Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: Kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme

Júlia Kovári, Orsolya Barabás, Enikõ Takács, Angéla Békési, Zsófia Dubrovay, Veronika Pongrácz, Imre Zagyva, Timea Imre, Pál Szabó, Beáta G. Vértessy

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 °C, 23 °C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg2+ binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.

Original languageEnglish
Pages (from-to)17932-17944
Number of pages13
JournalJournal of Biological Chemistry
Volume279
Issue number17
DOIs
Publication statusPublished - Apr 23 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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