Gigliobianco Tiziana, Lakaye Bernard, Makarchikov Alexander F, Wins Pierre, Bettendorff Lucien
Center for Cellular and Molecular Neurobiology, University of Liège, Liège, Belgium.
BMC Microbiol. 2008 Jan 23;8:16. doi: 10.1186/1471-2180-8-16.
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.
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.
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.
三磷酸硫胺素（ThTP）存在于大多数生物体中，可能在细胞应激反应中发挥作用。在大肠杆菌中，ThTP会在氨基酸饥饿时积累，但其合成机制仍存在争议。有人提出ThTP是由一种依赖ATP的特异性硫胺二磷酸激酶合成的。然而，也已知脊椎动物腺苷酸激酶1以非常低的速率催化ThTP的合成，并且有人推测这种酶在体内负责ThTP的合成。
我们在此表明，细菌的腺苷酸激酶与脊椎动物的一样，能够催化ThTP的合成，但速率比ATP合成低106倍以上。这种活性太低，无法解释在大肠杆菌氨基酸饥饿期间观察到的ThTP的高积累速率。此外，来自热敏CV2菌株的细菌在37摄氏度时积累大量ThTP（占总硫胺素的>50%），尽管腺苷酸激酶完全失活且细胞ATP随后下降。
这些结果清楚地表明，腺苷酸激酶在体内不负责ThTP的合成。此外，它们表明在ATP水平非常低的严重能量应激下，大肠杆菌会积累大量ThTP，这一观察结果不支持ThTP合成的ATP依赖机制。
