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See detailAn alternative role of FoF1-ATP synthase in Escherichia coli: synthesis of thiamine triphosphate
Gigliobianco, Tiziana ULiege; GANGOLF, Marjorie ULiege; Lakaye, Bernard ULiege et al

in Scientific Reports (2013), 3(1071),

In E. coli, thiamine triphosphate (ThTP), a putative signaling molecule, transiently accumulates in response to amino acid starvation. This accumulation requires the presence of an energy substrate ... [more ▼]

In E. coli, thiamine triphosphate (ThTP), a putative signaling molecule, transiently accumulates in response to amino acid starvation. This accumulation requires the presence of an energy substrate yielding pyruvate. Here we show that in intact bacteria ThTP is synthesized from free thiamine diphosphate (ThDP) and Pi, the reaction being energized by the proton-motive force (Dp) generated by the respiratory chain. ThTP production is suppressed in strains carrying mutations in F1 or a deletion of the atp operon. Transformation with a plasmid encoding the whole atp operon fully restored ThTP production, highlighting the requirement for FoF1-ATP synthase in ThTP synthesis. Our results show that, under specific conditions of nutritional downshift, FoF1-ATP synthase catalyzes the synthesis of ThTP, rather than ATP, through a highly regulated process requiring pyruvate oxidation. Moreover, this chemiosmotic mechanism for ThTP production is conserved from E. coli to mammalian brain mitochondria. [less ▲]

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See detailThiamine triphosphatase and the CYTH family of proteins
Bettendorff, Lucien ULiege; Wins, Pierre

in FEBS Journal (2013), 280(24), 64436455

The CYTH superfamily of proteins was named after its two founding members, the CYaB adenylyl cyclase from Aeromonas hydrophila and the human 25 kDa THiamine triphosphatase (ThTPase). Members of this ... [more ▼]

The CYTH superfamily of proteins was named after its two founding members, the CYaB adenylyl cyclase from Aeromonas hydrophila and the human 25 kDa THiamine triphosphatase (ThTPase). Members of this superfamily of proteins exist in all organisms including bacteria, archaea, fungi, plants and animals (except birds) and can be traced back to the Last Universal Common Ancestor. Their sequences include several charged residues involved in divalent cation and triphosphate binding. Indeed, all members of the CYTH family that have been characterized act on triphosphorylated substrates and require at least one divalent metal cation for catalysis. In most cases, the enzyme-substrate complex adopts a tunnel-like (ß-barrel) conformation. The Nitrosomonas europaea and E.coli CYTH proteins are specific inorganic triphosphatases. We propose that inorganic triphosphate, the simplest triphosphate compound, is the primitive substrate of CYTH proteins. Other enzyme activities such as adenylate cyclase (in A. hydrophila and Y. pestis), mRNA triphosphatase (in fungi and protozoans) and ThTPase (in metazoans) are secondary acquisitions. ThTPase activity is not limited to mammals, but sea anemone and zebrafish CYTH proteins are already specific ThTPases and the acquisition of this enzyme activity is linked to the presence of a Trp (W53 in mammalian ThTPases) residue involved in the binding of the thiazole heterocycle of the thiamine molecule. Furthermore, we propose a conserved catalytic mechanism between a bacterial inorganic triphosphatase and metazoan ThTPases, based on a catalytic dyad comprising a Lys and a Tyr residue, explaining the alkaline pH optimum of these enzymes. [less ▲]

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See detailCritical Review: Biological functions of thiamine derivatives: Focus on non-coenzyme roles
Bettendorff, Lucien ULiege; Wins, Pierre

in OA Biochemistry (2013), 1(1), 10

Thiamine (vitamin B1) is mainly known for its diphosphorylated derivatives (ThDP), an essential coenzyme in energy metabolism. However non-coenzyme roles have been suggested for this vitamin for many ... [more ▼]

Thiamine (vitamin B1) is mainly known for its diphosphorylated derivatives (ThDP), an essential coenzyme in energy metabolism. However non-coenzyme roles have been suggested for this vitamin for many years. Such roles have remained hypothetical, but recent data from various sources have shed a new light on this hypothesis. First, the existence of other phosphorylated thiamine derivatives, most prominently thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP) can reach significant levels in E. coli, respectively during amino acid starvation and energy stress. Though much less is known about these compounds in animals, mammalian cells contain a highly specific soluble thiamine triphosphatase controlling cytosolic ThTP concentrations. Second, there is now growing evidence in favour of the existence of thiamine-binding proteins with specific roles in the nervous system, possibly in the regulation of in neurotransmitter release. Thiamine and some of its synthetic precursors with higher bioavailability have beneficial effects in several models of Alzheimer’s disease and may be beneficial for patients suffering from Alzheimer's or Parkinson's diseases. These effects might be related to non-coenzyme roles of thiamine, possibly involving thiamine-binding proteins. [less ▲]

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See detailStructural Determinants of Specificity and Catalytic Mechanism in mammalian 25-kDa Thiamine Triphosphatase
Delvaux, David; Kerff, Frédéric ULiege; Murty, Mamidanna R.V.S. et al

in Biochimica et Biophysica Acta - General Subjects (2013), 1830

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine ... [more ▼]

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. Methods: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. Results: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. Conclusion: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. General significance: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins. [less ▲]

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See detailUp-regulation of 2-oxoglutarate dehydrogenase as a stress response
Graf, Anastasia; Trofimova, Lidia; Loshinskaja, Alexandra et al

in International Journal of Biochemistry & Cell Biology (2013), 45

2-Oxoglutarate dehydrogenase multienzyme complex (OGDHC) operates at a metabolic cross-road, mediating Ca2+- and ADP-dependent signals in mitochondria. Here, we test our hypothesis that OGDHC plays a ... [more ▼]

2-Oxoglutarate dehydrogenase multienzyme complex (OGDHC) operates at a metabolic cross-road, mediating Ca2+- and ADP-dependent signals in mitochondria. Here, we test our hypothesis that OGDHC plays a major role in the neurotransmitter metabolism and associated stress response. This possibility was assessed using succinyl phosphonate (SP), a highly specific and efficient in vivo inhibitor of OGDHC. Animals exposed to toxicants (SP, ethanol or MnCl2), trauma or acute hypoxia showed intrinsic up-regulation of OGDHC in brain and heart. The known mechanism of the SP action as OGDHC inhibitor pointed to the up-regulation triggered by the enzyme impairment. The animal behavior and skeletal muscle or heart performance were tested to correlate physiology with the OGDHC regulation and associated changes in the glutamate and cellular energy status. The SP-treated animals exhibited interdependent changes in the brain OGDHC activity, glutamate level and cardiac autonomic balance, suggesting the neurotransmitter role of glutamate to be involved in the changed heart performance. Energy insufficiency after OGDHC inhibition was detectable neither in animals up to 25 mg/kg SP, nor in cell culture during 24 h incubation with 0.1 mM SP. However, in animals subjected to acute ethanol intoxication SP did evoke energy deficit, decreasing muscular strength and locomotion and increasing the narcotic sleep duration. This correlated with the SP-induced decrease in NAD(P)H levels of the ethanol-exposed neurons. Thus, we show the existence of natural mechanisms to up-regulate mammalian OGDHC in response to stress, with both the glutamate neurotransmission and energy production potentially involved in the OGDHC impact on physiological performance. [less ▲]

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See detailHigh inorganic triphosphatase activities in bacteria and mammalian cells: Identification of the enzymes involved.
Kohn, Grégory ULiege; Delvaux, David ULiege; Lakaye, Bernard ULiege et al

in PLoS ONE (2012), 7(9), 43879

Background: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this ... [more ▼]

Background: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. [less ▲]

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See detailOverexpression of CD39 in mouse airways promotes bacteria induced inflammation
Theatre, Emilie ULiege; Frederix, Kim; Guilmain, William et al

in Journal of Immunology (2012), 189(4), 1966-1974

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of ... [more ▼]

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases.We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8+ T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X1 or P2X7 receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation. [less ▲]

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See detailSimultaneous versus solitary pharmacological manipulation of NMDA- and AMPA- receptors: effects of new drugs on contextual learning and its extinction
Vignisse, Julie ULiege; Steinbusch, Harry W.M.; Griegoriev, Vladimir et al

Poster (2012, July 17)

Both the attenuation of the NMDA-receptor mediated transmission via low affinity blockade mechanism, and the stimulation of AMPA receptor-mediated signaling were shown to result in beneficial ... [more ▼]

Both the attenuation of the NMDA-receptor mediated transmission via low affinity blockade mechanism, and the stimulation of AMPA receptor-mediated signaling were shown to result in beneficial neurobiological effects, such as an enhancement of memory and neurogenesis. We aimed to compare the effects of acute pharmacological manipulations of these mechanisms, exerted simultaneously or solely in mice, on learning of two mouse tasks with distinct predominant dependency on either glutamate receptor subtype. In a step-down avoidance task, memantine, low affinity NMDA receptor blocker (5 mg/kg), but not ampakine QQX (5 mg/kg) increased memory scores. In contrast, extinction of contextual fear conditioning was significantly enhanced by the latter, but not by the first drug. Among four new isothiourea derivates used at the doses 0.5-1 mg/kg, one compound that showed a maximal potency with respect to both glutamatergic mechanisms, as well as dimebon (1 mg/kg), had the most prominent memory enhancing effects. Thus, simultaneous low affinity blocade of the NMDA receptor and stimulation of AMPA-mediated transmission can result in eminent pro-cognitive activities. These data point to the importance of multi-target drug mechanism in the regulation of cognitive functions and suggest its potential for clinical implications. [less ▲]

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See detailChapter 17: Thiamin
Bettendorff, Lucien ULiege

in Erdman, Jr, John W.; MacDonald, Ian A.; Zeisel, Steven H. (Eds.) Present Knowledge in Nutrition (2012)

Thiamin (vitamin B1) was the first vitamin characterized and its discovery was at the origin of the concept of vitamin. Thiamin deficiency mainly affects the nervous system and causes two classical ... [more ▼]

Thiamin (vitamin B1) was the first vitamin characterized and its discovery was at the origin of the concept of vitamin. Thiamin deficiency mainly affects the nervous system and causes two classical diseases, beriberi (a polyneuritic syndrome) and Wernicke-Korsakoff syndrome (anterograde amnesia resulting from brain lesions in alcoholics). Thiamin transport through the membranes of intestinal and other cells requires specific carriers. As the process is rather slow, various lipid-soluble thiamin precursors with better bioavailability have been developed. In the cytosol, thiamin is pyrophosphorylated to thiamin diphosphate (ThDP), an indispenable cofactor in cell energy metabolism. Therefore, thiamin deficiency causes decreased cofactor function, leading to neuronal death. In addition, non-cofactor roles of the triphosphorylated derivatives thiamin triphosphate (ThTP) and adenosine thiamin triphosphate (AThTP) may play a role in metabolic regulation and may contribute to the pathology of thiamin deficiency-induced brain lesions. Current research interests are focused on the metabolism and role of thiamin derivatives (especially in catalysis by ThDP-dependent enzymes) and the biochemical and pathophysiological mechanisms by which thiamin deficiency induces specific brain lesions and may be implicated in other disorders such as Alzheimer’s disease and diabetes. [less ▲]

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See detailChapter 5: The chemistry, biochemistry and metabolism of thiamin (vitamin B1)
Bettendorff, Lucien ULiege

in Preedy, Victor R. (Ed.) B Vitamins and Folate: Chemistry, Analysis, Function and Effects (2012)

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See detailConsequences of the α-Ketoglutarate Dehydrogenase Inhibition for Neuronal Metabolism and Survival: Implications for Neurodegenerative Diseases
Trofimova, Lidia K.; Araùjo, Wagner L.; Strokina, Anastasiia A. et al

in Current Medicinal Chemistry (2012), 19

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See detailMolecular evolution of the CYTH superfamily of proteins
Bettendorff, Lucien ULiege; Delvaux, David ULiege; Kohn, Grégory ULiege et al

in FEBS Journal (2012), 279(Suppl. s1), 438

Molecular evolution of the CYTH superfamily of proteins L. Bettendorff, D. Delvaux, G. Kohn, P. Wins, B. Lakaye GIGA-Neurosciences, University of Liège, Belgium The CYTH superfamily of proteins was named ... [more ▼]

Molecular evolution of the CYTH superfamily of proteins L. Bettendorff, D. Delvaux, G. Kohn, P. Wins, B. Lakaye GIGA-Neurosciences, University of Liège, Belgium The CYTH superfamily of proteins was named after the two founding members, the CYaB adenylyl cyclase from Aeromonas hydrophila and the human 25-kDa THiamine triphosphatase (ThTPase). Members of this superfamily of proteins exist in all organisms including bacteria, archaea, plants and animals (except in birds) and can be traced back to the Last Universal Common Ancestor. They are characterized by a consensus sequence including several charged residues involved in divalent cation and triphosphate binding. Indeed, all members of the CYTH family that are characterized act on triphosphate derivatives and require at least one divalent cation for catalysis. The Nitrosomonas europaea (1) and E.coli CYTH proteins are specific inorganic triphosphatases. We propose that inorganic triphosphate (PPPi), the most simple triphosphate compound that can be imagined, is the primitive substrate of CYTH proteins. Other enzyme activities such as adenylate cyclase (in A. hydrophila), mRNA triphosphatase (in fungi and protozoans) and ThTPase (in metazoans) activities are secondary acquisitions. We show that ThTPase activity is not limited to mammals, but Sea anemone and Zebrafish CYTH proteins are already specific ThTPases and the acquisition of this enzyme activity is linked to the presence of a Trp (W53 in mammalian ThTPases) residue involved in the binding of the thiazole heterocycle of the thiamine molecule. The importance of W53 for the specificity of mammalian ThTPases is confirmed by site-directed mutagenesis. Furthermore, we propose a conserved catalytic mechanism between inorganic triphosphatases and ThTPases, based on a catalytic dyad comprising a Lys and a Tyr residue, explaining the alkaline pH optimum of CYTH proteins. (1) Delvaux et al. J. Biol. Chem 286 (2011) 34023-35 [less ▲]

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See detailThiamine : a link with Alzheimer’s disease ?
Bettendorff, Lucien ULiege

Scientific conference (2012)

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See detailChapter 17: Thiamin (E-book)
Bettendorff, Lucien ULiege

in Erdman, Jr, John W.; MacDonald, Ian A.; Zeisel, Steven H. (Eds.) Present Knowledge in Nutrition (E-book) (2012)

Thiamin (vitamin B1) was the first vitamin characterized and its discovery was at the origin of the concept of vitamin. Thiamin deficiency mainly affects the nervous system and causes two classical ... [more ▼]

Thiamin (vitamin B1) was the first vitamin characterized and its discovery was at the origin of the concept of vitamin. Thiamin deficiency mainly affects the nervous system and causes two classical diseases, beriberi (a polyneuritic syndrome) and Wernicke-Korsakoff syndrome (anterograde amnesia resulting from brain lesions in alcoholics). Thiamin transport through the membranes of intestinal and other cells requires specific carriers. As the process is rather slow, various lipid-soluble thiamin precursors with better bioavailability have been developed. In the cytosol, thiamin is pyrophosphorylated to thiamin diphosphate (ThDP), an indispenable cofactor in cell energy metabolism. Therefore, thiamin deficiency causes decreased cofactor function, leading to neuronal death. In addition, non-cofactor roles of the triphosphorylated derivatives thiamin triphosphate (ThTP) and adenosine thiamin triphosphate (AThTP) may play a role in metabolic regulation and may contribute to the pathology of thiamin deficiency-induced brain lesions. Current research interests are focused on the metabolism and role of thiamin derivatives (especially in catalysis by ThDP-dependent enzymes) and the biochemical and pathophysiological mechanisms by which thiamin deficiency induces specific brain lesions and may be implicated in other disorders such as Alzheimer’s disease and diabetes. [less ▲]

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See detailThe effect of thiamin tetrahydrofurfuryl disulfide on behavior of juvenile DBA/2J mice
Hills, Judith I.; Golub, Mari S.; Bettendorff, Lucien ULiege et al

in Neurotoxicology & Teratology (2012), 34

Due to genetic defects or illness some individuals require higher amounts of thiamin than are typically provided by the diet. Lipid-soluble thiamin precursors can achieve high blood levels of thiamin and ... [more ▼]

Due to genetic defects or illness some individuals require higher amounts of thiamin than are typically provided by the diet. Lipid-soluble thiamin precursors can achieve high blood levels of thiamin and result in increased concentrations in the central nervous system. High intakes of thiamin have been reported as beneficial in children with autism and attention deficit/hyperactivity disorder. The current study examined the effect of thiamin tetrahydrofurfuryl disulfide (TTFD), a lipophilic precursor, on behavior in the juvenile male DBA/2J mouse. Mice given by oral gavage deionized water or deionized water providing 100mg or 340mg TTFD/kg body weight daily for 17days, starting at postnatal day 18, were tested for effects on operant learning, social interaction, general activity level, and prepulse inhibition of acoustic startle, as well as effects on growth and select organ weights. Results indicate lower activity and altered social interaction at both treatment levels and decreased acoustic startle at the 100mg/kg level. Compared to controls, percent weight gain was lower in the TTFD-treatment groups, but percent body length increase was not affected by TTFD treatment. TTFD treatment did not influence percent organ weights as percentage of body weights. TTFD treatment resulted in increased whole brain thiamin concentrations. These results support the concept that lipophilic thiamin precursors provided during early development can affect a number of behavioral parameters. In clinical trials with children with behavior disorders, attention should be given to preventing possible adverse gastrointestinal irritant effects associated with TTFD therapy. [less ▲]

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See detailUne invitée surprise
Binet, Audrey; Delvaux, David ULiege; Bettendorff, Lucien ULiege

Learning material (2011)

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See detailA specific inorganic triphosphatase from Nitrosomonas europaea: structure and catalytic mechanism
Delvaux, David ULiege; Murty, Mamidana R.V.S; Gabelica, Valérie ULiege et al

in Journal of Biological Chemistry (2011), 286

The CYTH superfamily of proteins is named after its two founding members, the CyaB adenylyl cyclase from Aeromonas hydrophila and the human 25-kDa thiamine triphosphatase. Because these proteins often ... [more ▼]

The CYTH superfamily of proteins is named after its two founding members, the CyaB adenylyl cyclase from Aeromonas hydrophila and the human 25-kDa thiamine triphosphatase. Because these proteins often form a closed β-barrel, they are also referred to as “Triphosphate Tunnel Metalloenzymes” (TTM). Functionally, they are characterized by their ability to bind triphosphorylated substrates and divalent metal ions. These proteins exist in most organisms and catalyze different reactions, depending on their origin. Here we investigate structural and catalytic properties of the recombinant TTM protein from Nitrosomonas europaea (NeuTTM), a 19-kDa protein. Crystallographic data show that it crystallizes as a dimer and that, in contrast to other TTM proteins, it has an open β-barrel structure. We demonstrate that NeuTTM is a highly specific inorganic triphosphatase, hydrolyzing tripolyphosphate (PPPi) with high catalytic efficiency in the presence of Mg2+. These data are supported by native mass spectrometry analysis showing that the enzyme binds PPPi (and Mg-PPPi) with high affinity (Kd < 1.5 μM), while it has a low affinity for ATP or thiamine triphosphate. In contrast to Aeromonas and Yersinia CyaB proteins, NeuTTM has no adenylyl cyclase activity, but it shares several properties with other enzymes of the CYTH superfamily, e.g. heat-stability, alkaline pH optimum and inhibition by Ca2+ and Zn2+ ions. We suggest a catalytic mechanism involving a catalytic dyad formed by K52 and Y28. The present data provide the first characterization of a new type of phosphohydrolase (unrelated to pyrophosphatases or exopolyphosphatases), able to hydrolyze inorganic triphosphate with high specificity. [less ▲]

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