The refined structure of dUTPase from Escherichia coli

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, E.C. 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is involved in nucleotide metabolism and DNA synthesis. A crystal of the recombinant E. coli enzyme, precipitated from polyethylene glycol mixtures in the presence of succinate at pH 4.2, was used to collect synchrotron diffraction data to 19 Angstrom resolution, in space group R3, a = b = 86.62, c = 62.23 Angstrom. Mercury and platinum derivative data were collected at wavelengths to optimize the anomalous contribution. The resulting 2.2 Angstrom MIRAS phases differed from the final set by 40 degrees on average and produced an excellent map which was easy to interpret. The model contains 132 water molecules and refined to an R value of 13.7%. 136 residues have clear electron density out of 152 expected from the gene sequence. The 16 C-terminal residues are presumably disordered in the crystal lattice. The monomer is a 'jelly-roll' type, containing mostly beta-sheet and only one short helix. The molecule is a tight trimer. A long C-terminal arm extends from one subunit and encompasses the next one within the trimer contributing to its beta-sheet. Conserved sequence motifs common among dUTPases, previously suggested to compose the active site and confirmed in a recent study of the dUDP complex, are located at subunit-subunit interfaces along the threefold axis, in parts of the beta-sheet and in loop regions. A similar molecular architecture has recently been found in two other trimeric dUTPases.