Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase
- Authors
- Delvaux, D., Kerff, F., Murty, M.R., Lakaye, B., Czerniecki, J., Kohn, G., Wins, P., Herman, R., Gabelica, V., Heuze, F., Tordoir, X., Marée, R., Matagne, A., Charlier, P., De Pauw, E., and Bettendorff, L.
- ID
- ZDB-PUB-130703-34
- Date
- 2013
- Source
- Biochimica et biophysica acta. General subjects 1830(10): 4513-4523 (Journal)
- Registered Authors
- Keywords
- CyaB, ThTPase, triphosphate tunnel metalloenzyme, thiamine triphosphate, tripolyphosphate, divalent cation
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- Biocatalysis
- Circular Dichroism
- Crystallography, X-Ray
- Humans
- Models, Molecular
- Molecular Docking Simulation
- Molecular Sequence Data
- Protein Conformation
- Sequence Homology, Amino Acid
- Spectrometry, Mass, Electrospray Ionization
- Substrate Specificity
- Thiamin-Triphosphatase/chemistry
- Thiamin-Triphosphatase/metabolism*
- PubMed
- 23707715 Full text @ BBA General Subjects
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.
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.