Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase

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• 作者：David Delvaux ; Fr¨¦d¨¦ric Kerff ; Mamidanna R.V.S. Murty ; Bernard Lakaye ; Jan Czerniecki ; Gregory Kohn ; Pierre Wins ; Rapha?l Herman ; Val¨¦rie Gabelica ; Fabien Heuze ; Xavier Tordoir ; Rapha?l Mar¨¦e ; Andr¨¦ Matagne ; Paulette Charlier ; Edwin De Pauw ; Lucien Bettendorff
• 关键词：CD ; circular dichroism ; PPPi ; inorganic tripolyphosphate ; PPPase ; tripolyphosphatase ; ThDP ; thiamine diphosphate ; ThMP ; thiamine monophosphate ; ThTP ; thiamine triphosphate ; ThTPase ; thiamine triphosphatase ; TTM ; triphosphate tunnel metalloenzyme
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：2013
• 出版时间：October, 2013
• 年：2013
• 卷：1830
• 期：10
• 页码：4513-4523
• 全文大小：1474 K

文摘

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.