Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

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

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

Distrribution of specific thiamine triphosphatase in biological objects

peer reviewe

A study of the quantitative content of thiamine triphosphate in different biological objects by high-performance liquid chromatography

peer reviewe

Thiamine diphosphate adenylyl transferase from : functional characterization of the enzyme synthesizing adenosine thiamine triphosphate-2

<p><b>Copyright information:</b></p><p>Taken from "Thiamine diphosphate adenylyl transferase from : functional characterization of the enzyme synthesizing adenosine thiamine triphosphate"</p><p>http://www.biomedcentral.com/1471-2091/8/17</p><p>BMC Biochemistry 2007;8():17-17.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC1976097.</p><p></p>s-maleate, pH 7.0; 50 mM Tris-HCl, pH 7.5; (■) 50 mM Bis-Tris-propane; (□) 100 mM Bis-Tris-propane

Thiamine diphosphate adenylyl transferase from : functional characterization of the enzyme synthesizing adenosine thiamine triphosphate-0

<p><b>Copyright information:</b></p><p>Taken from "Thiamine diphosphate adenylyl transferase from : functional characterization of the enzyme synthesizing adenosine thiamine triphosphate"</p><p>http://www.biomedcentral.com/1471-2091/8/17</p><p>BMC Biochemistry 2007;8():17-17.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC1976097.</p><p></p> mM ADP. The flow rate was 0.5 ml/min

Thiamine diphosphate adenylyl transferase from : functional characterization of the enzyme synthesizing adenosine thiamine triphosphate-5

<p><b>Copyright information:</b></p><p>Taken from "Thiamine diphosphate adenylyl transferase from : functional characterization of the enzyme synthesizing adenosine thiamine triphosphate"</p><p>http://www.biomedcentral.com/1471-2091/8/17</p><p>BMC Biochemistry 2007;8():17-17.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC1976097.</p><p></p

83) Study of the role of ionogenic amino-acid residues in catalytic activity of thiamine triphosphatase from bovine kidney by means of chemical modification

peer reviewe

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals

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

Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme

peer reviewedThiamine 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