Structural Insights into the Catalytic Mechanism of Phosphate Ester Hydrolysis by dUTPase*♦

dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and α,β-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution. Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site. Results provide an understanding for the catalytic role of conserved residues in dUTPases.

Due to the chemical reactivity of DNA, numerous mutagenic and carcinogenic modifications to mammalian genome occur daily (1). Preserving DNA integrity is of vital importance. The ubiquitous enzyme dUTPase performs a key role in preventing uracil incorporation into DNA by catalyzing the cleavage of dUTP into dUMP and pyrophosphate (2). This reaction contributes to thymidylate biosynthesis and strictly controls cellular dUTP/dTTP ratios (3).
Targeting enzymes of de novo thymidylate biosynthesis by fluorouracil or methotrexate is a widely used approach in anticancer chemotherapy (4). These drugs perturb the cellular dUTP/dTTP pool resulting in synthesis of highly uracil-substituted DNA. Uracil-DNA transforms base-excision repair into a hyperactive cycle inducing DNA double-strand breaks and thy-mine-less cell death. This pathway preferentially kills cells actively synthesizing DNA, such as tumor and virus-infected cells. Moreover, in human cancer cell lines, dUTPase overexpression leads to development of fluorouracil resistance (5). Therefore, the clinical benefits of an anticancer therapy based on inducing thymine-less cell death may ultimately depend on the critical interplay among several enzymes involved in thymidylate metabolism. The recent finding of activation of dUT-Pase gene expression in p53 mutant tumor cells has reinforced the notion that this enzyme plays a significant role in tumor development and survival (6). The potential initiative role of dUTPase antagonism in thymine-less cell death has prompted investigations into the enzymatic mechanism.
Detailed description of the catalytic mechanism is central to enzymology (7)(8)(9). Although several dUTPase crystal structures at atomic to moderate resolution have already been published (10 -14), mechanistic issues remain obscure. Using water labeled with 18 O, it was established that the water oxygen gets bonded to dUMP and not to the pyrophosphate, arguing for initiation of the reaction by nucleophilic attack at the ␣-phosphorus (15). A strictly conserved aspartate residue (Asp 90 in the Escherichia coli enzyme) in a conserved sequence motif of dUTPases was suggested to activate a water molecule for the nucleophile attack (12,13,15). However, since the presumed catalytic water molecule could not be identified in the structures available to date, there are no reliable structural data about protein side chains involved in the hydrolytic reaction. Moreover, the possibility of a protein atom executing the initial nucleophilic attack could not have been excluded. The binding site of the Mg 2ϩ co-factor was also not localized in the previously reported structures preventing structural insights into its functional role. Recently, the presence of Mg 2ϩ was shown to facilitate substrate binding to both E. coli (16) and Drosophila dUTPases (17) (K m decreases by ϳ0.5-1 order of magnitude) and to increase the value of k cat 2-3-fold.
In the present work, we addressed the unresolved mechanistic issues by three-dimensional structural investigations and kinetic/mutagenesis studies. We determined crystal structures of the wild type E. coli dUTPase:␣,␤-imino-dUTP:Mg 2ϩ , wild type dUTPase:dUMP, Asp 90 3 Asn mutant dUTPase:dUTP: Mg 2ϩ and Asp 90 3 Asn mutant dUTPase:␣,␤-imino-dUTP: Mg 2ϩ complexes (at 1.9-, 1.6-, 1.95-, and 1.7-Å resolution) complexes to identify conformational changes of protein side chains and their interactions with the ligand. A kinetic analysis was performed for the reaction of ␣,␤-imino-dUTP hydrolysis by wild type dUTPase, as well as for the reaction of dUTP hydrolysis by Asp 90 3 Asn mutant dUTPase. Stepwise comparisons among these structures and the apoenzyme structure (11) together with the kinetic results revealed essential interactions required for catalysis and allowed novel insights into the mechanism.
The Asp 90 3 Asn mutant E. coli enzyme was created by QuikChange (Strategene) with mutagenic primers 5Ј-cctggtaggattgatcAACtctgactatcagggccag-3Ј and 5Ј-ctggccctgatagtcagaGTTgatcaatcctaccagg-3Ј on plasmid pET3a-dut (19). Wild type and Asp 90 3 Asn mutant E. coli dUT-Pase was expressed and purified as described previously (16). Enzyme kinetic parameters toward dUTP were measured by the continuous spectrophotometric assay as described in Refs. 17 and 20. ␣,␤-Imino-dUTP hydrolysis was followed in reaction mixtures of 3 mg/ml E. coli dUTPase and 1-10 mM ␣,␤-imino-dUTP in 0.1 M Tris buffer, pH 7.8, 5 mM MgCl 2 , also containing 400 mM sodium acetate, at room temperature. The decrease of ␣,␤-imino-dUTP levels and production of dUMP was followed in aliquots taken at different time points to be analyzed either on a Mono Q anion exchange column or by thin layer chromatography (17).
Catalytic competence of the enzyme in the crystal phase was checked by competition experiments with the native substrate dUTP. Crystals of wild type dUTPase containing ␣,␤-imino-dUTP were washed with reservoire solution and dUTP was added at 10 mM concentration. Hydrolysis was followed by discontinuous thin layer chromatography activity test (24).
Structure Determination and Refinement-Structures of wild type and Asp 90 3 Asn mutant dUTPases, in complexes with ␣,␤-imino-dUTP:Mg 2ϩ , dUTP:Mg 2ϩ , and dUMP, were solved by molecular replacement (MOLREP (25)). The apo-dUTPase structure (11) was used as search model for all the four presently determined structures to exclude any model bias with respect to the ligand position. The asymmetric unit contained one monomer. In each case, the resultant initial maps were of exceptional quality, allowing residues 1-136 and entire ligands to be built with ease.
Generation of monomer libraries for the ligands (␣,␤-imino-dUTP, dUTP) and refinement were carried out using CCP4i and Refmac5 (22). Positional and B-factor refinement rounds were altered with manual rebuilding steps using the graphics program O (26), and ARP/wARP (27) was used for water building. Residues belonging to the C-terminal Motif V (residues 137-152) are hardly visible even in the final maps and are therefore mostly omitted from the model. A summary of the crystallographic data collection and refinement statistics is given in Table II.
Coordinates and structure factor data have been deposited in the Protein Data Bank with accession codes 1RN8, 1RNJ, 1SYL, and 1SEH, for the wild-type enzyme-␣,␤-imino-dUTP complex, the Asp 90 3 Asn enzyme-␣,␤-imino-dUTP complex, the Asp 90 3 Asn enzyme-dUTP complex, and the wild type enzyme-dUMP complex, respectively.
Simulated annealed omit electron density maps (sigma-weighted 2F o Ϫ F c maps) were calculated using CNS v1.1, omitting the entity in question and a 3.5-Å region surrounding it. The figures are restricted to show only the entity in question.

RESULTS
The Experimental Approach-Previous dUTPase complex structures identified the protein fold and the overall characteristics of active site architecture; however, they failed to provide an understanding of the catalytic mechanism (10,(12)(13)(14). This failure was probably due to the fact that these structures represented dead-end enzyme-inhibitor complexes. To produce the relevant enzyme-substrate complexes with lifetimes allowing crystallographic characterization, we considered the following potentials.
First, the substrate dUTP was replaced by the substrate analogue ␣,␤-imino-dUTP. Imino analogues of nucleotide phosphates are commonly used as isosteric mimics of the natural substrates (31,32) both with respect to bond distances (1.63 and 1.68 Å, for P-O-P and P-N-P moieties, respectively) and bond angles (128.7°and 127.2°, for P-O-P and P-N-P, respectively) (33). The lower electronegativity of the nitrogen atom results in less reactivity of the phosphate ester imino analogues. Nevertheless, several enzymes (e.g. small GTPase p21 (34), sarcoplasmic reticulum ATPase (35), and alkaline phosphatase (36)) were reported to catalyze imino-linkage cleavage, arguing for some reactivity associated with the P-N-P moiety. Fig. 1A demonstrates that dUTPase is also capable of hydrolyzing the imino-linkage of ␣,␤-imino-dUTP, while no detectable hydrolysis of the ␤-␥ bond occurs, i.e. the exclusive specificity of dUTPases for ␣-␤ bond cleavage is retained in the reaction with the imino analogue as well. Table I summarizes kinetic and ligand binding data for the dUTPase-catalyzed dUTP and ␣,␤-imino-dUTP hydrolysis. The very low k cat value in the ␣,␤-imino-dUTP hydrolysis reaction rendered K m determination unreliable; therefore the previously determined K d value is presented for comparison (37). Assuming rapid equilibrium reactions, as suggested by an earlier kinetic analysis (15), K m and K d values are expected to be the same. The very close agreement in the nucleotide interaction data (K m , K d , cf. Table I) for dUTP and the analogue ␣,␤-imino-dUTP strongly suggests highly similar accommodation patterns for these two hydrolysable nucleotides in the dUTPase active site, although the rate of hydrolysis differs by almost 6 orders of magnitude. Based on these kinetic and ligand binding data, both the enzyme-dUTP and the enzyme-␣,␤-imino-dUTP complex will be termed in the present work as enzyme-substrate complexes (Fig. 1B).
Second, the dUTPase catalytic co-factor Mg 2ϩ ion (15,16), not yet localized in any previously reported crystal structure (10 -14, 38, 39), was also included during complex formation. Mg 2ϩ binding in the enzyme:␣,␤-imino-dUTP/dUTP complexes was promoted by novel crystallization conditions at neutral or slightly basic pH values, since deprotonation of side chains has a well known positive effect on metal ion coordination capability. Earlier crystallization solvents contained either acidic buffers (11,12,38) or high concentrations of metal-chelating citrate (13,39).
Third, a site-directed mutation was designed to exchange the strictly conserved aspartate side chain (Asp 90 ) within the active site into an asparagine residue. This mutation was expected to decrease the reaction rate and allow for a structural analysis of the enzyme-dUTP complex in the presence of the co-factor Mg 2ϩ ion.
Structure of Enzyme-Substrate Complex-dUTPases show exquisite substrate specificity with respect to base, sugar, and phosphate chain moieties of dUTP that is essential to prevent unintended hydrolysis of other high energy-containing nucleotides. Most dUTPases are homotrimers ( Fig. 2A) with five conserved sequence motifs in each monomer ( The catalytic water molecule initiating nucleophile attack and the Mg 2ϩ ion coordinated to the triphosphate chain are shown as determined in the crystal structure (D). In the reaction products, the Mg 2ϩ -pyrophosphate interaction is hypothetic, the hypothesis being based upon the apparent absence of the Mg 2ϩ from the dUTPase:dUMP structure. C, D, and E, active-site close-ups of apoenzyme, dUTPase:␣,␤-imino-dUTP:Mg 2ϩ , and dUTPase:dUMP structures, respectively. In the subunit-color-coded ribbon model of the protein, the side chain and/or main chain atoms of some conserved residues (Tyr 93 , Asp 90 , Leu 88 from monomer A, Ala 29 , Asp 32 , Arg 71 , Ser 72 , Gly 73 , and Gln 119 from monomer B) important in active site architecture are also shown, together with some significant water molecules. For denominations of these residues and waters, omitted from this figure to avoid small print details, please refer to Fig. 3A that presents the active site in the same orientation. The ligands and the non-carbon protein atoms are in atom coloring bonds model (carbon, dark gray; oxygen, red; phosphorus, orange; nitrogen, blue; magnesium, purple). The apoenzyme structure was taken from the literature (11). among dUTPases from retroviruses and bacteria to man (10,(12)(13)(14).
In agreement with previous studies (10,(12)(13)(14), each ␤-pleated subunit forms jellyroll topology and contributes the C-terminal ␤-strand to the neighboring subunit. The three active sites of the homotrimer are located in clefts between neighboring monomers and recruit conserved motifs from different subunits in a 3-fold symmetric pattern ( Fig. 2A). A ␤-hairpin formed by conserved Motif III from monomer A accommodates the uracil and sugar rings. A conserved tyrosine (Tyr 93 ) stacks to the 2Ј-deoxyribose ring and sterically excludes ribonucleotides. Phosphate chain recognition is provided by conserved Motifs I, II, and IV of monomer B (Figs. 1, D and E, and 3). Therefore, active site architecture ultimately depends on correct oligomerization of the homotrimer. To our present knowledge, this organization of ligand binding sites is unique among proteins.
The present structure localizes the catalysis-assisting physiological co-factor Mg 2ϩ , not seen in any previously reported structure, and the entire triphosphate chain of the hydrolyz- The trimer is shown with ribbon model of color-coded (code retained throughout the present study, where applicable) subunits and bonds model of the nucleotide ligand molecule in the three active sites. Mg 2ϩ -ions are represented as purple balls. B, Sequence alignments of dUTPases. Conserved residues within the five (I-V) dUTPase motifs are in bold white on black background. Arrowhead points at the conserved aspartate in Motif III, Asp90 in the E. coli sequence, which was selected for the Asn mutation.
able substrate ␣,␤-imino-dUTP in the enzyme active site with low crystallographic B-factors, indicating a well ordered assembly (cf. Table II, first data column). Detailed analysis of the catalytically competent enzyme conformation provides novel determinant features of the enzymatic mechanism. All three phosphate groups are within the coordination sphere of the well defined metal ion that also receives coordination through water molecules coordinated by Asp 32 and Gln 119 of subunit B (Figs. 3A and 4A). The ␣-phosphate oxygens are contacted by N⑀2 of the same Gln 119 , and the main chain NH of Ser 72 from subunit B, while Arg 71 of subunit B reaches toward the ␤and ␥-phosphates (Figs. 3A and 4A). Some water molecules are well localized (with temperature factors 17, 8, 18, 14, and 12 Å 2 , for W1, W2, W4, W15, and W21, respectively, to be compared with the average temperature factor of 17.4 Å 2 ) around the phosphate oxygens. These waters play a crucial role in mediating interactions between the protein residues and the phosphate oxygens.
Comparison of apoenzyme and enzyme-substrate complex shows only minor conformational shifts in the active site, and most of these are involved in residues of subunit B, responsible for phosphate accommodation. The movement of Arg 116 and Arg 71 by 1.23 and 0.88 Å, respectively, provide phosphate chain interactions, while Gln 119 adopts an altered conformation for proper hydrogen-bonding geometry. The catalytic site in the apoenzyme already presents side chain conformations adequate for binding of the uracil and deoxyribose moieties, alleviating the need for significant changes induced by the incoming dUTP (Figs. 1, C and D, and 3D).
Identification of the Nucleophile Responsible for Attack at the ␣-Phosphorus-The present dUTPase-substrate complex structure allows identification of the attacking nucleophile. A search along the axis of the ␣-phosphorus-imino nitrogen bond located a single possible candidate for in-line nucleophile attack: a water oxygen (termed W cat , numbered as W5 in the 1RN8 coordinate file, red arrow in Figs. 1D and 3, A and B, cf. also Fig  4A) coordinated to Asp 90 in monomer A (previously suggested to be a catalytic residue (12)). No other protein or water atoms were found within 4.0 Å to the ␣-phosphorus, emphasizing the special role of the proximal water (W cat ). A simulated annealed omit map, calculated with omission of entities in the 3.5 Å radius region around this water, was created for its clear localization (Fig. 3A). Both carboxyl oxygens of Asp 90 may participate in hydrogen-bonding to W cat . AspO␦2 is also within Hbonding distance to the deoxyribose 3Ј-OH group, while AspO␦1 is H-bonded to monomer B Ala 29 main chain NH and another water (W4) that contacts monomer B Gln119 through W1 (Figs. 3A and 4A). The monomer B Ala 29 main chain Hbond may play a role in orienting the Asp 90 side chain. No other side chain atoms were closer than 4.0 Å to this putative catalytic water. However, another protein atom (Leu88 main chain carbonyl O) is also within H-bonding distance to W cat as discussed below. This arrangement suggested that replacing only one side chain oxygen of Asp 90 with an amino group might drastically affect enzyme function. The Asp 90 3 Asn mutant was constructed and proved to be largely compromised in catalytic efficiency (Table I). k cat /K m for the mutant was determined to be 850 M Ϫ1 s Ϫ1 as compared with 3.5 ϫ 10 7 M Ϫ1 s Ϫ1 for wild type, a decrease of almost 5 orders of magnitude. Importantly, K m was the same for both mutant and wild type enzymes (0.45 versus 0.5 M) ( Table I), indicating that substrate accommodation is the same in both species. The crystal structure of the Asp 90 3 Asn mutant in complex with Mg 2ϩ and ␣,␤-imino-dUTP was solved at 1.7-Å resolution and is shown as superimposed on the wild type complex structure (Fig. 3B, cf. also Fig 3F, and Table II). The superimposed structures are practically indistinguishable (r.m.s. 1 for fitting of all atoms is 0.12), except for the catalytic water electron density (Fig. 3, A,  B, E, and F) that is missing from the mutant complex structure. A straightforward interpretation of this result is that replacing AspO␦2 with an NH 2 group with altered H-bonding characteristics adversely interferes with W cat coordination in the Asp 90 3 Asn mutant. A rotation of AsnO␦1 (the corresponding atom for AspO␦1) into the AspO␦2 position could theoretically provide proper coordination for W cat , but this rotation is impeded by the AsnO␦1-monomer B Ala 29 main chain H-bond, also present with AspO␦1 in the wild type complex. 1 The abbreviation used is: r.m.s., root mean square. The very low enzymatic activity of the Asp 90 3 Asn mutant rendered it possible to determine the crystal structure of the mutant enzyme:dUTP:Mg 2ϩ complex (Figs. 3, C and G , Table  II). In this structure, substrate and Mg 2ϩ accommodation patterns are highly similar to those realized in the enzyme:␣,␤imino-dUTP:Mg 2ϩ complex (Fig. 3, C, F, and G). All atoms of the imino analogue and the physiological substrate, including the ␣-phosphate group where nucleophilic attack occurs, are well superimposable (r.m.s. is 0.179).
These structures, together with the kinetic data, clearly demonstrate that the water molecule termed W cat provides the attacking nucleophile oxygen. Although the structures that identified W cat have been determined for the ␣,␤-imino-dUTP complexes of wild type and Asp 90 3 Asn mutant dUTPases, the catalytic incompetence of the Asp 90 3 Asn mutant was proved in the physiological reaction (dUTP hydrolysis). Therefore, the Asp 90 residue plays a determinant role in dUTP hydrolysis, too.  Table I and three-dimensional structures in Fig. 3, B, E, and F). Consequently the attenuated catalytic efficiency of the mutant enzyme is most probably due to lack of proper coordination to the catalytic water molecule in the physiological reaction. In addition, phosphate chain conformations in the dUTPase:␣,␤-imino-dUTP:Mg 2ϩ and dUTPase: dUTP:Mg 2ϩ complexes are identical (Fig. 3C), indicating that the nucleophile attack mechanism should be similar in both cases.
Interestingly, the catalytic water molecule seems to be present in the apoenzyme structure (1EUW (11)), as well (W334 at a distance of 0.86 Å to the W cat position, cf. also Fig 3D). It is coordinated to Asp 90 O␦2, as well as to the Leu 88 main chain carbonyl oxygen, just as it is found in the presently determined enzyme:␣,␤-imino-dUTP:Mg 2ϩ complex (Fig. 3D). In the latter complex, W cat clearly adopts a closely co-linear location to the scissile bond to carry out nucleophilic attack on the ␣-phos-phate. In the enzyme-product complex structure, as detailed below (Table II, Figs. 1E, 3D, and 4B), the W cat proximal water (W49) has again the same coordination pattern. In summary, W cat (or the W cat proximal water) coordinates to the same protein atom ligands (Asp 90 O␦2 and Leu 88 main chain O) in all these structures. We propose that these two protein ligands, with the possible participation of other water molecules, create a binding site for W cat that is, in fact, one of the substrates required for the dUTP hydrolysis reaction (dUTP ϩ H 2 O ϭ dUMP ϩ PP i , cf. Fig 1B). This binding site is already available in the absence of the nucleotide substrate. After completion of the catalytic reaction, products (dUMP and PP i ) are expected to be exchanged for substrates (dUTP and H 2 O). Following the two-substrate analogy, the presence of W cat in the enzymeproduct complex may be interpreted as recruitment of a solvent water molecule in such an exchange reaction.
Completion of the Catalytic Cycle-The pyrophosphate group Interactions are shown only for the phosphate chain moiety of the ligand. Due to the close similarity of the nucleotide interactions in the three enzyme-substrate complexes determined in the present study (cf. Fig. 3 and Table I), the map was selected to show the actual distances as found in the wild type dUTPase:␣,␤-imino-dUTP: Mg 2ϩ (X ϭ N) complex where W cat is also present. In the Asp 90 3 Asn mutant dUT-Pase:␣,␤-imino-dUTP:Mg 2ϩ (X ϭ N) and Asp90 3 Asn mutant dUTPase:dUTP: Mg 2ϩ (X ϭ O) complex, the only significant differences are that (i) W cat is absent and Asp 90 O␦2 becomes AsnN␦2 and (ii) in the Asp 90 3 Asn mutant dUTPase:dUTP: Mg 2ϩ (X ϭ O) complex, the X-Ser 72 O␥ interaction is absent. Changes in all other distances are within Ϯ0.2 Å. at the entrance of the active site is expected to exit freely as it can easily make numerous contacts with the bulk solvent. The metal ion may facilitate pyrophosphate discharge since it is more likely to bind to the pyrophosphate (charge minus 4) as compared with dUMP (charge minus 2 at physiological pH). Accordingly, no electron density could be assigned to Mg 2ϩ in the enzyme-product structure, although the dUTPase-dUMP complex has been crystallized in the presence of the metal ion (Table II and Figs. 3D and 4B). The C-terminal Motif V from monomer C, mostly disordered in our present and also in previously reported structures, may also participate in pyrophosphate escape via charge stabilization with its strictly conserved Arg 141 side chain. Preferential discharge of the pyrophosphate moiety over the dUMP is also strengthened by x-ray crystallographic investigations carried out on dUTPase crystals soaked into substrate containing solution that retained dUMP, but not the pyrophosphate moiety, in the enzyme active site (14).
Figs. 3D and 4, A and B, indicate that the reaction product dUMP retains most of the binding interactions found with the substrate with respect to the sugar and base moieties. However, some side chains providing interactions with the triphosphate moiety (e.g. monomer B side chains Asp 32 , Arg 71 , Ser 72 , and Gln 119 ) are shifted back to their positions occupied in the apoenzyme (Fig. 3D). This shift may contribute to the significant decrease of dUMP binding affinity to dUTPase, when compared with dUDP, dUTP, and ␣,␤-imino-dUTP (15,37). Less tight binding of dUMP as compared with dUTP facilitates exchange of product with substrate in the active site whenever substrate is available in the bulk phase.

DISCUSSION
The side-by-side three-dimensional structural analysis of the apoenzyme, as well as substrate, and product complexes delineated atomic interactions crucial to the catalytic mechanism of dUTPase. The conclusions were based on structures and kinetic data determined with the imino substrate analogue, as well as the physiological substrate and the physiological product. Several results indicate that the reaction pathway may well be of similar character with both dUTP and ␣,␤-imino-dUTP. First, a water molecule in the immediate vicinity to the presently determined W cat is localized already in the apoenzyme structure and is also present in the enzyme-dUMP complex (Fig. 3D). Second, the binding position of the physiological substrate dUTP, most importantly the entire phosphate chain conformation, is equivalent to that of the imino analogue ( Fig.  3. C, F, and G). Third, the Asp 90 3 Asn mutation was shown to adversely affect W cat coordination and, parallel to this, to inactivate the enzyme in the physiological reaction. It is therefore reasonable to assume that (i) a water molecule coordinated to Asp 90 and Leu 88 is present with high probability in the active site of the wild type enzyme when dUTP is also bound, and (ii) this water molecule is poised for in-line attack, well within 4.0 Å to the ␣-phosphorus. Given such an arrangement, it can be assumed that this specific water will act as the nucleophile in dUTP hydrolysis.
The Metal Ion Site-The localization of the site for the catalysis-assisting Mg 2ϩ provided significant novel insights, as well. Its important role in providing catalytically competent accommodation of the substrate in the dUTPase active site, as judged by k cat and K m determinations in the presence and absence of the metal ion, was well established in a number of earlier studies on dUTPases from diverse sources (15)(16)(17)24). It was also well known that divalent metal ions usually contribute to enzymatic reactions of nucleotides by coordinating the otherwise quite flexible phosphate chain. Consequently, absence of the active site Mg 2ϩ from the earlier determined crystal structures prevented the definition of the catalytically competent phosphate chain conformation, thereby rendering the identification of the nucleophile attacker impossible. The data on the dUTPase:dUDP:Sr 2ϩ complex were a significant step forward in this problem (10). However, due to the diminished catalytic competence of the Sr 2ϩ substitution as well as the altered coordination geometry of the Sr 2ϩ (coordination number 8) as compared with Mg 2ϩ (coordination number 6), the character of the nucleophile attack could not be determined before. In fact, in the dUTPase:dUDP:Sr 2ϩ complex, the critical position of the ␣-phosphorus is 3.2 Å away from its competent site as determined in our present dUTPase:dUTP:Mg 2ϩ and Mg 2ϩ :␣,␤-imino-dUTP:dUTPase structures. The coordination of Mg 2ϩ to all the three phosphate groups is not very common in other nucleotide-protein structures where the metal ion is frequently seen as coordinating to only two phosphate groups (41)(42)(43)(44). In the dUTPase structures, the triple phosphate coordination of Mg 2ϩ contributes to an increasingly compact phosphate chain conformation presumably optimal for catalysis.
Role of Conserved Residues-The enzymatic reaction is facilitated by non-covalent bonding interactions and involves residues from different monomers, as well as several water molecules bound within the active sites. All residues proposed to interact with ligand and W cat are strictly conserved among dUTPase sequences. The contacts and role of some of these residues, like those of the ␤-hairpin, Ile 89 , Asp 90 , Tyr 93 , Ser 72 , Arg 71 , Arg 116 , and Gln 119 , were already suggested based on previously determined crystal structures of E. coli, equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and Homo sapiens dUTPases (10,(12)(13)(14). However, as previous studies failed to define a catalytically competent substrate conformation, the relevant roles of conserved residues need to be reinterpreted in the light of our present data.
In general, the conclusions drawn from our structures concerning the role of Gly 73 main chain nitrogen and Arg 71 guanidino group in coordination and charge distribution of the phosphate chain, especially the ␤and ␥-phosphates correspond well with suggestions supported by previously determined structures of dUTPases from various sources (10,(12)(13)(14). However, the interaction between the phosphate chain and Arg 71 became water-mediated in our structures. This difference might be ascribed to the effective charge stabilization provided by the Mg 2ϩ in our structures. Due to the presence of Mg 2ϩ , the triphosphate chain adopts a more compact conformation that increases its distance from the Arg 71 guanidino group. The weaker charge stabilization effect of the Arg 71 side chain is probably compensated for by the Mg 2ϩ ion.
The compact character of the phosphate chain in the present structures also shifts the interaction between the Ser 72 main chain NH and an oxygen in the ␤-phosphate group to another oxygen in the ␣-phosphate group. This shift may contribute to activation of the ␣-phosphorus for the nucleophile attack and might stabilize the developing charge in the transition state.
The present data provide detailed characterization of role of Asp 32 as well. In the previous retroviral dUTPase:dUDP:Sr 2ϩ complex structure (10), one of the carboxyl oxygens of this conserved residue (Asp 20 in the retroviral sequence) was within H-bonding distance to one of the eight coordinating ligands of the Sr 2ϩ ion. The large size of Sr 2ϩ , its altered coordination geometry, and poor catalytic competence, however, attenuated the relevance of the Mg 2ϩ 3 Sr 2ϩ substitution for mechanistic conclusions. In the present structures, both side chain oxygens of Asp 32 take part in coordination of two water molecules in the coordination sphere of the physiological metal ion that has six ligands.
The Catalytic Water-We presented a description of the catalytic pathway via identification of the nucleophile agent and characterization of interactions responsible for building up the required arrangement and initiating the reaction. The key residue here, Asp 90 , was previously shown to contribute to substrate binding via H-bonding to the OH group of the ribose. A general base-like role was also suggested, but in the absence of the identification of the nucleophile attacker atom, this role could not have been clarified. In the present structures, one of the side chain oxygens of Asp 90 (O␦2) provides close coordination to the catalytic water, and Asp 90 O␦1 is also nearby (Fig.  4A). Importantly, the present structural data also show that the proper orientation of the critical Asp 90 side chain is provided by an interaction between one of its other carboxyl oxygens (O␦1) and the main chain NH of the conserved Ala 29 from the neighboring subunit. This connection is strictly retained in all other dUTPase structures, as well, but no importance was ascribed to it. It also explains why the Asp 90 3 Asn mutant is so much attenuated in catalytic efficiency and ineffective in coordinating the W cat . In the mutant, the Ala 29 main chain NH retains its interaction with O␦1 of the Asn 90 residue, leaving only the nitrogen atom of the mutant (N␦2) for the role of close coordination to the catalytic water molecule. The nitrogen atom, however, cannot fulfill this role efficiently.
We also identified a second protein atom ligand of W cat . The main chain carbonyl oxygen of Leu 88 , a conserved hydrophobic residue together with Asp 90 creates a well defined binding site for this water that is the second substrate of the enzyme.
At last, significance of Gln 119 (Motif IV) has also been discovered in this study. Its side chain oxygen (O⑀1) coordinates a water molecule (W1) that provides H-bonding in the water network around W cat . The Gln 119 side chain amino group participates in H-bonding to one of the oxygens on the reaction center ␣-phosphorous.
The Character of Phosphate Ester Hydrolysis-Available data for non-enzymatic phosphate diester hydrolysis reactions are in favor of a mechanism with significant associative character, since the metaphosphate transition state required for the dissociative mechanism is highly unstable when an additional bulky ligand is present (45,46). Our present results for the dUTPase-catalyzed reaction are in agreement with this expectation. In our structure, the distance between the entering oxygen and the phosphorus reaction center is 3.6 Å with an experimental error of 0.164 Å based on the Luzzati plot (47), while a minimum of 4.9 Å is considered to be required for the dissociative mechanism (cf. Ref. 48). The present data show co-linearity of W cat with the scissile bond for in-line attack that can also be reconciled with a mechanism of significant associative character.