title: Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase.
abstract: 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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: GIGA-Neurosciences, Université de Liège, Avenue de l'Hôpital 1, Liège, Belgium.
authors.forename: David
authors.initials: D
authors.lastname: Delvaux
authors.forename: Frédéric
authors.initials: F
authors.lastname: Kerff
authors.forename: Mamidanna R V S
authors.initials: MR
authors.lastname: Murty
authors.forename: Bernard
authors.initials: B
authors.lastname: Lakaye
authors.forename: Jan
authors.initials: J
authors.lastname: Czerniecki
authors.forename: Gregory
authors.initials: G
authors.lastname: Kohn
authors.forename: Pierre
authors.initials: P
authors.lastname: Wins
authors.forename: Raphaël
authors.initials: R
authors.lastname: Herman
authors.forename: Valérie
authors.initials: V
authors.lastname: Gabelica
authors.forename: Fabien
authors.initials: F
authors.lastname: Heuze
authors.forename: Xavier
authors.initials: X
authors.lastname: Tordoir
authors.forename: Raphaël
authors.initials: R
authors.lastname: Marée
authors.forename: André
authors.initials: A
authors.lastname: Matagne
authors.forename: Paulette
authors.initials: P
authors.lastname: Charlier
authors.forename: Edwin
authors.initials: E
authors.lastname: De Pauw
authors.forename: Lucien
authors.initials: L
authors.lastname: Bettendorff
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013837:Thiamin-Triphosphatase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D013837:Thiamin-Triphosphatase
keywords: CD; CyaB, ThTPase; Divalent cation; PPP(i); PPPase; TTM; ThDP; ThMP; ThTP; ThTPase; Thiamine triphosphate; Triphosphate tunnel metalloenzyme; Tripolyphosphate; circular dichroism; inorganic tripolyphosphate; thiamine diphosphate; thiamine monophosphate; thiamine triphosphatase; thiamine triphosphate; triphosphate tunnel metalloenzyme; tripolyphosphatase