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Frontiers in Neuroscience 2019The profile of 122 metabolites in the cerebrospinal fluid (CSF) of patients suffering from Alzheimer's disease (AD) and controls was studied. Among the 122 metabolites...
The profile of 122 metabolites in the cerebrospinal fluid (CSF) of patients suffering from Alzheimer's disease (AD) and controls was studied. Among the 122 metabolites analyzed, 61 could be detected. Statistically significant differences between the AD and control group were only detected for metabolites of the glycolysis. Thus, accurate quantification of 11 glycolytic metabolites was done. We detected a significant reduction of five of them, namely phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate, pyruvate and dihydroxyacetone phosphate in the AD CSF compared to controls. These results correlate with the known reduction of glucose metabolism in the brain of patients with AD and indicate that metabolic analysis of the central carbon metabolism can be a potential tool in AD diagnostic. Although the Receiver operating characteristic (ROC) analyses of the metabolites do not reach the level of the diagnostic informativity of AD biomarkers, the combination of specific glycolysis metabolites with the established biomarkers may lead to an improvement in sensitivity and specificity.
PubMed: 31496932
DOI: 10.3389/fnins.2019.00871 -
The Journal of Biological Chemistry Feb 2020The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C precursors...
The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C precursors (such as ribose) during growth on other (non-C) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PP-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PP-PFKs of and C indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of , which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PP-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.
Topics: Clostridiales; Clostridium thermocellum; Dihydroxyacetone Phosphate; Escherichia coli; Fructose-Bisphosphate Aldolase; Fructosephosphates; Kinetics; Pentose Phosphate Pathway; Pentoses; Phosphofructokinase-1; Phosphotransferases; Ribose; Sugar Phosphates; Transaldolase; Xylose
PubMed: 31871051
DOI: 10.1074/jbc.RA119.011239 -
Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells.The Journal of Biological Chemistry May 2020Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell...
Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.
Topics: A549 Cells; Diphosphoglyceric Acids; Glucose; Glyceric Acids; Glycolysis; HeLa Cells; Humans; Kinetics; Lactic Acid; Neoplasm Proteins; Neoplasms; Phosphoglycerate Kinase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32217690
DOI: 10.1074/jbc.RA119.012312 -
Cellular and Molecular Life Sciences :... Jul 2022Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency,...
Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.
Topics: Amino Acids; Cytosol; Humans; Kinetics; Substrate Specificity; Sugars; Transaminases
PubMed: 35834009
DOI: 10.1007/s00018-022-04439-3 -
IScience Dec 2022D-Tagatose is a promising low-calorie sugar-substituting sweetener in the food industry. Most ingested D-tagatose is fermented by intestinal microorganisms. Until now,...
D-Tagatose is a promising low-calorie sugar-substituting sweetener in the food industry. Most ingested D-tagatose is fermented by intestinal microorganisms. Until now, has been considered incapable of growing on D-tagatose. Here, we discovered a gene cluster involved in D-tagatose utilization in . The chromosome of the intestinal probiotic Nissle 1917 contains a six-gene cluster encoding the ABC transporter, D-tagatose kinase, D-tagatose-bisphosphate aldolase, and putative aldose 1-epimerase. The functionality of the gene cluster was experimentally validated. Based on single-gene deletions, D-tagatose dissimilation occurs via D-tagatose 6-phosphate to D-tagatose 1,6-bisphosphate to D-glyceraldehyde 3-phosphate plus dihydroxyacetone phosphate. Remarkably, this gene cluster was located in 93% of the completely sequenced genomes of the B2 phylogroup, which contains the majority of extraintestinal pathogenic and adherent-invasive strains prevalent in patients with inflammatory bowel disease. This highlights the importance of understanding the clinical significance of D-tagatose in microbiota alterations.
PubMed: 36479150
DOI: 10.1016/j.isci.2022.105655 -
RSC Advances Jan 2023To realize sustainable societies, the production of organic compounds not based on fossil resources should be developed. Thus, C1 chemistry, utilizing one-carbon...
To realize sustainable societies, the production of organic compounds not based on fossil resources should be developed. Thus, C1 chemistry, utilizing one-carbon compounds as starting materials, has been of increasing importance. In particular, the formose reaction is promising because the reaction produces sugars (monosaccharides) from formaldehyde under basic conditions. On the other hand, since microwave (MW) induces the rotational motion of molecules, MW irradiation often improves the selectivity and efficiency of reactions. In this study, the formose reaction under MW irradiation was thus investigated under various conditions. The formose reaction proceeded very fast using 1.0 mol per kg formaldehyde and 55 mmol per kg calcium hydroxide (Ca(OH)) as a catalyst at a high set temperature (150 °C) for a short time (1 min) to form preferentially specific hexose and heptose. The major products were isolated by thin layer chromatography and characterized by mass spectroscopy and NMR. These characterization data elucidated that the hexose and heptose were 2-hydroxymethyl-1,2,4,5-tetrahydroxy-3-pentanone (C6*) and 2,4-bis(hydroxymethyl)-1,2,4,5-tetrahydroxy-3-pentanone (C7*), respectively. On the basis of these observations, as well as density functional theory calculations, a plausible reaction pathway was also discussed; once 1,3-dihydroxyacetone is formed, consecutive aldol reactions favorably occur to form C6* and C7*.
PubMed: 36756559
DOI: 10.1039/d2ra07249a -
Magma (New York, N.Y.) Feb 2021To enhance detection of the products of hyperpolarized [2-C]dihydroxyacetone metabolism for assessment of three metabolic pathways in the liver in vivo. Hyperpolarized...
OBJECTIVES
To enhance detection of the products of hyperpolarized [2-C]dihydroxyacetone metabolism for assessment of three metabolic pathways in the liver in vivo. Hyperpolarized [2-C]DHAc emerged as a promising substrate to follow gluconeogenesis, glycolysis and the glycerol pathways. However, the use of [2-C]DHAc in vivo has not taken off because (i) the chemical shift range of [2-C]DHAc and its metabolic products span over 144 ppm, and (ii) H decoupling is required to increase spectral resolution and sensitivity. While these issues are trivial for high-field vertical-bore NMR spectrometers, horizontal-bore small-animal MR scanners are seldom equipped for such experiments.
METHODS
Real-time hepatic metabolism of three fed mice was probed by H-decoupled C-MR following injection of hyperpolarized [2-C]DHAc. The spectra of [2-C]DHAc and its metabolic products were acquired in a 7 T small-animal MR scanner using three purpose-designed spectral-spatial radiofrequency pulses that excited a spatial bandwidth of 8 mm with varying spectral bandwidths and central frequencies (chemical shifts).
RESULTS
The metabolic products detected in vivo include glycerol 3-phosphate, glycerol, phosphoenolpyruvate, lactate, alanine, glyceraldehyde 3-phosphate and glucose 6-phosphate. The metabolite-to-substrate ratios were comparable to those reported previously in perfused liver.
DISCUSSION
Three metabolic pathways can be probed simultaneously in the mouse liver in vivo, in real time, using hyperpolarized DHAc.
Topics: Animals; Carbon Isotopes; Dihydroxyacetone; Gluconeogenesis; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Mice; Protons
PubMed: 32910316
DOI: 10.1007/s10334-020-00884-y -
Current Research in Neurobiology 2022Triosephosphate isomerase deficiency (TPI Df) is a rare, aggressive genetic disease that typically affects young children and currently has no established treatment. TPI...
Triosephosphate isomerase deficiency (TPI Df) is a rare, aggressive genetic disease that typically affects young children and currently has no established treatment. TPI Df is characterized by hemolytic anemia, progressive neuromuscular degeneration, and a markedly reduced lifespan. The disease has predominately been studied using invertebrate and models, which lack key aspects of the human disease. While other groups have generated mammalian mutant strains, specifically with the mouse these do not recapitulate key characteristic phenotypes of the human disease. Reported here is the generation of a novel murine model of TPI Df. CRISPR-Cas9 was utilized to engineer the most common human disease-causing mutation, and mice were also isolated as a frame-shifting deletion. mice experience a markedly shortened lifespan, postural abnormalities consistent with extensive neuromuscular dysfunction, hemolytic anemia, pathological changes in spleen, and decreased body weight. There is a ∼95% reduction in TPI protein levels in animals compared to wild-type littermates, consistent with decreased TPI protein stability, a known cause of TPI Df. This work illustrates the capability of mice to serve as a mammalian model of human TPI Df. This work will allow for advancement in the study of TPI Df within a model with physiology similar to humans. The development of the model reported here will enable mechanistic studies of disease pathogenesis and, importantly, efficacy testing in a mammalian system for emerging TPI Df treatments.
PubMed: 36405628
DOI: 10.1016/j.crneur.2022.100062 -
The Journal of Nutrition Apr 2020The association between high selenium (Se) intake and metabolic disorders such as type 2 diabetes has raised great concern, but the underlying mechanism remains unclear.
BACKGROUND
The association between high selenium (Se) intake and metabolic disorders such as type 2 diabetes has raised great concern, but the underlying mechanism remains unclear.
OBJECTIVE
Through targeted metabolomics analysis, we examined the liver sugar and acylcarnitine metabolism responses to supranutritional selenomethionine (SeMet) supplementation in pigs.
METHODS
Thirty-six castrated male pigs (Duroc-Landrace-Yorkshire, 62.0 ± 3.3 kg) were fed SeMet adequate (Se-A, 0.25 mg Se/kg) or SeMet supranutritional (Se-S, 2.5 mg Se/kg) diets for 60 d. The Se concentration, biochemical, gene expression, enzyme activity, and energy-targeted metabolite profiles were analyzed.
RESULTS
The Se-S group had greater fasting serum concentrations of glucose (1.9-fold), insulin (1.4-fold), and free fatty acids (FFAs,1.3-fold) relative to the Se-A group (P < 0.05). The liver total Se concentration was 4.2-fold that of the Se-A group in the Se-S group (P < 0.05), but expression of most selenoprotein genes and selenoenzyme activity did not differ between the 2 groups. Seven of 27 targeted sugar metabolites and 4 of 21 acylcarnitine metabolites significantly changed in response to high SeMet (P < 0.05). High SeMet supplementation significantly upregulated phosphoenolpyruvate carboxy kinase (PEPCK) activity by 64.4% and decreased hexokinase and succinate dehydrogenase (SDH) activity by 46.5-56.7% (P < 0.05). The relative contents of glucose, dihydroxyacetone phosphate, α-ketoglutarate, fumarate, malate, erythrose-4-phosphate, and sedoheptulose-7-phosphate in the Se-S group were 21.1-360% greater than those in the Se-A group (P < 0.05). The expression of fatty acid synthase (FASN) and the relative contents of carnitine, hexanoyl-carnitine, decanoyl-carnitine, and tetradecanoyl-carnitine in the Se-S group were 35-97% higher than those in the Se-A group (P < 0.05).
CONCLUSIONS
Dietary high SeMet-induced hyperglycemia and hyperinsulinemia were associated with suppression of sugar metabolism and elevation of lipid synthesis in pig livers. Our research provides novel insights into high SeMet intake-induced type 2 diabetes.
Topics: Animals; Carnitine; Diabetes Mellitus, Type 2; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Homeostasis; Hyperglycemia; Hyperinsulinism; Lipids; Liver; Male; Metabolomics; Models, Animal; Oxidation-Reduction; RNA, Messenger; Selenium; Selenomethionine; Selenoproteins; Sugars; Sus scrofa
PubMed: 32060554
DOI: 10.1093/jn/nxz317 -
Biochemistry May 2022The cationic K120 and K204 side chains lie close to the C-2 carbonyl group of substrate dihydroxyacetone phosphate (DHAP) at the active site of glycerol-3-phosphate...
The cationic K120 and K204 side chains lie close to the C-2 carbonyl group of substrate dihydroxyacetone phosphate (DHAP) at the active site of glycerol-3-phosphate dehydrogenase (GPDH), and the K120 side chain is also positioned to form a hydrogen bond to the C-1 hydroxyl of DHAP. The kinetic parameters for unactivated and phosphite dianion-activated GPDH-catalyzed reduction of glycolaldehyde and acetaldehyde (AcA) show that the transition state for the former reaction is stabilized by 5 kcal/mole by interactions of the C-1 hydroxyl group with the protein catalyst. The K120A and K204A substitutions at wild-type GPDH result in similar decreases in , but is only affected by the K120A substitution. These results are consistent with 3 kcal/mol stabilizing interactions between the K120 or K204 side chains and a negative charge at the C-2 oxygen at the transition state for hydride transfer from NADH to DHAP. This stabilization resembles that observed at oxyanion holes for other enzymes. There is no detectable rescue of the K204A variant by ethylammonium cation (EtNH), compared with the efficient rescue of the K120A variant. This is consistent with a difference in the accessibility of the variant enzyme active sites to exogenous EtNH. The K120A/K204A substitutions cause a (6 × 10)-fold increase in the promiscuity of wild-type GPDH for catalysis of the reduction of AcA compared to DHAP. This may reflect conservation of the active site for an ancestral alcohol dehydrogenase, whose relative activity for catalysis of reduction of AcA increases with substitutions that reduce the activity for reduction of the specific substrate DHAP.
Topics: Catalysis; Catalytic Domain; Dihydroxyacetone Phosphate; Glycerolphosphate Dehydrogenase; Kinetics
PubMed: 35502876
DOI: 10.1021/acs.biochem.2c00053