Production of l-ribose from l-arabinose by co-expression of l-arabinose isomerase and d-lyxose isomerase in Escherichia coli.

l-Ribose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs. However, it is a non-natural and expensive rare sugar. Recently, the enzymatic synthesis of l-ribose has attracted considerable attention owing to its considerable advantages over chemical approaches. In this work, a new strategy was developed for the production of l-ribose from the inexpensive starting material l-arabinose. The l-arabinose isomerase (l-AIase) gene from Alicyclobacillus hesperidum and the d-lyxose isomerase (d-LIase) gene from Thermoflavimicrobium dichotomicum were cloned and co-expressed in Escherichia coli, resulting in recombinant cells harboring the vector pCDFDuet-Alhe-LAI/Thdi-DLI. The co-expression system exhibited optimal activity at a temperature of 70 °C and pH 6.0, and the addition of Co2+ enhanced the catalytic activity by 27.8-fold. The system containing 50 g L-1 of recombinant cells were relatively stable up to 55 °C. The co-expression system (50 g L-1 of recombinant cells) afforded 20.9, 39.7, and 50.3 g L-1 of l-ribose from initial l-arabinose concentrations of 100, 300, and 500 g L-1, corresponding to conversion rate of 20.9%, 13.2%, and 10.0%, respectively. Overall, this study provides a viable approach for producing l-ribose from l-arabinose under slightly acidic conditions using a co-expression system harboring l-AIase and d-LIase genes.

A plant/fungal-type phosphoenolpyruvate carboxykinase located in the parasite mitochondrion ensures glucose-independent survival of Toxoplasma gondii.

Toxoplasma gondii is considered to be one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host cell types of essentially any warm-blooded organism. Our earlier work demonstrated that the acute (tachyzoite) stage of T. gondii depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not yet been resolved. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for tachyzoite survival. TgPEPCKnet can also be ablated in a glycolysis-deficient mutant, while TgPEPCKmt is refractory to deletion. Consistent with this, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCKmt in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function ofTgPEPCKmt in cohesive operation of glycolysis and the tricarboxylic acid cycle in a normal glucose-replete milieu. Conversely, we found that the otherwise integrative enzyme pyruvate carboxylase (TgPyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of T. gondii tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.

The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels.

Inactivation of the TCA cycle enzyme, fumarate hydratase (FH), drives a metabolic shift to aerobic glycolysis in FH-deficient kidney tumors and cell lines from patients with hereditary leiomyomatosis renal cell cancer (HLRCC), resulting in decreased levels of AMP-activated kinase (AMPK) and p53 tumor suppressor, and activation of the anabolic factors, acetyl-CoA carboxylase and ribosomal protein S6. Reduced AMPK levels lead to diminished expression of the DMT1 iron transporter, and the resulting cytosolic iron deficiency activates the iron regulatory proteins, IRP1 and IRP2, and increases expression of the hypoxia inducible factor HIF-1α, but not HIF-2α. Silencing of HIF-1α or activation of AMPK diminishes invasive activities, indicating that alterations of HIF-1α and AMPK contribute to the oncogenic growth of FH-deficient cells.

Abnormalities in glucose uptake and metabolism in imatinib-resistant human BCR-ABL-positive cells.

The development of imatinib resistance has become a significant therapeutic problem in which the etiology seems to be multifactorial and poorly understood. As of today, clinical criteria to predict the development of imatinib resistance in chronic myelogenous leukemia (CML), other than rebound of the myeloproliferation, are under development. However, there is evidence that the control of glucose-substrate flux is an important mechanism of the antiproliferative action of imatinib because imatinib-resistant gastrointestinal stromal KIT-positive tumors reveal highly elevated glucose uptake in radiologic images. We used nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry to assess (13)C glucose uptake and metabolism (glycolysis, TCA cycle, and nucleic acid ribose synthesis) during imatinib treatment in CML cell lines with different sensitivities to imatinib. Our results show that sensitive K562-s and LAMA84-s BCR-ABL-positive cells have decreased glucose uptake, decreased lactate production, and an improved oxidative TCA cycle following imatinib treatment. The resistant K562-r and LAMA84-r cells maintained a highly glycolytic metabolic phenotype with elevated glucose uptake and lactate production. In addition, oxidative synthesis of RNA ribose from (13)C-glucose via glucose-6-phosphate dehydrogenase was decreased, and RNA synthesis via the nonoxidative transketolase pathway was increased in imatinib-resistant cells. CML cells which exhibited a (oxidative/nonoxidative) flux ratio for nucleic acid ribose synthesis of >1 were sensitive to imatinib. The resistant K562-r and LAMA84-r exhibited a (oxidative/nonoxidative) flux ratio of <0.7. The changes in glucose uptake and metabolism were accompanied by intracellular translocation of GLUT-1 from the plasma membrane into the intracellular fraction in sensitive cells treated with imatinib, whereas GLUT-1 remained located at the plasma membrane in LAMA84-r and K562-r cells. The total protein load of GLUT-1 was unchanged among treated sensitive and resistant cell lines. In summary, elevated glucose uptake and nonoxidative glycolytic metabolic phenotype can be used as sensitive markers for early detection of imatinib resistance in BCR-ABL-positive cells.

Nutrient-gene interaction: tracer-based metabolomics.

Understanding nutrient-gene interaction requires tools for both the study of nutrigenomics and the characterization of phenotype. Metabolomics or metabolite profiling is a powerful tool for characterizing metabolic phenotype, and tracer-based metabolomics is a subset of metabolomics that focuses on metabolite distribution and flux determination using tracers. In this review, the characterizations of metabolic phenotype by metabolite profiling and by metabolic flux measurements are compared. The rationale and methodologies of tracer-based metabolomics are explained. Tracer-based metabolomics provides a relational database of metabolites linked by the relationship of shared metabolic pathways, common substrates, and cofactors. Such a collection of flux measurements provides precise and accurate information on the operation of the cellular metabolic network and its response to genetic and nutrient environment changes. Nutrient-gene interaction can be studied using the concept of constraint-based modeling, which states that the observed metabolic phenotype is a consequence of constraints from genetic factors and the nutrient environment. Thus, genetic inheritance (genomic constraints) confers a wide range of possible phenotypes whereas selection by metabolic (structural and pathway relationship) and environmental (physical environment and nutrient availability) constraints determines the final observed phenotype. The study of the contribution from nutrient and genetic factors to the survival advantage of cancer cells using flux measurements is a critical first step in our understanding of the relationship between nutrient intake and cancer risk.

Metabolic control analysis aimed at the ribose synthesis pathways of tumor cells: a new strategy for antitumor drug development.

Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with lower concentrations of drug, if an enzyme with a high control coefficient on tumor growth is being inhibited. Here we measure glucose-6-phosphate dehydrogenase (G6PDH) control coefficient on in vivo tumor growth using mice bearing Ehrlich ascites tumor cells. We used dehydroepiandrosterone-sulphate (DHEA-S), an uncompetitive inhibitor of this enzyme and the in situ cytochemical method to measure the enzyme activity changes that accompany changes on tumor cell growth. This method ensures that the enzyme activity determined is the one existing in the in situ conditions and enables computing a control coefficient in in situ conditions. From the data obtained on tumor cell number and the in situ enzyme activities in absence and presence of DHEA-S, a control coefficient of 0.41 for G6PDH on tumor cell growth was computed. This value is approximately the half of the transketolase control coefficient value of 0.9 previously reported. Moreover, the use of in situ methods to assess enzyme activities, applied for first time for the calculation of control coefficients in this study, opens new avenues to the use of uncompetitive inhibitors for the measurement of in situ control coefficients.

Trm7p catalyses the formation of two 2'-O-methylriboses in yeast tRNA anticodon loop.

The genome of Saccharomyces cerevisiae encodes three close homologues of the Escherichia coli 2'-O-rRNA methyltransferase FtsJ/RrmJ, designated Trm7p, Spb1p and Mrm2p. We present evidence that Trm7p methylates the 2'-O-ribose of nucleotides at positions 32 and 34 of the tRNA anticodon loop, both in vivo and in vitro. In a trm7Delta strain, which is viable but grows slowly, translation is impaired, thus indicating that these tRNA modifications could be important for translation efficiency. We discuss the emergence of a family of three 2'-O-RNA methyltransferases in Eukaryota and one in Prokaryota from a common ancestor. We propose that each eukaryotic enzyme is located in a different cell compartment, in which it would methylate a different RNA that can adopt a very similar secondary structure.

Wheat germ extract decreases glucose uptake and RNA ribose formation but increases fatty acid synthesis in MIA pancreatic adenocarcinoma cells.

The fermented wheat germ extract with standardized benzoquinone composition has potent tumor propagation inhibitory properties. The authors show that this extract induces profound metabolic changes in cultured MIA pancreatic adenocarcinoma cells when the [1,2-13C2]glucose isotope is used as the single tracer with biologic gas chromatography-mass spectrometry. MIA cells treated with 0.1, 1, and 10 mg/mL wheat germ extract showed a dose-dependent decrease in cell glucose consumption. uptake of isotope into ribosomal RNA (2.4%, 9.4%, and 28.0%), and release of 13CO2. Conversely, direct glucose oxidation and ribose recycling in the pentose cycle showed a dose-dependent increase of 1.2%, 20.7%, and 93.4%. The newly synthesized fraction of cell palmitate and the 13C enrichment of acetyl units were also significantly increased with all doses of wheat germ extract. The fermented wheat germ extract controls tumor propagation primarily by regulating glucose carbon redistribution between cell proliferation-related and cell differentiation-related macromolecules. Wheat germ extract treatment is likely associated with the phosphorylation and transcriptional regulation of metabolic enzymes that are involved in glucose carbon redistribution between cell proliferation-related structural and functional macromolecules (RNA, DNA) and the direct oxidative degradation of glucose, which have devastating consequences for the proliferation and survival of pancreatic adenocarcinoma cells in culture.

Genistein inhibits nonoxidative ribose synthesis in MIA pancreatic adenocarcinoma cells: a new mechanism of controlling tumor growth.

Genistein is a plant isoflavonoid bearing potent tumor growth-regulating characteristics. This effect of genistein has been attributed partially to its tyrosine kinase-regulating properties, resulting in cell-cycle arrest and limited angiogenesis. Genistein has been used in chemotherapy-resistant cases of advanced leukemia with promising results. Here we demonstrate that genistein primarily affects nucleic acid synthesis and glucose oxidation in tumor cells using the [1,2-(13)C2]glucose isotope as the single tracer and gas chromatography/mass spectrometry to follow various intracellular glucose metabolites. The ribose fraction of RNA demonstrated a rapid 4.6%, 16.4%, and 46.3% decrease in isotope uptake through the nonoxidative branch of the pentose cycle and a sharp 4.8%. 24.6%, and 48% decrease in 13CO2 release from glucose after 2, 20, and 200 micromol/L genistein treatment, respectively. Fatty acid synthesis and the 13C enrichment of acetyl units were not significantly affected by genistein treatment. De novo glycogen synthesis from media glucose was not detected in cultured MIA cells. It can be concluded from these studies that genistein controls tumor growth primarily through the regulation of glucose metabolism, specifically targeting glucose carbon incorporation into nucleic acid ribose through the nonoxidative steps of the pentose cycle, which represents a new paradigm for the antiproliferative action of a plant phytochemical.

Nonoxidative pentose phosphate pathways and their direct role in ribose synthesis in tumors: is cancer a disease of cellular glucose metabolism?

Pentose phosphate pathways (PPP) are considered important in tumor proliferation processes because of their role in supplying tumor cells with reduced NADP and carbons for intracellular anabolic processes. Direct involvement of PPP in tumor DNA/RNA synthesis is not considered as significant as in lipid and protein syntheses. Currently, PPP activity in tumor cells is measured by lactate production, which shows a moderate activity: about 4% to 7% compared with glycolysis. Recent data generated in our laboratory indicate that PPP are directly involved in ribose synthesis in pancreatic adenocarcinoma cells, through oxidative steps (< 31%) and transketolase reactions (69%). These findings raise serious questions about the adequacy of lactate in measuring PPP activity in tumors. We hypothesize that ribose, not lactate, is the major product of PPP in tumor cells. Control of both oxidative and nonoxidative PPP may be critical in the treatment of cancer. PPP are substantially involved in the proliferation of human tumors, which raises the prospect of new treatment strategies targeting specific biochemical reactions of PPP by hormones related to glucose metabolism, controlling thiamine intake, the cofactor of the nonoxidative transketolase PPP reaction, or treating cancer patients with antithiamine analogues.

Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation.

This study investigates the significance of the glucose-6-phosphate dehydrogenase (G6PD) catalyzed oxidative and the transketolase (TK) catalyzed nonoxidative pentose cycle (PC) reactions in the tumor proliferation process by characterizing tumor growth patterns and synthesis of the RNA ribose moiety in the presence of respective inhibitors of G6PD and TK. Mass spectra analysis of 13C-labeled carbons revealed that these PC reactions contribute to over 85% of de novo ribose synthesis in RNA from [1,2-(13)C]glucose in cultured Mia pancreatic adenocarcinoma cells, with the fraction synthesized through the TK pathway predominating (85%). Five days of treatment with the TK inhibitor oxythiamine (OT) and the G6PD inhibitor dehydroepiandrosterone-sulfate (0.5 microM each) exerted a 39 and a 23% maximum inhibitory effect on cell proliferation in culture, which was increased to 60% when the two drugs were administered in combination. In vivo testing of 400 mg/kg OT or dehydroepiandrosterone-sulfate in C57BL/6 mice hosting Ehrlich's ascitic tumor cells revealed a 90.4 and a 46% decrease in the final tumor mass after 3 days of treatment. RNA ribose fractional synthesis through the TK reaction using metabolites directly from glycolysis declined by 9.1 and 23.9% after OT or the combined treatment, respectively. Nonoxidative PC reactions play a central regulating role in the carbon-recruiting process toward de novo nucleic acid ribose synthesis and cell proliferation in vitro and in vivo. Therefore, enzymes or substrates regulating the nonoxidative synthesis of ribose could also be the sites to preferentially target tumor cell proliferation by new anticancer drugs.

Production of D-ribose by fermentation.

The production of D-ribose by fermentation has received much attention lately, possibly because of the use of this pentose to synthesize antiviral and anticancer drugs. This review briefly outlines the methods that have been used to synthesize D-ribose since it was identified in yeast RNA, and focuses in particular on the latest developments in D-ribose fermentation, which have led to D-ribose yields that exceed 90 g/1. Furthermore, the various transketolase-deficient D-ribose-producing mutants that are used, and the biochemical and genetic rationales applied to select them or to enhance their D-ribose productivities, are dealt with. Attention is also drawn to the unusual pleiotropic characteristics of the mutant strains, as well as to the industrial and academic applications of D-ribose.

Incorporation of glucose carbon into ribonucleotides of axenic Entamoeba histolytica.

Axenic Entamoeba histolytica grown with (UL-14C)glucose in TP-S-1 medium were capable of biosynthesizing ribose from labeled glucose. RNA isolated by phenol extraction was hydrolyzed to the ribonucleotide level by alkaline hydrolysis. The hydrolysate, chromatographed on ion exchange resins, yielded AMP, GMP, and UMP, but not CMP containing labeled glucose carbon. The present nucleotide composition of the isolated amebal RNA was, respectively, as follows, CMP, 0.20; GMP, 0.22; AMP, 0.30; UMP, 0.29. The location of all the radiolabel in each ribonucleotide was the ribose moiety. The relative specific incorporation of glucose carbon into AMP, GMP, and UMP was 0.47, 0.05, and 0.10, respectively. These results suggest that the bulk of amebal nucleic acid precursors are obtained as preformed nucleosides and/or nucleotides from TP-S-1 medium. The mean RNA content per milliliter packed cells of amebae was 4.2 +/- 0.2 mg.

Adenylation and ADP-ribosylation in the mouse 1-cell embryo.

The incorporation of [3H]AAadenosine into cold trichloroacetic acid (TCA) insoluble material by the mouse 1-cell embryo has been studied. Incorporation of label was high immediately after fertilization, then decreased over the next 7 h with the sharpest decline occurring 3-5 h after fertilization. A small maximum was observed at the time of pronuclear DNA synthesis. Actinomycin D at a concentration which inhibited the cleavage of 1-cell embryos by 50% had little effect on this incorporation, which in the period 1-6 h post-fertilization was shown by autoradiography to be confined to the ooplasm of the newly fertilized ovum. [3H]Adenosine and poly ([3H]A) were released from embryo RNA labelled 1-3 h after fertilization with [3H]adenosine by digestion with a mixture of ribonucleases A and T1. The poly ([3H]A) segments were hydrolysed by alkali to 3'-[3H]AMP and [3H]adenosine ([3H]AMP/[3H]adenosine = 5/1), and by snake venom phosphodiesterase to 5'-[3H]AMP but very little [3H]adenosine. These results suggest that adenylation of RNA occurs soon after fertilization, that this is a cytoplasmic event, and that most of the newly synthesized poly ([3H]A) segments are joined to pre-existing poly (A) tracts. The unusual polynucleotide, poly (ADP-ribose), identified by its resistance to alkali and the release of 2'-(5''-phosphoribosyl)-5'[3H]AMP on incubation with snake venom phosphodiesterase, was also found in the ribonuclease digest.

Metabolic consequences of a block in the synthesis of 5-keto-D-fructose in a mutant of Gluconobacter cerinus.

A mutant of Gluconobacter cerinus var. ammoniacus, IFO 3267, has been isolated which is deficient with respect to fructose 5-dehydrogenase, the enzyme catalyzing the oxidation of d-fructose to 5-keto-d-fructose (5 KF). Growth of this mutant on fructose as the sole carbon source was impaired unless the culture medium was supplemented with 5 KF. Significant randomization of the 1 and 6 positions of fructose has been reported previously for the wild-type organism during growth on this ketohexose. The pattern of (3)H incorporation into the C5 position of ribonucleic acid-ribose when the mutant was grown on [1-(3)H]fructose and [6-(3)H]fructose in the presence of 5 KF indicated that such randomization did not occur in this variant. The randomization observed in the wild type is, therefore, a consequence of the partial oxidation of fructose to the symmetrical 5 KF intermediate prior to its conversion to pentose. When the mutant was grown on [1-(3)H]fructose in the presence of unlabeled 5 KF, [5-(3)H]fructose appeared in the culture medium. Thus, 5 KF served as the oxidant for the nicotinamide adenine dinucleotide phosphate, reduced form, generated during growth on fructose.