Molecular Characterization of a Specific Thiamine Triphosphatase Widely Expressed in Mammalian Tissues

Abstract

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis. In this report we purified a soluble thiamine triphosphatase (ThTPase; EC 3.6.1.28) from calf brain. The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity. Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBank TM data base. A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652. The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced. The human open reading frame was expressed in E. coli as a GST fusion protein. Transformed bacteria had a high isopropyl-␤-D-thiogalactopyranoside-inducible ThTPase activity. The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP. The mRNA was expressed in most human tissues but at relatively low levels. This is the first report of a molecular characterization of a specific ThTPase.

Structure–function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins

“…The founders of this family are the bacterial adenylate cyclase enzyme CyaB (Sismeiro et al 1998) and the mammalian enzyme thiamine triphosphatase (Lakaye et al 2002). Whereas CYTH domains were predicted to exist in many organisms in all three domains of the universal phylogenetic tree, there was no relationship noted at the time between the CYTH domain and the Cet1-like RNA triphosphatases.…”

Novel Triphosphate Phosphohydrolase Activity of Clostridium thermocellum TTM, a Member of the Triphosphate Tunnel Metalloenzyme Superfamily

“…This notion has been eclipsed by the finding that the heretofore unique tertiary structure and active site of yeast RNA triphosphatase are recapitulated in the crystal structures of archaeal and bacterial proteins of unknown biochemical function, including proteins from Pyrococcus (Protein Data Bank (PDB) accession codes 1YEM and 2DC4), Vibrio (2ACA), and Nitrosomonas (2FBL) (1). The Cet1-like archaeal/bacterial proteins are usually annotated as belonging to the so-called CYTH family (19), which is defined by its two biochemically characterized founding members, an Aeromonas hydrophila adenylate cyclase CyaB and a mammalian thiamine triphosphatase (20,21). A crystal structure of a Cet1-like adenylate cyclase from Yersinia (2FJT) has been reported recently (22).…”

Properties of chicken liver membrane-associated thiamine triphosphatase

“…Результаты исследований, прове-денных за последнее десятилетие, указывают на существование более фундаментальной био-логической роли ТТР, общей для клеток раз-личных типов и специализации, которая может иметь отношение к сигнальным (регуляторным) механизмам [10]. В тканях млекопитающих при обычных физиологических условиях концен-трация ТТР поддерживается на низком уровне (порядка 30-400 пмоль/л) благодаря экспрессии растворимой тиаминтрифосфатазы (ТТРаза, 3.6.1.28) -Mg 2+ -зависимого энзима с Мм 25 кДа, обладающего абсолютной субстратной специ-фичностью [11][12][13]. Хотя растворимая ТТРаза экспериментально найдена только у млекопи-тающих, методами биоинформатики существо-вание протеинов-гомологов также предсказано у костных рыб, земноводных и рептилий, тогда как у птиц, насекомых, червей, прокариот, про-стейших, грибов и растений аминокислотные и нуклеотидные последовательности, гомологич-ные ТТРазе с Мм 25 кДа, не выявлены [14,15].…”

“…В настоящее время растворимая ТТРаза, первоначально полученная в гомоген-ном виде из головного мозга [12], а затем из по-чек [30] быка, охарактеризована на молекуляр-ном уровне [13,31,32]. Это Mg 2+ -зависимый мономерный протеин с Mм ~ 25 кДа, проявляю-щий максимальную активность при рН 8,5-9,0.…”