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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.

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro dd -carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide ( d -α-aminopymelyl-e- d -alanyl- d -alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A β-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of d -α-aminopymelyl-e- d -alanyl- d -alanine resulted in an adduct between PBP4a and a d -α-aminopimelyl-e- d -alanine dipeptide and an unbound d -alanine, i.e. the products of acylation of PBP4a by d -α-aminopymelyl-e- d -alanyl- d -alanine with the release of a d -alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.

Particle induced X-ray emission (PIXE) method is used in the field of archeometry and specially to investigate pigment colored multilayers. The tilting of the sample with respect to the incident proton beam direction allows to modify the relative contribution of each layer to the fluorescence signal. The experimental results coupled to computer simulations lead to semi-quantitative information about the thickness, the position and the composition of the successive layers.