References of "Van Schaftingen, Emile"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailFailure to eliminate a phosphorylated glucose analog leads to neutropenia in patients with G6PT and G6PC3 deficiency
Veiga-da-Cunha, Maria; Chevalier, Nathalie; Stephenne, Xavier et al

in Proceedings of the National Academy of Sciences of the United States of America (2019), 116(4), 1241-1250

Neutropenia presents an important clinical problem in patients with G6PC3 or G6PT deficiency, yet why neutropenia occurs is unclear. We discovered that G6PC3 and G6PT collaborate to dephosphorylate a ... [more ▼]

Neutropenia presents an important clinical problem in patients with G6PC3 or G6PT deficiency, yet why neutropenia occurs is unclear. We discovered that G6PC3 and G6PT collaborate to dephosphorylate a noncanonical metabolite (1,5anhydroglucitol-6-phosphate; 1,5AG6P) which is produced when glucose-phosphorylating enzymes erroneously act on 1,5-anhydroglucitol, a food-derived polyol present in blood. In patients or mice with G6PC3 or G6PT deficiency, 1,5AG6P accumulates and inhibits the first step of glycolysis. This is particularly detrimental in neutrophils, since their energy metabolism depends almost entirely on glycolysis. Consistent with our findings, we observed that treatment with a 1,5anhydroglucitol-lowering drug treats neutropenia in G6PC3deficient mice. Our findings highlight that the elimination of noncanonical side products by metabolite-repair enzymes makes an important contribution to mammalian physiology. [less ▲]

Detailed reference viewed: 90 (5 UL)
Full Text
Peer Reviewed
See detailNit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione
Peracchi, Alessio; Veiga-da-Cunha, Maria; Kuhara, Tomiko et al

in Proceedings of the National Academy of Sciences of the United States of America (2017), 1613736114

The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase ... [more ▼]

The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable “repair enzyme” to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria. [less ▲]

Detailed reference viewed: 67 (0 UL)
Full Text
Peer Reviewed
See detailA conserved phosphatase destroys toxic glycolytic side products in mammals and yeast
Collard, François; Baldin, Francesca; Gerin, Isabelle et al

in Nature Chemical Biology (2016), 12(8), 601-607

Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that ... [more ▼]

Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that may be toxic. In some cases, ‘metabolite repair enzymes’ eliminating side products have been identified. We show that mammalian glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, two core glycolytic enzymes, produce 4-phosphoerythronate and 2-phospho-L-lactate, respectively. 4-Phosphoerythronate strongly inhibits an enzyme of the pentose phosphate pathway, whereas 2-phospho-L-lactate inhibits the enzyme producing the glycolytic activator fructose 2,6-bisphosphate. We discovered that a single, widely conserved enzyme, known as phosphoglycolate phosphatase (PGP) in mammals, dephosphorylates both 4-phosphoerythronate and 2-phospho-L-lactate, thereby preventing a block in the pentose phosphate pathway and glycolysis. Its yeast ortholog, Pho13, similarly dephosphorylates 4-phosphoerythronate and 2-phosphoglycolate, a side product of pyruvate kinase. Our work illustrates how metabolite repair enzymes can make up for the limited specificity of metabolic enzymes and permit high flux in central metabolic pathways. [less ▲]

Detailed reference viewed: 62 (1 UL)
Full Text
Peer Reviewed
See detailEnzyme complexity in intermediary metabolism.
Van Schaftingen, Emile; Veiga-da-Cunha, Maria; Linster, Carole UL

in Journal of inherited metabolic disease (2015), 38(4), 721-7

A good appraisal of the function of enzymes is essential for the understanding of inborn errors of metabolism. However, it is clear now that the 'one gene, one enzyme, one catalytic function' rule ... [more ▼]

A good appraisal of the function of enzymes is essential for the understanding of inborn errors of metabolism. However, it is clear now that the 'one gene, one enzyme, one catalytic function' rule oversimplifies the actual situation. Genes often encode several related proteins, which may differ in their subcellular localisation, regulation or function. Furthermore, enzymes often show several catalytic activities. In some cases, this is because they are multifunctional, possessing two or more different active sites that catalyse different, physiologically related reactions. In enzymes with broad specificity or in multispecificity enzymes, a single type of catalytic site performs the same reaction on different physiological substrates at similar rates. Enzymes that act physiologically in only one reaction often show nonetheless substrate promiscuity: they act at low rates on compounds that resemble their physiological substrate(s), thus forming non-classical metabolites, which are in some cases eliminated by metabolite repair. In addition to their catalytic role, enzymes may have moonlighting functions, i.e. non-catalytic functions that are most often not related with their catalytic activity. Deficiency in such functions may participate in the phenotype of inborn errors of metabolism. Evolution has also made that some enzymes have lost their catalytic activity to become allosteric proteins. [less ▲]

Detailed reference viewed: 115 (5 UL)
Full Text
Peer Reviewed
See detailOccurrence and subcellular distribution of the NAD(P)HX repair system in mammals.
Marbaix, Alexandre Y.; Tyteca, Donatienne; Niehaus, Tom D. et al

in The Biochemical journal (2014), 460(1), 49-58

Hydration of NAD(P)H to NAD(P)HX, which inhibits several dehydrogenases, is corrected by an ATP-dependent dehydratase and an epimerase recently identified as the products of the vertebrate Carkd ... [more ▼]

Hydration of NAD(P)H to NAD(P)HX, which inhibits several dehydrogenases, is corrected by an ATP-dependent dehydratase and an epimerase recently identified as the products of the vertebrate Carkd (carbohydrate kinase domain) and Aibp (apolipoprotein AI-binding protein) genes respectively. The purpose of the present study was to assess the presence of these enzymes in mammalian tissues and determine their subcellular localization. The Carkd gene encodes proteins with a predicted mitochondrial propeptide (mCARKD), a signal peptide (spCARKD) or neither of them (cCARKD). Confocal microscopy analysis of transfected CHO (Chinese-hamster ovary) cells indicated that cCARKD remains in the cytosol, whereas mCARKD and spCARKD are targeted to the mitochondria and the endoplasmic reticulum respectively. Unlike the other two forms, spCARKD is N-glycosylated, supporting its targeting to the endoplasmic reticulum. The Aibp gene encodes two different proteins, which we show to be targeted to the mitochondria (mAIBP) and the cytosol (cAIBP). Quantification of the NAD(P)HX dehydratase and epimerase activities in rat tissues, performed after partial purification, indicated that both enzymes are widely distributed, with total activities of approximately 3-10 nmol/min per g of tissue. Liver fractionation by differential centrifugation confirmed the presence of the dehydratase and the epimerase in the cytosol and in mitochondria. These data support the notion that NAD(P)HX repair is extremely widespread. [less ▲]

Detailed reference viewed: 91 (7 UL)
Full Text
Peer Reviewed
See detailEthylmalonyl-CoA decarboxylase, a new enzyme involved in metabolite proofreading
Linster, Carole UL; Noël, Gaëtane; Stroobant, Vincent et al

in Journal of Biological Chemistry (2011), 286(50), 42992-3003

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few ... [more ▼]

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria. [less ▲]

Detailed reference viewed: 108 (5 UL)
Full Text
Peer Reviewed
See detailExtremely conserved ATP- or ADP-dependent enzymatic system for nicotinamide nucleotide repair
Marbaix, Alexandre Y.; Noël, Gaëtane; Detroux, Aline M. et al

in Journal of Biological Chemistry (2011), 286(48), 41246-52

The reduced forms of NAD and NADP, two major nucleotides playing a central role in metabolism, are continuously damaged by enzymatic or heat-dependent hydration. We report the molecular identification of ... [more ▼]

The reduced forms of NAD and NADP, two major nucleotides playing a central role in metabolism, are continuously damaged by enzymatic or heat-dependent hydration. We report the molecular identification of the eukaryotic dehydratase that repairs these nucleotides and show that this enzyme (Carkd in mammals, YKL151C in yeast) catalyzes the dehydration of the S form of NADHX and NADPHX, at the expense of ATP, which is converted to ADP. Surprisingly, the Escherichia coli homolog, YjeF, a bidomain protein, catalyzes a similar reaction, but using ADP instead of ATP. The latter reaction is ascribable to the C-terminal domain of YjeF. This represents an unprecedented example of orthologous enzymes using either ADP or ATP as phosphoryl donor. We also show that eukaryotic proteins homologous to the N-terminal domain of YjeF (apolipoprotein A-1-binding protein (AIBP) in mammals, YNL200C in yeast) catalyze the epimerization of the S and R forms of NAD(P)HX, thereby allowing, in conjunction with the energy-dependent dehydratase, the repair of both epimers of NAD(P)HX. Both enzymes are very widespread in eukaryotes, prokaryotes, and archaea, which together with the ADP dependence of the dehydratase in some species indicates the ancient origin of this repair system. [less ▲]

Detailed reference viewed: 77 (4 UL)
Full Text
Peer Reviewed
See detailVitamin C. Biosynthesis, recycling and degradation in mammals.
Linster, Carole UL; Van Schaftingen, Emile

in FEBS Journal (2007), 274(1), 1-22

Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this ... [more ▼]

Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate. [less ▲]

Detailed reference viewed: 97 (2 UL)
Full Text
Peer Reviewed
See detailGlucuronate, the precursor of vitamin C, is directly formed from UDP-glucuronate in liver
Linster, Carole UL; Van Schaftingen, Emile

in FEBS Journal (2006), 273(7), 1516-27

The conversion of UDP-glucuronate to glucuronate, usually thought to proceed by way of glucuronate 1-phosphate, is a site for short-term regulation of vitamin C synthesis by metyrapone and other ... [more ▼]

The conversion of UDP-glucuronate to glucuronate, usually thought to proceed by way of glucuronate 1-phosphate, is a site for short-term regulation of vitamin C synthesis by metyrapone and other xenobiotics in isolated rat hepatocytes. Our purpose was to explore the mechanism of this effect in cell-free systems. Metyrapone and other xenobiotics stimulated, by approximately threefold, the formation of glucuronate from UDP-glucuronate in liver extracts enriched with ATP-Mg, but did not affect the formation of glucuronate 1-phosphate from UDP-glucuronate or the conversion of glucuronate 1-phosphate to glucuronate. This and other data indicated that glucuronate 1-phosphate is not an intermediate in glucuronate formation from UDP-glucuronate, suggesting that this reaction is catalysed by a 'UDP-glucuronidase'. UDP-glucuronidase was present mainly in the microsomal fraction, where its activity was stimulated by UDP-N-acetylglucosamine, known to stimulate UDP-glucuronosyltransferases by enhancing the transport of UDP-glucuronate across the endoplasmic reticulum membrane. UDP-glucuronidase and UDP-glucuronosyltransferases displayed similar sensitivities to various detergents, which stimulated at low concentrations and generally inhibited at higher concentrations. Substrates of glucuronidation inhibited UDP-glucuronidase activity, suggesting that the latter is contributed by UDP-glucuronosyltransferase(s). Inhibitors of beta-glucuronidase and esterases did not affect the formation of glucuronate, arguing against the involvement of a glucuronidation-deglucuronidation cycle. The sensitivity of UDP-glucuronidase to metyrapone and other stimulatory xenobiotics was lost in washed microsomes, even in the presence of ATP-Mg, but it could be restored by adding a heated liver high-speed supernatant or CoASH. In conclusion, glucuronate formation in liver is catalysed by a UDP-glucuronidase which is closely related to UDP-glucuronosyltransferases. Metyrapone and other xenobiotics stimulate UDP-glucuronidase by antagonizing the inhibition exerted, presumably indirectly, by a combination of ATP-Mg and CoASH. [less ▲]

Detailed reference viewed: 77 (4 UL)
Full Text
Peer Reviewed
See detailA spectrophotometric assay of D-glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase
Linster, Carole UL; Van Schaftingen, Emile

in Protein Expression & Purification (2004), 37(2), 352-60

Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E. coli BL21(DE3)pLysS cells and purified to near-homogeneity. The kinetic properties of the two enzymes were ... [more ▼]

Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E. coli BL21(DE3)pLysS cells and purified to near-homogeneity. The kinetic properties of the two enzymes were investigated. The isomerase was found to be inhibited by EDTA and to be stimulated by Zn(2+), Co(2+), and Mn(2+), but not by Mg(2+) or Ca(2+). Both enzymes were used to develop a sensitive spectrophotometric assay, in which D-glucuronate is converted to D-mannonate with concomitant oxidation of NADH to NAD(+). The sensitivity of this assay permits the detection of less than 1 nmol D-glucuronate. This assay can also be used to determine the concentration of beta-glucuronides and glucuronate 1-phosphate after enzymatic hydrolysis of these compounds with beta-glucuronidase or alkaline phosphatase. [less ▲]

Detailed reference viewed: 82 (2 UL)
Full Text
Peer Reviewed
See detailRapid stimulation of free glucuronate formation by non-glucuronidable xenobiotics in isolated rat hepatocytes
Linster, Carole UL; Van Schaftingen, Emile

in Journal of Biological Chemistry (2003), 278(38), 36328-33

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the ... [more ▼]

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle. [less ▲]

Detailed reference viewed: 96 (4 UL)