title: Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme.
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 degrees C, 23 degrees C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg(2+) 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.
authors.affiliation: Institute of Enzymology, Biological Research Center (BRC), Hungarian Academy of Sciences, POB 7, H-1518, Budapest, Hungary.
authors.forename: Júlia
authors.initials: J
authors.lastname: Kovári
authors.forename: Orsolya
authors.initials: O
authors.lastname: Barabás
authors.forename: Enikõ
authors.initials: E
authors.lastname: Takács
authors.forename: Angéla
authors.initials: A
authors.lastname: Békési
authors.forename: Zsófia
authors.initials: Z
authors.lastname: Dubrovay
authors.forename: Veronika
authors.initials: V
authors.lastname: Pongrácz
authors.forename: Imre
authors.initials: I
authors.lastname: Zagyva
authors.forename: Timea
authors.initials: T
authors.lastname: Imre
authors.forename: Pál
authors.initials: P
authors.lastname: Szabó
authors.forename: Beáta G
authors.initials: BG
authors.lastname: Vértessy
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004260:DNA Repair; D019281:Dimerization; D004331:Drosophila melanogaster / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D007477:Ions; D007700:Kinetics; D008027:Light; D008274:Magnesium / Q000737:chemistry; D013058:Mass Spectrometry; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014965:X-Rays
chemical_list: D007477:Ions; D011994:Recombinant Proteins; D014357:Trypsin; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D008274:Magnesium; D013941:Thymine