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See detailAdenylate Kinase-Independent Thiamine Triphosphate Accumulation under Severe Energy Stress in Escherichia Coli
Gigliobianco, Tiziana ULg; Lakaye, Bernard ULg; Makarchikov, Alexander F et al

in BMC Microbiology (2008), 8

BACKGROUND: Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism ... [more ▼]

BACKGROUND: Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. RESULTS: Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine) at 37 degrees C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. CONCLUSION: These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis. [less ▲]

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See detailDiscovery of a natural thiamine adenine nucleotide
Bettendorff, Lucien ULg; Wirtzfeld, Barbara; Makarchikov, Alexander F et al

in Nature Chemical Biology (2007), 3(4), 211-212

Several important cofactors are adenine nucleotides with a vitamin as the catalytic moiety. Here, we report the discovery of the first adenine nucleotide containing vitamin B1: adenosine thiamine ... [more ▼]

Several important cofactors are adenine nucleotides with a vitamin as the catalytic moiety. Here, we report the discovery of the first adenine nucleotide containing vitamin B1: adenosine thiamine triphosphate (AThTP, 1), or thiaminylated ATP. We discovered AThTP in Escherichia coli and found that it accumulates specifically in response to carbon starvation, thereby acting as a signal rather than a cofactor. We detected smaller amounts in yeast and in plant and animal tissues. [less ▲]

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See detailThiamine Diphosphate Adenylyl Transferase from E. Coli: Functional Characterization of the Enzyme Synthesizing Adenosine Thiamine Triphosphate
Makarchikov, Alexander F; Brans, Alain ULg; Bettendorff, Lucien ULg

in BMC Biochemistry (2007), 8

BACKGROUND: We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a ... [more ▼]

BACKGROUND: We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. RESULTS: Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP) and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5-7.0 and a high Km for ThDP (5 mM), suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65). CONCLUSION: This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells. [less ▲]

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See detailPig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme
Szyniarowski, Piotr; Lakaye, Bernard ULg; Czerniecki, Jan ULg et al

in Biochimica et Biophysica Acta - General Subjects (2005), 1725(1), 93-102

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian ... [more ▼]

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis. [less ▲]

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See detailDistrribution of specific thiamine triphosphatase in biological objects
Makarchikov, Alexander F; Bettendorff, Lucien ULg

in News of Biomedical Sciences (2005), 2

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See detailA study of the quantitative content of thiamine triphosphate in different biological objects by high-performance liquid chromatography
Makarchikov, Alexander F; Bettendorff, Lucien ULg

in News of Biomedical Sciences (2004), 2

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See detailNeuronal localization of the 25-kDa specific thiamine triphosphatase in rodent brain
Czerniecki, Jan ULg; Chanas, Grazyna; Verlaet, Myriam ULg et al

in Neuroscience (2004), 125(4), 833-840

Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but little is known about its physiological role. In vertebrate tissues, ThTP may act as a phosphate ... [more ▼]

Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but little is known about its physiological role. In vertebrate tissues, ThTP may act as a phosphate donor for the phosphorylation of certain proteins; this may be part of a new signal transduction pathway. We have recently characterized a highly specific 25-kDa thiamine triphosphatase (ThTPase) that is expressed in most mammalian tissues. The role of this enzyme may be the control of intracellular concentrations of ThTP. As the latter has been considered to be a neuroactive form of thiamine, we have studied the distribution of ThTPase mRNA and protein in rodent brain using in situ hybridization and immunohistochemistry. With both methods, we found the strongest staining in hippocampal pyramidal neurons, as well as cerebellar granule cells and Purkinje cells. Some interneurons were also labeled and many ThTPase mRNA-positive and immunoreactive cells were distributed throughout cerebral cortical gray matter and the thalamus. White matter was not significantly labeled. ThTPase immunoreactivity seems to be located mainly in the cytoplasm of neuronal perikarya. Immunocytochemical data using dissociated cultured cells from hippocampal and cerebellum showed that the staining was more intense in neurons than in astrocytes. The protein was rather uniformly located in the perikarya and dendrites, suggesting that ThTP and ThTPase may play a general role in neuronal metabolism rather than a specific role in excitability. There was no apparent correlation between ThTPase expression and selective vulnerability of certain brain regions to thiamine deficiency. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved. [less ▲]

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See detailExpression of 25 kDa thiamine triphosphatase in rodent tissues using quantitative PCR and characterization of its mRNA
Lakaye, Bernard ULg; Verlaet, Myriam ULg; Dubail, Johanne ULg et al

in International Journal of Biochemistry & Cell Biology (2004), 36(10), 2032-2041

Thiamine triphosphate (ThTP) is found in most organisms, but its biological role remains unclear. In mammalian tissues, cellular ThTP concentrations remain low, probably because of hydrolysis by a ... [more ▼]

Thiamine triphosphate (ThTP) is found in most organisms, but its biological role remains unclear. In mammalian tissues, cellular ThTP concentrations remain low, probably because of hydrolysis by a specific 25 kDa thiamine triphosphatase (ThTPase). The aim of the present study was to use quantitative PCR, for comparing the 25 kDa ThTPase mRNA expression in various mouse tissues with its enzyme activities. ThTPase mRNA was expressed at only a few copies per cell. The highest amount of mRNA was found in testis, followed by lung and muscle, while the highest enzyme activities were found in liver and kidney. The poor correlation between mRNA levels and enzyme activities might result either from tissue-specific post-transcriptional regulation of mRNA processing and/or translation or from the regulation of enzyme activities by post-translational mechanisms. Purified recombinant human ThTPase was phosphorylated by casein kinase 11, but this phosphorylation did not modify the enzyme activity. However, the characterization of the 3'-untranslated mRNA region revealed a unique, highly conserved, 200-nucleotide sequence that might be involved in translational control. In situ hybridization studies in testis suggest a predominant localization of ThTPase mRNA in poorly differentiated spermatogenic cells. This is the first study demonstrating a cell-specific 25 kDa ThTPase mRNA expression, suggesting that this enzyme might be related to the degree of differentiation or the metabolic state of the cell. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailHuman recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties
Lakaye, Bernard ULg; Makarchikov, Alexander F; Wins, Pierre et al

in International Journal of Biochemistry & Cell Biology (2004), 36(7), 1348-1364

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 ... [more ▼]

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix. [less ▲]

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See detailThiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals
Makarchikov, Alexander F; Lakaye, Bernard ULg; Gulyai, I. E. et al

in Cellular and Molecular Life Sciences : CMLS (2003), 60(7), 1477-1488

In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain ... [more ▼]

In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency. [less ▲]

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See detailMolecular characterization of a specific thiamine triphosphatase widely expressed in mammalian tissues
Lakaye, Bernard ULg; Makarchikov, Alexander F; Antunes, Adelio F et al

in Journal of Biological Chemistry (2002), 277(16), 13771-13777

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian ... [more ▼]

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis. In this report we purified a soluble thiamine triphosphatase (ThTPase; EC 3.6.1.28) from calf brain. The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity. Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBank(TM) data base. A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652. The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced. The human open reading frame was expressed in E. coli as a GST fusion protein. Transformed bacteria had a high isopropyl-beta-D-thiogalactopyranoside-inducible ThTPase activity. The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP. The mRNA was expressed in most human tissues but at relatively low levels. This is the first report of a molecular characterization of a specific ThTPase. [less ▲]

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See detailStudy of the role of ionogenic amino-acid residues in catalytic activity of thiamine triphosphatase from bovine kidney by means of chemical modification
Makarchikov, Alexander F; Luchko, A. A.; Bettendorff, Lucien ULg et al

in News of Biomedical Sciences (2002), 3

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See detailAdenylate kinase 1 knockout mice have normal thiamine triphosphate levels
Makarchikov, Alexander F; Wins, Pierre; Janssen, E. et al

in Biochimica et Biophysica Acta-Molecular Cell Research (2002), 1592(2), 117-121

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling ... [more ▼]

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP + ATP double left right arrow ThTP + ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase I (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase. (C) 2002 Elsevier Science B.V All rights reserved. [less ▲]

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