Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli

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

Adenosine thiamine triphosphate accumulates in Escherichia coli cells in response to specific conditions of metabolic stress

<p>Abstract</p> <p>Background</p> <p><it>E. coli </it>cells are rich in thiamine, most of it in the form of the cofactor thiamine diphosphate (ThDP). Free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP) and the newly discovered adenosine thiamine triphosphate (AThTP). While, ThTP accumulation requires oxidation of a carbon source, AThTP slowly accumulates in response to carbon starvation, reaching ~15% of total thiamine. Here, we address the question whether AThTP accumulation in <it>E. coli </it>is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress.</p> <p>Results</p> <p>In minimal M9 medium, <it>E. coli </it>cells produce AThTP not only when energy substrates are lacking but also when their metabolization is inhibited. Thus AThTP accumulates in the presence of glucose, when glycolysis is blocked by iodoacetate, or in the presence lactate, when respiration is blocked by cyanide or anoxia. In both cases, ATP synthesis is impaired, but AThTP accumulation does not appear to be a direct consequence of reduced ATP levels. Indeed, in the CV2 <it>E. coli </it>strain (containing a thermolabile adenylate kinase), the ATP content is very low at 37°C, even in the presence of metabolizable substrates (glucose or lactate) and under these conditions, the cells produce ThTP but not AThTP. Furthermore, we show that ThTP inhibits AThTP accumulation. Therefore, we conclude that a low energy charge is not sufficient to trigger AThTP accumulation and the latter can only accumulate under conditions where no ThTP is synthesized. We further show that AThTP production can also be induced by the uncoupler CCCP but, unexpectedly, this requires the presence of pyruvate or a substrate yielding pyruvate (such a D-glucose or L-lactate). Under the conditions described, AThTP production is not different when RelA or SpoT mutants are used.</p> <p>Conclusions</p> <p>In <it>E. coli</it>, AThTP accumulates in response to two different conditions of metabolic stress: lack of energy substrates (or inhibition of their metabolization) and uncoupled pyruvate oxidation. Both conditions prevent bacterial growth. There is no obvious link with the stringent response or catabolite repression.</p

Product inhibition of mammalian thiamine pyrophosphokinase is an important mechanism for maintaining thiamine diphosphate homeostasis

peer reviewedBackground: Thiamine diphosphate (ThDP), an indispensable cofactor for oxidative energy metabolism, is syn- thesized through the reaction thiamine + ATP ⇆ ThDP + AMP, catalyzed by thiamine pyrophosphokinase 1 (TPK1), a cytosolic dimeric enzyme. It was claimed that the equilibrium of the reaction is in favor of the for- mation of thiamine and ATP, at odds with thermodynamic calculations. Here we show that this discrepancy is due to feedback inhibition by the product ThDP. Methods: We used a purified recombinant mouse TPK1 to study reaction kinetics in the forward (physiological) and for the first time also in the reverse direction. Results: Keq values reported previously are strongly underestimated, due to the fact the reaction in the forward direction rapidly slows down and reaches a pseudo-equilibrium as ThDP accumulates. We found that ThDP is a potent non-competitive inhibitor (Ki ≈ 0.4 μM) of the forward reaction. In the reverse direction, a true equi- librium is reached with a Keq of about 2 × 10− 5, strongly in favor of ThDP formation. In the reverse direction, we found a very low Km for ThDP (0.05 μM), in agreement with a tight binding of ThDP to the enzyme. General significance: Inhibition of TPK1 by ThDP explains why intracellular ThDP levels remain low after administration of even very high doses of thiamine. Understanding the consequences of this feedback inhibition is essential for developing reliable methods for measuring TPK activity in tissue extracts and for optimizing the therapeutic use of thiamine and its prodrugs with higher bioavailability under pathological conditions

Thiamine Status in Humans and Content of Phosphorylated Thiamine Derivatives in Biopsies and Cultured Cells

Background Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. Methodology and Principal Findings Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ~60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. Conclusions and Significance The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation

Biological functions of thiamine derivatives: Focus on non-coenzyme roles

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

Thiamine triphosphate: a ubiquitous molecule in search of a physiological role

Thiamine triphosphate (ThTP) was discovered over 60 years ago and it was long thought to be a specifically neuroactive compound. Its presence in most cell types, from bacteria to mammals, would suggest a more general role but this remains undefined. In contrast to thiamine diphosphate (ThDP), ThTP is not a coenzyme. In E. coli cells, ThTP is transiently produced in response to amino acid starvation, while in mammalian cells, it is constitutively produced at a low rate. Though it was long thought that ThTP was synthesized by a ThDP:ATP phosphotransferase, more recent studies indicate that it can be synthesized by two different enzymes: (1) adenylate kinase 1 in the cytosol and (2) FoF1-ATP synthase in brain mitochondria. Both mechanisms are conserved from bacteria to mammals. Thus ThTP synthesis does not seem to require a specific enzyme. In contrast, its hydrolysis is catalyzed, at least in mammalian tissues, by a very specific cytosolic thiamine triphosphatase (ThTPase), controlling the steady-state cellular concentration of ThTP. In some tissues where adenylate kinase activity is high and ThTPase is absent, ThTP accumulates, reaching ≥ 70% of total thiamine, with no obvious physiological consequences. In some animal tissues, ThTP was able to phosphorylate proteins, and activate a high-conductance anion channel in vitro. These observations raise the possibility that ThTP is part of a still uncharacterized cellular signaling pathway. On the other hand, its synthesis by a chemiosmotic mechanism in mitochondria and respiring bacteria might suggest a role in cellular energetics

Mechanism of Thiamine Transport in Neuroblastoma Cells. Inhibition of a High Affinity Carrier by Sodium Channel Activators and Dependence of Thiamine Uptake on Membrane Potential and Intracellular Atp

Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a saturable high affinity mechanism (Km = 35 nM). This was blocked by low concentrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 microM) and batrachotoxin (IC50 = 0.9 microM). These effects were not antagonized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the mechanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (Km = 0.8 mM), insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibited (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intracellular ATP concentration was found. When cells grown in a medium of low thiamine concentration (6 nM) were exposed to 100 nM extracellular thiamine, a 3-fold increase in intracellular thiamine diphosphate was observed after 2 h while the concomitant increase in intracellular free thiamine was barely significant. These data suggest a secondary active transport of thiamine, the main driving force being thiamine phosphorylation rather than the sodium gradient

Thiamine triphosphatase and the CYTH family of proteins

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

Biochemistry of thiamine and thiamine phosphate compounds

peer reviewedaudience: researcher, professional, student, popularizatio