Metabolic significance of inorganic triphosphate, thiamine triphosphate and their hydrolyzing enzymes

Our laboratory has been interested for many years in thiamine triphosphate (ThTP), an unusual triphosphate derivative of thiamine (vitamin B1) found in nearly all organisms. In mammalian tissues, ThTP is hydrolyzed by a very specific cytosolic thiamine triphosphatase (ThTPase) belonging to an ancient superfamily of proteins called CYTH. Several members of this superfamily have been characterized and they all have in common that they act on triphosphorylated substrates. Some bacterial members (the N. europeae specifically) hydrolyze inorganic triphosphate (PPPi) which raises the question of the physiological significance of this compound.We first studied the tripolyphosphatase activity in mammals and in bacteria and we showed that it is widely distributed in all organisms. We attempted to identify the enzymes responsible for this activity. We showed that the E. coli CYTH enzyme, ygiF, although highly specific for PPPi plays only a minor role in the cytosolic PPPase activity, while most of the activity is due to inorganic pyrophosphatase. In animal tissues, most PPPase activity is due to the short-chain exopolyphosphatase prune, which hydrolyzes PPPi with high catalytic efficiency. We hypothesize that PPPi may be formed as a by-product of metabolism and the major role of PPPase activities may be to keep PPPi concentrations very low to avoid toxic effects linked to interference with Ca2+ metabolism. In order to check this hypothesis, it would be important to have a specific and sensitive method for measuring intracellular PPPi concentrations. Such a technique is presently not available.In the second part of our work, we tried to manipulate intracellular ThTP concentrations in order to get insight into the physiological role of this compound. Expression of mammalian ThTPase in E. coli prevented ThTP accumulation, but did not affect the growth of the bacteria. We then reduced ThTPase expression in zebrafish embryos using morpholino oligomers. This led to severe malformations of the embryos. Finally, we attempted to produce a ThTPase knockout mouse. However, we found that the spermatogenesis of ThTPase-null sperm cells was impaired and the chimerae were unable to transmit. In conclusion, our results suggest that ThTPase inactivation results in heavy developmental consequences, possibly as a result of ThTP toxicity.