Thiamine and its phosphorylated derivatives are common cellular constituents in all living forms studied so far [1]. While the role of thiamine diphosphate (ThDP) as a cofactor for more than 25 enzymes is well documented [2], we have so far little information concerning the possible role(s) of other thiamine derivatives.
No known biological role has been documented for thiamine monophosphate (ThMP), but recent results suggest a role for thiamine triphosphate (ThTP). Although it is only a minor component (0.1 – 1 %) of total thiamine in most tissues, ThTP was found in all organisms investigated so far [1]. In plants and in bacteria, the appearance of ThTP seems to be a response to specific conditions of cellular stress [1,3]. In E. coli for instance, the initial accumulation of ThTP appears to be required for optimal growth in media containing a carbon source but no amino acids.
Recently, we identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP). This compound was first discovered in E. coli, but it is also present in low amounts in plants and animals [4]. Like ThTP, AThTP appears to be a signal produced in bacteria in response to some form of cellular stress; however, the two compounds are formed under different conditions and generally do not accumulate simultaneously. Both are hardly detectable when the bacteria are grown in rich media under optimal conditions. When the bacteria are transferred to minimal M9 medium, AThTP appears in the absence of any carbon source and it quickly disappears when glucose is added, suggesting that it is produced in response to carbon starvation. In contrast, ThTP synthesis requires the presence of an energy substrate such as glucose.
Although the presence of ThTP in many tissues has been known for over 50 years, the mechanism of its enzymatic synthesis remains unclear. In particular, no significant net synthesis of ThTP could be detected so far using cell-free extracts of E. coli. In contrast, we observed a synthesis of AThTP from ADP and ThDP in soluble fractions from sonicated bacteria. Here, we describe the partial purification and some kinetic properties of a high molecular weight enzyme (or enzyme complex) catalyzing the synthesis of AThTP in E. coli.
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