Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme
ABSTRACT Thiamine 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.
- SourceAvailable from: Abdoulaye Bâ[Show abstract] [Hide abstract]
ABSTRACT: In the literature, previous descriptions of the role of thiamine (B1 vitamin) focused mostly on its biochemical functions as a coenzyme precursor of some key enzymes of the carbohydrate metabolism. This report reviews recent developments on the metabolic and structural role of thiamine, e.g., the coenzyme and noncoenzyme functions of the vitamin. Taking into account analysis of our experimental data relating to the effects of thiamine deficiency on developing central nervous system (CNS) and data available in literature, we seek to establish a clear difference between the metabolic and structural role of thiamine. Our experimental data indicate that the specific and nonspecific effects express two diametrically diverse functions of thiamine in development: the nonspecific effects show up the metabolic consequences of thiamine deficiency resulting in apoptosis and severe cellular deficit; inversely, the specific effects announced the structural consequences of thiamine deficiency, described as cellular membrane damage, irregular and ectopic cells. The review highlights the existence of noncoenzyme functions of this vitamin through its interactions with biological membranes.Cellular and Molecular Neurobiology 11/2008; 28(7):923-31. DOI:10.1007/s10571-008-9297-7 · 2.20 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: 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.BMC Microbiology 02/2008; 8:16. DOI:10.1186/1471-2180-8-16 · 2.98 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Mammalian soluble thiamine triphosphatase (ThTPase) is a 25-kDa cytosolic enzyme that specifically catalyzes the conversion of thiamine triphosphate (ThTP) to thiamine diphosphate and has an absolute requirement for divalent cations. We have investigated the kinetic properties of recombinant mouse thiamine triphosphatase (mThTPase) and determined its solution structure by NMR spectroscopy. Residues responsible for binding Mg(2+) and ThTP were determined from NMR titration experiments. The binding of Mg(2+) induced only a minor local conformational change, whereas ThTP binding was found to cause a more global conformational change. We derived a structural model for the mThTPase.ThTP.Mg(2+) ternary complex and concluded from this that whereas free mThTPase has an open cleft fold, the enzyme in the ternary complex adopts a tunnel fold. Our results provide a functional rationale for a number of conserved residues and suggest an essential role for Mg(2+) in catalysis. We propose a mechanism underlying the high substrate specificity of mThTPase and discuss the possible role of water molecules in enzymatic catalysis.Journal of Biological Chemistry 05/2008; 283(16):10939-48. DOI:10.1074/jbc.M709675200 · 4.60 Impact Factor