The genetic engineering of the pentose phosphate pathway in C. glutamicum improved shinorine production by 8.3-fold in total, and could be applied to produce the other chemicals derived from sedoheptulose-7-phosphate.     

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.     

Regulation of photosynthetic carbon metabolism. The effect of inorganic phosphate on stromal sedoheptulose-1,7-bisphosphatase

The activation and steady-state kinetics of wheat chloroplast sedoheptulose-1,7-bisphosphatase at several concentrations of inorganic phosphate are examined. Inorganic phosphate competitively inhibits substrate binding to both the active and inactive forms of the enzyme and reduces the rate of enzyme activation. Modulation of the apparent Km of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase for their substrates by inorganic phosphate is discussed in terms of the control of intermediate pool sizes in the reductive pentose phosphate pathway and of the flux of fixed carbon towards starch synthesis or export from the chloroplast.     

Riboneogenesis in yeast

Glucose is catabolized in yeast via two fundamental routes, glycolysis and the oxidative pentose phosphate pathway, which produces NADPH and the essential nucleotide component ribose-5-phosphate. Here, we describe riboneogenesis, a thermodynamically driven pathway that converts glycolytic intermediates into ribose-5-phosphate without production of NADPH. Riboneogenesis begins with synthesis, by the combined action of transketolase and aldolase, of the seven-carbon bisphosphorylated sugar sedoheptulose-1,7-bisphosphate. In the pathway's committed step, sedoheptulose bisphosphate is hydrolyzed to sedoheptulose-7-phosphate by the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity we identified based on metabolomic analysis of the corresponding knockout strain. The crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate reveals that the substrate binds in the closed furan form in the active site. Sedoheptulose-7-phosphate is ultimately converted by known enzymes of the nonoxidative pentose phosphate pathway to ribose-5-phosphate. Flux through SHB17 increases when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells.     

Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LC-MS/MS: application to two new inherited defects of metabolism

We describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify pentose phosphate pathway intermediates (triose-3-phosphates, tetrose-4-phosphate, pentose-5-phosphate, pentulose-5-phosphates, hexose-6-phosphates and sedoheptulose-7-phosphate (sed-7P)) in bloodspots, fibroblasts and lymphoblasts. Liquid chromatography was performed using an ion pair loaded C(18) HPLC column and detection of the sugar phosphates was carried out by tandem mass spectrometry using an electron ion spray source operating in the negative mode and multiple reaction monitoring. Reference values for the pentose phosphate pathway intermediates in blood spots, fibroblasts and lymphoblasts were established. The method was applied to cells from patients affected with a deficiency of transaldolase. The transaldolase-deficient cells showed an increased concentration of sedoheptulose-7-phosphate. (Bloodspots: 5.19 and 5.43 micromol/L [0.49-3.33 micromol/L]; fibroblasts 7.43 and 26.46 micromol/mg protein [0.31-1.14 micromol/mg protein]; lymphoblasts 16.03 micromol/mg protein [0.61-2.09 micromol/mg protein].) The method was also applied to study enzymes of the pentose phosphate pathway by incubating fibroblasts or lymphoblasts homogenates with ribose-5-phosphate or 6-phosphogluconate and the subsequent analysis of the formed sugar phosphates.     

Elementary modes analysis of photosynthate metabolism in the chloroplast stroma.

We briefly review the metabolism of the chloroplast stroma, and describe the structural modelling technique of elementary modes analysis. The technique is applied to a model of chloroplast metabolism to investigate viable pathways in the light, in the dark, and in the dark with the addition of sedoheptulose-1,7-bisphosphatase (normally inactive in the dark). The results of the analysis show that it is possible for starch degradation to enhance photosynthetic triose phosphate export in the light, but the reactions of the Calvin cycle alone are not capable of providing a sustainable flux from starch to triose phosphate in the dark. When reactions of the oxidative pentose phosphate pathway are taken into consideration, triose phosphate export in the dark becomes possible by the operation of a cyclic pathway not previously described. The effect of introducing sedoheptulose-1,7-bisphosphatase to the system are relatively minor and, we predict, innocuous in vivo. We conclude that, in contrast with the traditional view of the Calvin cycle and oxidative pentose phosphate pathway as separate systems, they are in fact, in the context of the chloroplast, complementary and overlapping components of the same system.     

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.     

Reversible inhibition of the calvin cycle and activation of oxidative pentose phosphate cycle in isolated intact chloroplasts by hydrogen peroxide

Hydrogen peroxide (6x10^-4 M) causes a 90% inhibition of CO₂-fixation in isolated intact chloroplasts. The inhibition is reversed by adding catalase (2500 U/ml) or DTT (10 mM). If hydrogen peroxide is added to a suspension of intact chloroplasts in the light, the incorporation of carbon into hexose- and heptulose bisphosphates and into pentose monophosphates is significantly increased, whereas; carbon incorporation into hexose monophosphates and ribulose 1,5-bisphosphate is decreased. At the same time formation of 6-phosphogluconate is dramatically stimulated, and the level of ATP is increased. All these changes induced by hydrogen peroxide are reversed by addition of catalase or DTT. Additionally, the conversion of [¹⁴C]glucose-6-phosphate into different metabolites by lysed chloroplasts in the dark has been studied. In presence of hydrogen peroxide, formation of ribulose-1,5-bisphosphate is inhibited, whereas formation of other bisphosphates,of triose phosphates, and pentose monophosphates is stimulated. Again, DTT has the opposite effect. The release of ¹⁴CO₂ from added [¹⁴C]glucose-6-phosphate by the soluble fraction of lysed chloroplasts via the reactions of oxidative pentose phosphate cycle is completely inhibited by DTT (0.5 mM) and re-activated by comparable concentrations of hydrogen peroxide. These results indicate that hydrogen peroxide interacts with reduced sulfhydryl groups which are involved in the light activation of enzymes of the Calvin cycle at the site of fructose- and sedoheptulose bisphophatase, of phosphoribulokinase, as well as in light-inactivation of oxidative pentose phosphate cycle at the site of glucose-6-phosphate dehydrogenase.Abbreviations ADPG ADP-glucose - DHAP dihydroxyacetone phosphate - DTT dithiothreitol - FBP fructose-1,6-bisphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HMP hexose monophosphates (fructose-6-phosphate, glucose-6-phosphate, glucose-1-phosphate) - 6-PGI 6-phosphogluconate - PMP pentose monophosphates (xylulose-5-phosphate, ribose-5-phosphate, ribulose-5-phosphate) - RuBP ribulose-1,5-bisphosphate - S7P sedoheptulose-7-phosphate - SBP sedoheptulose-1,7-bisphosphate Dedicated to Prof. Dr. W. Simonis on the occasion of his 70th birthday     

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants. Correspondence to: M. Ciriacy     

Ribose-5-phosphate biosynthesis in Methanocaldococcus jannaschii occurs in the absence of a pentose-phosphate pathway

Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6-2H2]- and [U-13C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of D-arabino-3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the D-ara-bino-3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from D-arabino-3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.     

A newly discovered metabolic diseases due to defects in the pentose pathway

Two previously unreported inborn errors of metabolism occur in the reversible part of the pentose phosphate pathway. Deficiency of ribose-5-phosphate isomerase has been described in one patient who suffered from a progressive leukoencephalopathy and developmental delay. Transaldolase deficiency has been diagnosed in 11 patients from 6 families in which the probands presented in the newborn and antenatal period with hepatospIenomegaly, hemolytic anaemia, hepatic fibrosis, kidney problems. Enzymes deficiency results in accumulations in body fluids erythritol, arabitol, ribitol, sedoheptitol, sedoheptulose, sedoheptulose-7-phosphate. Isomerase and transaldolase activity can be determined in leukocytes or fibroblasts.     

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.     

The interdependence of glycolytic and pentose cycle intermediates in ad libitum fed rats

Equilibrium constants for reactions catalyzed by ribulose-5-phosphate 3-epimerase, [sigma xylulose-5-P]/[sigma ribulose-5-P] = 1.82, ribose-5-phosphate isomerase, [sigma Rib-5-P]/[sigma ribulose-5-P] = 1.20, transaldolase, [sigma erythrose-4-P] [sigma Fru-6-P]/[sigma sedoheptulose-7-P] [sigma glyceraldehyde 3-P] = 0.37, and transketolase, [sigma Fru-6-P] [sigma glyceraldehyde 3-P]/[sigma erythrose-4-P] [sigma xylulose-5-P] = 29.7 and [sigma Rib-5-P] [sigma xylulose-5-P]/[sigma sedoheptulose-7-P] [sigma glyceraldehyde 3-P] = 0.48, were redetermined under physiological conditions. The equilibrium constant for the combined glucose-6-P dehydrogenase and 6-phosphoglucono-gamma-lactonase reaction, [6-phosphogluconate3-] [NADPH] [H+]2/[Glc-6-P2-] [NADP+], was found to be at least 1 X 10(-9). Using these redetermined equilibrium constants, calculated values of pentose cycle intermediates, based on near equilibrium assumptions and the tissue content of Fru-6-P and glyceraldehyde 3-P, were found to be in good agreement with measured values for male Wistar rats injected with saline, 20 mumol/g pyruvate, 20 mumol/g gluconate, and 20 mumol/g ribose. Measured and calculated values for pentose cycle intermediates in saline injected animals were ribulose-5-P; 3.8 +/- 0.4 and 2.4 +/- 0.1 nmol/g; xylulose-5-P, 5.9 +/- 0.6 nmol/g and 4.3 +/- 0.2 nmol/g; sedoheptulose-7-P, 41.5 +/- 2.4 and 37.6 +/- 2.9 nmol/g; and combined sedopheptulose-7-P and Rib-5-P, 43.0 +/- 2.8 nmol/g and 40.5 +/- 3.0 nmol/g; liver content of erythrose-4-P was less than the detection limits of the assay, 2 nmol/g. Calculated erythrose-4-P was 0.23 +/- 0.01 nmol/g. Liver content of 6-phosphogluconate was 8.5 +/- 0.7 nmol/g. The free cytosolic [NADP+]/[NADPH] ratio calculated from the 6-phosphogluconate dehydrogenase redox couple, 0.0030 +/- 0.0002, was also in good agreement with that calculated from the malic enzyme redox couple, 0.0051 +/- 0.0007, and the isocitrate dehydrogenase redox couple, 0.0066 +/- 0.0008. These data indicate the interdependence of the liver content of glycolytic intermediates and pentose cycle intermediates in ad libitum fed rats.     

Quantitative measurement of the L-type pentose phosphate cycle with [2-14C]glucose and [5-14C]glucose in isolated hepatocytes.

1. Investigations of the mechanism of the non-oxidative segment of the pentose phosphate cycle in isolatd hepatocytes by prediction-labelling studies following the metabolism of [2-14C]-, [5-14C]- and [4,5,6-14C]glucose are reported. The 14C distribution patterns in glucose 6-phosphate show that the reactions of the L-type pentose pathway in hepatocytes. 2. Estimates of the quantitative contribution of the L-type pentose cycle are the exclusive form of the pentose cycle to glucose metabolism have been made. The contribution of the L-type pentose cycle to the metabolism of glucose lies between 22 and 30% in isolated hepatocytes. 3. The distribution of 14C in the carbon atoms of glucose 6-phosphate following the metabolism of [4,5,6-14C]- and [2-14C]glucose indicate that gluconeogenesis from triose phosphate and non-oxidative formation of pentose 5-phosphate do not contribute significantly to randomization of 14C in isolated hepatocytes. The transaldolase exchange reaction between fructose 6-phosphate and glyceraldehyde 3-phosphate is very active in these cells.     

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.     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly regulated by the ratio of NADPH/NADP⁺, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP⁺ ratio. With a ratio of NADPH/NADP⁺ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited.This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP⁺ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions.Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP⁺ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP⁺.It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP⁺ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolite unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP⁺ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

NADPH production in the oxidative pentose phosphate pathway as source of reducing equivalents in glycolysis of human red cells in vitro.

Studies have been carried out on human erythrocytes in vitro to clarify the deficit of pyruvate formation under conditions when 2,3 DPG is degraded. The results lead to the conclusion that there exist a cross connection between the glycolytic and the oxidative pentose phosphate pathway which is mediated by the NADP/NADPH couple. NADPH serves as additional reducing equivalent in the reaction of the LDH. In the absence of glucose the pool of the metabolites of the pentose phosphate pathway is able to supply glucose-6-phosphate for the production of NADPH by recombination. The reaction of NADPH at the LDH is probably of significance under in vivo conditions.     

Revisiting the 13C-label distribution of the non-oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence

The currently applied reaction structure in stoichiometric flux balance models for the nonoxidative branch of the pentose phosphate pathway is not in accordance with the established ping-pong kinetic mechanism of the enzymes transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). Based upon the ping-pong mechanism, the traditional reactions of the nonoxidative branch of the pentose phosphate pathway are replaced by metabolite specific, reversible, glycolaldehyde moiety (C(2)) and dihydroxyacetone moiety (C(3)) fragments producing and consuming half-reactions. It is shown that a stoichiometric model based upon these half-reactions is fundamentally different from the currently applied stoichiometric models with respect to the number of independent C(2) and C(3) fragment pools in the pentose phosphate pathway and can lead to different label distributions for (13)C-tracer experiments. To investigate the actual impact of the new reaction structure on the estimated flux patterns within a cell, mass isotopomer measurements from a previously published (13)C-based metabolic flux analysis of Saccharomyces cerevisiae were used. Different flux patterns were found. From a genetic point of view, it is well known that several micro-organisms, including Escherichia coli and S. cerevisiae, contain multiple genes encoding isoenzymes of transketolase and transaldolase. However, the extent to which these gene products are also actively expressed remains unknown. It is shown that the newly proposed stoichiometric model allows study of the effect of isoenzymes on the (13)C-label distribution in the nonoxidative branch of the pentose phosphate pathway by extending the half-reaction based stoichiometric model with two distinct transketolase enzymes instead of one. Results show that the inclusion of isoenzymes affects the ensuing flux estimates.