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

Poster (2011)

Exponentially growing E. coli cells contain high amounts of thiamine diphosphate (ThDP), a well-known cofactor, but only ~20% is bound to apoenzymes. We showed that the abundant free ThDP is the precursor ... [more ▼]

Exponentially growing E. coli cells contain high amounts of thiamine diphosphate (ThDP), a well-known cofactor, but only ~20% is bound to apoenzymes. We showed that the abundant free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP) and the newly discovered adenosine thiamine triphosphate (AThTP). Both compounds are produced under different conditions of stress. ThTP transiently accumulates in response to amino acid starvation, but, in contrast to AThTP, its synthesis requires the presence of a carbon source yielding pyruvate. Under such conditions, the amount of cellular ThTP may reach up to 20–60% of total thiamine, while it is generally a minor compound (0.1 to 1% of total thiamine). The biological role of ThTP remains uncertain until now, though in E. coli, it seems to be required for rapid adaptation to amino acid starvation. ThTP accumulation requires inorganic phosphate and is inhibited by anoxia and cyanide. It is also blocked by low concentrations of the protonophore CCCP and is inhibited by low concentrations of DCCD, an inhibitor of FoF1-ATP synthase. These results suggest that ThTP is synthesized by a mechanism similar to oxidative phosphorylation, i. e. the reaction ThDP + Pi  ThTP catalyzed by FoF1-ATP synthase and energized by respiration. This hypothesis is supported by the finding that E. coli with FoF1-ATP synthase mutated in various subunits are unable to synthesize ThTP in minimal medium in response to glucose exposure. Moreover, a strain in which the atp operon was deleted is unable to synthesize ThTP, but transformation with a plasmid encoding the atp operon totally restores this capacity. We recently demonstrated a similar chemiosmotic mechanism for ThTP synthesis in rat brain mitochondria suggesting a conserved mechanism for ThTP synthesis between bacteria and mammals. [less ▲]

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See detailThiaminylated adenine nucleotides — chemical synthesis, structural characterization and natural occurrence
Frederich, Michel ULg; Delvaux, David ULg; Gigliobianco, Tiziana ULg et al

in FEBS Journal (2009), 276(12), 32563268

Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine ... [more ▼]

Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate (AThTP), produced in E. coli in response to carbon starvation. Here, we show that the chemical synthesis of AThTP leads to another new compound, adenosine thiamine diphosphate (thiaminylated ADP, AThDP), as a side product. The structure of both compounds was confirmed by mass spectrometry and 1H-, 13C- and 31P-NMR and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared to the conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting an U-shaped folding of adenosine thiamine derivatives. Such a structure where the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, AThTP may account for 15% of total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and in some cell lines. Using a HPLC method, we show for the first time that AThDP may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate. [less ▲]

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See detailRole of thiamine triphosphate and adenosine thiamine triphosphate in Escherichia coli
Gigliobianco, Tiziana ULg

Poster (2008)

Thiamine triphosphate (ThTP) is found in all organisms studied from bacteria to mammals. In E. coli, ThTP is accumulated as a result of amino acid starvation in the presence of a suitable carbon source ... [more ▼]

Thiamine triphosphate (ThTP) is found in all organisms studied from bacteria to mammals. In E. coli, ThTP is accumulated as a result of amino acid starvation in the presence of a suitable carbon source such as glucose, pyruvate and lactate. On the other hand, adenosine thiamine triphosphate (AThTP), that was recently discovered in E. coli, accumulates in the absence of a carbon source. While AThTP is synthesized in E. coli according to the reaction ThDP + ADP (ATP) ↔ AThTP + Pi (PPi) by a soluble high molecular mass nucleotidyl transferase, the mechanism of synthesis of ThTP remains unknown. Though both bacterial and mammalian adenylate kinases are able to catalyze ThTP synthesis at a very low rate in vitro, this reaction is not responsible for ThTP synthesis in vivo. AThTP is synthesized in E. coli incubated in minimal medium devoid of any carbon source. The addition of glucose leads to the immediate disappearance of AThTP and the appearance of ThTP. ThTP production is strongly impaired in the presence of protonophores, even when a high intracellular concentration of ATP is maintained through glycolysis. Our results suggest that ThTP accumulation requires a high protonmotive force rather than a high intracellular ATP concentration. On the other hand, AThTP is synthesized under conditions of limited energy availability, either in the absence of a carbon source or in the presence of a carbon source and metabolic inhibitors or uncouplers. Interestingly, ThTP and AThTP never accumulate simultaneously in high amounts. Our results suggest that in E. coli both compounds might therefore act as specific “alarmones”, responding to different conditions of cellular stress. [less ▲]

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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 detailAdenosine thiamine triphosphate, a new signal synthesized in response to specific forms of cellular stress in Escherichia coli
Gigliobianco, Tiziana ULg

Poster (2007)

Thiamine, also known as vitamin B1, and its different phosphorylated derivates are found in all known organisms. The main form of the vitamin is the coenzyme thiamine diphosphate (ThDP). Thiamine ... [more ▼]

Thiamine, also known as vitamin B1, and its different phosphorylated derivates are found in all known organisms. The main form of the vitamin is the coenzyme thiamine diphosphate (ThDP). Thiamine triphosphate (ThTP) is also found in low amounts in all organisms studied from bacteria to mammals. In E. coli ThTP accumulation is linked to amino acid starvation and the presence of a suitable carbon source. Recently a new thiamine derivative, adenosine thiamine triphosphate (AThTP) or thiaminylated ATP was discovered in our laboratory. AThTP is accumulated in E. coli during carbon starvation. When the cells are grown under optimal conditions (rich medium containing amino acids) neither ThTP nor AThTP is present in significant amounts. The synthesis of both compounds is inhibited by amino acids. In E. coli, AThTP accumulates in response to two different conditions of metabolic stress: lack of energy substrates (or inhibition of their metabolization) and uncoupled pyruvate oxidation. [less ▲]

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