[en] Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. Methods: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. Results: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. Conclusion: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. General significance: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins.
Research center :
Giga-Neurosciences - ULiège CIP - Centre d'Ingénierie des Protéines - ULiège Giga-Systems Biology and Chemical Biology - ULiège Physical Chemistry and Mass Spectrometry Laboratory
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Delvaux, David; Université de Liège - ULiège > GIGA-Neurosciences
Kerff, Frédéric ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Murty, Mamidanna R.V.S.
Lakaye, Bernard ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
Czerniecki, Jan
Kohn, Grégory ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
Wins, Pierre
Herman, Raphaël ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Gabelica, Valérie ; Université de Liège - ULiège > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.)
Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Charlier, Paulette ; Université de Liège - ULiège > Département des sciences de la vie > Cristallographie des macromolécules biologiques
De Pauw, Edwin ; Université de Liège - ULiège > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.)
Bettendorff, Lucien ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique
Structural Determinants of Specificity and Catalytic Mechanism in mammalian 25-kDa Thiamine Triphosphatase
Publication date :
2013
Journal title :
Biochimica et Biophysica Acta - General Subjects
ISSN :
0304-4165
eISSN :
1872-8006
Publisher :
Elsevier Science, Amsterdam, Netherlands
Volume :
1830
Pages :
4513-4523
Peer reviewed :
Peer Reviewed verified by ORBi
Name of the research project :
Belgian program of Interuniversity Attraction Poles initiated by the Federal Office for Scientific Technical and Cultural Affaires
Funders :
FRFC - Fonds de la Recherche Fondamentale Collective [BE] ULg FSR - Université de Liège. Fonds spéciaux pour la recherche [BE] BELSPO - Politique scientifique fédérale [BE] F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE]
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