Multidimensional NMR Identifies the Conformational Shift Essential for Catalytic Competence in the 60-kDa Drosophila melanogaster dUTPase Trimer

Zsófia Dubrovay, Zoltán Gáspári, Éva Hunyadi-Gulyás, Katalin F. Medzihradszky, András Perczel, Beáta G. Vértessy

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The catalytic mechanism of dUTP pyrophosphatase (dUTPase), responsible for the prevention of uracil incorporation into DNA, involves ordering of the flexible C terminus of the enzyme. This conformational shift is investigated by multidimensional NMR on the Drosophila enzyme. Flexible segments of the homotrimer give rise to sharp resonances in the 1H-15N heteronuclear single-quantum coherence (HSQC) spectra, which are clearly distinguishable from the background resonances of the well folded protein globule. Binding of the product dUMP or the analogues dUDP and α,β-imino-dUTP to the enzyme induces a conformational change reflected in the disappearance of eight sharp resonances. This phenomenon is interpreted as nucleotide binding-induced ordering of some residues upon the folded protein globule. Three-dimensional 15N-edited 1H-15N HSQC total correlation spectroscopy (TOCSY) and 1H-15N HSQC nuclear Overhauser effect spectroscopy measurements allowed clear assignment of these eight specific resonance peaks. The residues identified correspond to the conserved C-terminal sequence motif, indicating that (i) this conformational shift is amenable to NMR studies in solution even in the large trimeric molecule and (ii) formation of the closed enzyme conformer in the case of the Drosophila enzyme does not require the complete triphosphate chain of the substrate. NMR titration of the enzyme with the nucleotide ligands as well as kinetic data indicated significant deviation from the model of independent active sites within the homotrimer. The results suggest allosterism in the eukaryotic dUTPase.

Original languageEnglish
Pages (from-to)17945-17950
Number of pages6
JournalJournal of Biological Chemistry
Issue number17
Publication statusPublished - ápr. 23 2004


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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