The Refined Structure of dUTPase from Escherichia coli

Abstract

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 1.9 A resolution, in space group R3, a = b = 86.62, c = 62.23 A. Mercury and platinum derivative data were collected at wavelengths to optimize the anomalous contribution. The resulting 2.2 A MIRAS phases differed from the final set by 40 c 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/3-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 fl-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 fl-sheet and in loop regions. A similar molecular architecture has recently been found in two other trimeric dUTPases.

African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages

“…2B, the observed single activity peak overlapped with the protein peak, sedimenting with an apparent molecular mass of ϳ55 kDa, which corresponds, most probably, to a trimeric form of ASFV dUTPase. This result is in agreement with the trimeric structure described for dUTPases (14,31,35). Fractions 5 to 7 of the glycerol gradient were pooled and used for further in vitro analysis of ASFV dUTPase activity.…”

“…The prediction of a virus encoded dUTPase was tested by the multiple alignment of these sequences with some of the members of the dUTPase family ( Fig. 1A), including two of the dUTPases, the E. coli (14,31) and human (35) proteins, whose crystal structures have been described. Previously published comparisons of the primary structures of dUTPases from different sources (34) indicate the presence of five conserved motifs along the alignment, which are numbered and boxed in Fig.…”

Altered Subunit Communication in Subfamilies of Trimeric dUTPases

“…Apart from the Q106 polar residues at the top, the central channel of the E. coli enzyme is highly hydrophobic (c.f. [25]). The R104 long aliphatic side chains also provide van der Waals interactions with the W102 rings.…”

“…[25]). For ease of reference, these types are here termed as pairwise, armcrossing, and threefold interactions, respectively.…”

Structures of feline immunodeficiency virus dUTP pyrophosphatase and its nucleotide complexes in three crystal forms

“…However, in dUTPase the role of the P-loop motif has not been clearly established. In available crystal structures of Escherichia coli, human, FIV and EIAV dUTPase (Dauter et al, 1998(Dauter et al, , 1999Mol et al, 1996;Prasad et al, 1996), motif V is either poorly de®ned or completely disordered. In this paper, we report seven crystal structures of FIV dUTPase in three lattices, including ®ve complexes with dUMP, dUDP and dUTP.…”