Deoxyuridine 5-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes essential for hydrolysis of dUTP, thus preventing its incorporation into DNA. Although Epstein-Barr virus (EBV) dUTPase is monomeric, it has a high degree of similarity with the more frequent trimeric form of the enzyme. In both cases, the active site is composed of five conserved sequence motifs. Structural and functional studies of mutants based on the structure of EBV dUTPase gave new insight into the mechanism of the enzyme. A first mutant allowed us to exclude a role in enzymatic activity for the disulfide bridge involving the beginning of the disordered C terminus. Sequence alignments revealed two groups of dUTPases, based on the position in sequence of a conserved aspartic acid residue close to the active site. Single mutants of this residue in EBV dUTPase showed a highly impaired catalytic activity, which could be partially restored by a second mutation, making EBV dUTPase more similar to the second group of enzymes. Deletion of the flexible C-terminal tail carrying motif V resulted in a protein completely devoid of enzymatic activity, crystallizing with unhydrolyzed Mg 2Ű -dUTP complex in the active site. Point mutations inside motif V highlighted the essential role of lid residue Phe 273 . Magnesium appears to play a role mainly in substrate binding, since in absence of Mg 2Ű , the K m of the enzyme is reduced, whereas the k cat is less affected.Epstein-Barr virus, a human â„-herpesvirus, is the causative agent of infectious mononucleosis and establishes a lifelong persistent infection in over 90% of the world's population. EBV 3 is implicated in a number of cancers, such as Burkitt's lymphoma, undifferentiated nasopharyngeal carcinoma, or Hodgkin disease. The large DNA genome of this virus codes for about 86 proteins implicated in a large number of functions related to viral latency or the lytic cycle, during which the virus replicates.One protein of the lytic cycle is deoxyuridine 5Đ-triphosphate pyrophosphatase, a ubiquitous enzyme catalyzing the cleavage of dUTP into dUMP and pyrophosphate (PP i ). This enzyme not only provides the precursor for the formation of dTMP by thymidylate synthase, but it also has a crucial role in maintaining a low dUTP/dTTP ratio in the cell in order to limit the incorporation of deoxyuridylate into DNA by DNA polymerases. Based on their oligomerization state, dUTPases can be divided into three families.The first family of dUTPases contains homodimeric enzymes with the prototype structure of Trypanosoma cruzi dUTPase (1).dUTPases of the second and largest family form homotrimers. Their structure is unrelated to dimeric dUTPases. Trimeric dUTPases are found in most eukaryotes, in prokaryotes, in some DNA viruses, such as poxvirus, and in a number of retroviruses. Several x-ray structures of dUTPases of this family are available: Escherichia coli (2), Homo sapiens (3), equine infectious anemia virus (4), feline immunodeficiency virus (5), Mason-Pfizer monkey virus (6), Mycobacterium tuberculosis...