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.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions and related enzymes of the cycle in adipose tissue

1. Measurements were made of the activities of the enzymes of the pentose phosphate pathway concerned in both the oxidative (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and the non-oxidative (ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase) reactions of this pathway, together with hexokinase and phosphoglucose isomerase, in adipose tissue in a variety of nutritional and hormonal conditions. 2. Starvation for 2 days caused a significant decrease in the activities of all the enzymes of the pentose phosphate pathway, with the exception of glucose 6-phosphate dehydrogenase, when expressed as activity/2 fat-pads; only the activities of ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase were significantly decreased on the basis of activity/mg. of protein. Re-feeding with a high-carbohydrate or high-fat diet for 3 days restored the activity of all the enzymes of the pentose phosphate pathway to the range of the control values, with the exception of transketolase, which showed a marked ;overshoot' in rats re-fed with carbohydrate. Starvation for 3 days caused a marked decrease in the activities of glucose 6-phosphate dehydrogenase and transketolase. 3. On the basis of activity/two fat-pads, alloxan-diabetes caused a marked decrease, to about half the control value, in the activities of all the enzymes concerned in the pentose phosphate pathway, transketolase showing the smallest decrease; hexokinase and phosphoglucose isomerase activities were also decreased. Treatment with insulin for 3 and 7 days raised the activities to normal or supranormal values, transketolase showing the most marked ;overshoot' effect. On the basis of activity/mg. of protein the activity of none of the enzymes was significantly decreased in alloxan-diabetes; transketolase and transaldolase activities were raised above the control values. With insulin treatment for 3 or 7 days the activities of all the enzymes were significantly increased, except that of ribulose 5-phosphate epimerase at the shorter time-interval. Glucagon treatment did not alter any of the enzyme activities expressed on either basis. 4. Thyroidectomy caused a decrease of 30-40% in the activities of enzymes of the pentose phosphate pathway, except for transketolase activity, which fell to 50% of the control value. Little change occurred in adipose-tissue weight or protein content. 5. Adrenalectomy caused a decrease of 40% in the activity of glucose 6-phosphate dehydrogenase and of 20-30% in the activities of the remaining enzymes of the pentose phosphate pathway; hexokinase activity was also decreased. Treatment with cortisone for 3 days did not significantly raise the activity from that found in adrenalectomized rats. Treatment of normal rats with high doses of cortisone had no significant effect on the activities of the enzymes of the pentose phosphate pathway in adipose tissue. 6. The changes in enzyme activities are discussed in relation to: (a) the concept of constant-proportion groups of enzymes; (b) the known changes in the flux of glucose through alternative metabolic pathways; (c) the pattern of change found in liver with similar hormonal and dietary conditions.     

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.     

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.     

Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants

The compositions of intracellular pentose phosphate pathway enzymes have been examined in mutants of Pachysolen tannophilus NRRL Y-2460 which possessed enhanced D-xylose fermentation rates. The levels of oxidoreductive enzymes involved in converting D-xylose to D-xylulose via xylitol were 1.5–14.7-fold higher in mutants than in the parent. These enzymes were still under inductive control by D-xylose in the mutants. The D-xylose reductase activity (EC 1.1.1.21) which catalyses the conversion of D-xylose to xylitol was supported with either NADPH or NADH as coenzyme in all the mutant strains. Other enzyme specific activities that generally increased were: xylitol dehydrogenase (EC 1.1.1.9), 1.2–1.6-fold; glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 1.9–2.6-fold; D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9), 1.2–2.6-fold; and alcohol dehydrogenase (EC 1.1.1.1), 1.5–2.7-fold. The increase of enzymatic activities, 5.3–10.3-fold, occurring in D-xylulokinase (EC 2.7.1.17), suggested a pivotal role for this enzyme in utilization of D-xylose by these mutants. The best ethanol-producing mutant showed the highest ratio of NADH- to NADPH-linked D-xylose reductase activity and high levels of all other pentose phosphate pathway enzymes assayed.     

Natural and altered induction of the L-fucose catabolic enzymes in Klebsiella aerogenes.

Mutants of Klebsiella aerogenes W70 were isolated that had gained the ability to utilize the uncommon pentose D-arabinose as their sole source of carbon and energy. In contrast to the D-arabinose-negative, parent strain, these mutants were found to be either constitutive for certain enzymes of the L-fucose catabolic pathway or inducible for such enzymes when incubated in the presence of D-arabinose. The mutants used L-fucose isomerase to convert D-arabinose to D-ribulose, which is an intermediate and inducer of the ribitol catabolic pathway. The D-ribulokinase of the ribitol pathway was then induced. This enzyme catalyzed the phosphorylation of D-ribulose at the 5-carbon position. Mutants that were negative for D-ribulokinase could still dissimilate D-arabinose slowly by using all three enzymes, the isomerase, kinase, and aldolase, of the L-fucose pathway. Using condition negative mutants, we were able to demonstrate that the natural induction of the L-fucose pathway enzymes by L-fucose required the activity of a functional L-fucose isomerase and a functional L-fuculokinase but not an L-fuculose-1-phosphate aldolase. A metabolic intermediate, L-fuculose-1-phosphate, was thereby shown to be a probable inducer of at least the isomerase and kinase of the L-fucose catabolic pathway. Similar experiments, with D-arabinose-positive mutants, which were induced for the L-fucose pathway enzymes upon incubation with D-arabinose, revealed that the activities of the L-fucose isomerase and the L-fuculokinase were also required for the induction of the L-fucose enzymes. These D-arabinose-positive mutants apparently produced an altered regulatory protein that accepted both L-fuculose-1-phosphate and D-ribulose-1-phosphate as inducers. Examination of constitutive mutants revealed that L-fucose isomerase and L-fuculokinase were both synthesized constitutively, with the aldolase apparently under separate control.     

Genetic and biochemical studies of nitrate reduction in Aspergillus nidulans

1. In Aspergillus nidulans nitrate and nitrite induce nitrate reductase, nitrite reductase and hydroxylamine reductase, and ammonium represses the three enzymes. 2. Nitrate reductase can donate electrons to a wide variety of acceptors in addition to nitrate. These artificial acceptors include benzyl viologen, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, cytochrome c and potassium ferricyanide. Similarly nitrite reductase and hydroxylamine reductase (which are possibly a single enzyme in A. nidulans) can donate electrons to these same artificial acceptors in addition to the substrates nitrite and hydroxylamine. 3. Nitrate reductase can accept electrons from reduced benzyl viologen in place of the natural donor NADPH. The NADPH-nitrate-reductase activity is about twice that of reduced benzyl viologen-nitrate reductase under comparable conditions. 4. Mutants at six gene loci are known that cannot utilize nitrate and lack nitrate-reductase activity. Most mutants in these loci are constitutive for nitrite reductase, hydroxylamine reductase and all the nitrate-induced NADPH-diaphorase activities. It is argued that mutants that lack nitrate-reductase activity are constitutive for the enzymes of the nitrate-reduction pathway because the functional nitrate-reductase molecule is a component of the regulatory system of the pathway. 5. Mutants are known at two gene loci, niiA and niiB, that cannot utilize nitrite and lack nitrite-reductase and hydroxylamine-reductase activities. 6. Mutants at the niiA locus possess inducible nitrate reductase and lack nitrite-reductase and hydroxylamine-reductase activities. It is suggested that a single enzyme protein is responsible for the reduction of nitrite to ammonium in A. nidulans and that the niiA locus is the structural gene for this enzyme. 7. Mutants at the niiB locus lack nitrate-reductase, nitrite-reductase and hydroxylamine-reductase activities. It is argued that the niiB gene is a regulator gene whose product is necessary for the induction of the nitrate-utilization pathway. The niiB mutants either lack or produce an incorrect product and consequently cannot be induced. 8. Mutants at the niiribo locus cannot utilize nitrate or nitrite unless provided with a flavine supplement. When grown in the absence of a flavine supplement the activities of some of the nitrate-induced enzymes are subnormal. 9. The growth and enzyme characteristics of a total of 123 mutants involving nine different genes indicate that nitrate is reduced to ammonium. Only two possible structural genes for enzymes concerned with nitrate utilization are known. This suggests that only two enzymes, one for the reduction of nitrate to nitrite, the other for the reduction of nitrite to ammonium, are involved in this pathway.     

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.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

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.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known ;overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30-40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine-zinc-insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine-zinc-insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding.     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

In sycamore cells grown on nitrate as opposed to glutamate there is a higher pentose phosphate pathway carbon flux relative to glycolysis in the early stages of cell growth when nitrate assimilation is most active. The high pentose phosphate pathway activity compared with glycolysis in nitrate grown cells is accompanied by enhanced levels of hexokinase, pyruvate kinase, glucose-6-phosphate de-hydrogenase, 6-phosphogluconate dehydrogenase and transketolase. There is no significant increase in activity of the solely glycolytic enzyme, phosphofructokinase. It is suggested that the increased pentose phosphate pathway activity in nitrate grown cells is correlated with a demand by nitrite assimilation for NADPH.II=Jessup and Fowler, 1976 b     

Sedoheptulose-1,7-diphosphate as a source of pentose phosphates in heart muscle]

A possibility and some peculiarities of phosphatase degradation of sedoheptulose-1,7-diphosphate in the myocardium has been demonstrated. The reaction products are inorganic phosphate and sedoheptulose-7-phosphate; the latter product is the substrate of the transketolase reaction during pentose phosphates production. The process is largely localized in the soluble fraction of heart cells; in cellular organelles it is represented in a lesser degree. Possible regulatory mechanisms of pentose phosphate biosynthesis in animal tissues are discussed.     

Characterization of mutations that overcome the toxic effect of glucose on phosphoglucose isomerase less strains of Saccharomyces cerevisiae

Abstract Glucose inhibits growth of yeast phosphoglucose isomerase mutants in permissive media. Mutants insensitive to this effect were isolated by selection on media containing 2% fructose + 2% glucose. A nuclear, monogenic, recessive mutation named rgl was responsible for this phenotype. The mutants isolated belonged to two complementation groups and have been termed rgl1 and rgl2 . When the double mutants were grown on fructose, fermentation of fructose or glucose was similar to that of the parental pgi strain but was not measurable when grown on fructose + glucose. Under these conditions, respiration of glucose and to a lesser extent of fructose was enhanced. The double mutants pgi rgl did not grow on fructose + glucose in the presence of antimycin A or ethidium bromide and their cytochrome oxidase was no longer sensitive to glucose repression. The results are interpreted as an indication that in the double mutants the glucose may be channeled through the pentose phosphate pathway to respiration.     

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.     

Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress

We have isolated several mutants ofSaccharomyces cerevisiae that are sensitive to oxidative stress in a screen for elevated sensitivity to hydrogen peroxide. Two of the sixteen complementation groups obtained correspond to structural genes encoding enzymes of the pentose phosphate pathway. Allelism of thepos10 mutation (POS forperoxidesensitivity) to thezwf1/met1 mutants in the structural gene for glucose 6-phosphate dehydrogenase was reported previously. The second mutation,pos18, was complemented by transformation with a yeast genomic library. The open reading frame of the isolated gene encodes 238 amino acids. No detectable ribulose 5-phosphate epimerase activity was found in thepos18 mutant, suggesting that the corresponding structural gene is affected in this mutant. For that reason the gene was renamedRPE1 (forribulose 5-phosphateepimerase).RPE1 was localized to chromosome X. The predicted protein has a molecular mass of 25 966 Daltons, a codon adaptation index (CAI) of 0.32, and an isoelectric point of 5.82. Database searches revealed 32 to 37% identity with ribulose 5-phosphate epimerases ofEscherichia coli, Rhodospirillum rubrum, Alcaligenes eutrophus andSolanum tuberosum. We have characterizedRPE1 by testing enzyme activities inrpe1 deletion mutants and in strains that overexpressRPE1, and compared the hydrogen peroxide sensitivity ofrpe1 mutants to that of other mutants in the pentose phosphate pathway. Interestingly, all mutants tested (glucose 6-phosphate dehydrogenase, gluconate 6-phosphate dehydrogenase, ribulose 5-phosphate epimerase, transketolase, transaldolase) are sensitive to hydrogen peroxide.