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. 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 (1Friedberg E. Walker G. Siede W. DNA Mutagenesis and Repair. ASM Press, Washington, D. C.1995: 108-133Google Scholar). 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 (2Shlomai J. Kornberg A. J. Biol. Chem. 1978; 253: 3305-3312Abstract Full Text PDF PubMed Google Scholar). This reaction contributes to thymidylate biosynthesis and strictly controls cellular dUTP/dTTP ratios (3Pearl L.H. Savva R. Nat. Struct. Biol. 1996; 3: 485-487Crossref PubMed Scopus (39) Google Scholar). Targeting enzymes of de novo thymidylate biosynthesis by fluorouracil or methotrexate is a widely used approach in anticancer chemotherapy (4Aherne G.W. Browne S. Jackman A.L. Anticancer Development Guide: Antifolate Drugs in Cancer Therapy. Humana Press, Inc., Totowa, NJ1999: 409-421Google Scholar). 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 thymine-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 (5Canman C.E. Radany E.H. Parsels L.A. Davis M.A. Lawrence T.S. Maybaum J. Cancer Res. 1994; 54: 2296-2298PubMed Google Scholar). 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 dUTPase gene expression in p53 mutant tumor cells has reinforced the notion that this enzyme plays a significant role in tumor development and survival (6Pugacheva E.N. Ivanov A.V. Kravchenko J.E. Kopnin B.P. Levine A.J. Chumakov P.M. Oncogene. 2002; 21: 4595-4600Crossref PubMed Scopus (86) Google Scholar). 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 (7Benkovic S.J. Hammes-Schiffer S. Science. 2003; 301: 1196-1202Crossref PubMed Scopus (947) Google Scholar, 8Knowles J.R. Nature. 1991; 350: 121-124Crossref PubMed Scopus (494) Google Scholar, 9Schowen R.L. Gandour R.D. Schowen R.L. Transition States of Biochemical Processes. Plenum Press, New York1978: 77-113Crossref Google Scholar). Although several dUTPase crystal structures at atomic to moderate resolution have already been published (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 11Gonzalez A. Larsson G. Persson R. Cedergren-Zeppezauer E. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 767-774Crossref PubMed Scopus (28) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar), mechanistic issues remain obscure. Using water labeled with 18O, 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 α-phosphorus (15Larsson G. Nyman P.O. Kvassman J.O. J. Biol. Chem. 1996; 271: 24010-24016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). A strictly conserved aspartate residue (Asp90 in the Escherichia coli enzyme) in a conserved sequence motif of dUTPases was suggested to activate a water molecule for the nucleophile attack (12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 15Larsson G. Nyman P.O. Kvassman J.O. J. Biol. Chem. 1996; 271: 24010-24016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). 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 the nucleophilic attack could not have been The binding of the was not in the structures preventing structural into the of was shown to substrate binding to E. coli D. A. S. A. 2003; PubMed Scopus Google and dUTPases J. E. A. Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google by of and to the of the the mechanistic issues by structural investigations and determined crystal structures of the wild type E. coli wild type mutant and mutant complexes and complexes to of protein side chains and interactions with the A kinetic analysis was performed for the reaction of hydrolysis by wild type as as for the reaction of dUTP hydrolysis by mutant among structures and the A. Larsson G. Persson R. Cedergren-Zeppezauer E. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 767-774Crossref PubMed Scopus (28) Google with the kinetic essential interactions for and into the mechanism. and was from was by enzymatic of Larsson G. Nyman P.O. Chem. 1996; 4: PubMed Scopus Google and was shown to be by were of from or The mutant E. coli enzyme was by with mutagenic and on R. J. R. Nyman P.O. 2002; PubMed Scopus Google Scholar). type and mutant E. coli dUTPase was and as D. A. S. A. 2003; PubMed Scopus Google Scholar). kinetic dUTP were by the as in J. E. A. Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and PubMed Scopus Google hydrolysis was in reaction of E. coli dUTPase and in at The of and of dUMP was in at to be on a or by J. E. A. Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and were at by The for or or and in and was with of and data were into and of in in a data were at at wild type E. coli at wild type E. coli at and mutant E. coli at the data of the mutant E. coli was at the and were out Crystallogr. and Acta Crystallogr. D. Biol. Crystallogr. 1994; PubMed Scopus Google or W. J. Crystallogr. Scopus Google Scholar). of the enzyme in the crystal was by with the substrate of wild type dUTPase were with and dUTP was at was by A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Structure and of wild type and mutant in complexes with and were by A. A. J. Crystallogr. Scopus Google The A. Larsson G. Persson R. Cedergren-Zeppezauer E. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 767-774Crossref PubMed Scopus (28) Google was used as for the determined structures to with to the The the were of residues and to be with of for the and were out and Acta Crystallogr. D. Biol. Crystallogr. 1994; PubMed Scopus Google Scholar). and were with the Acta Crystallogr. A. 1991; PubMed Scopus Google Scholar), and Wilson K.S. Acta Crystallogr. D. Biol. Crystallogr. PubMed Google was used for water to the are in the and are from the A of the data and is in data and wild type in to the resolution mutant mutant is the of the and is the multiple from from are for a of the data that was from the is the of the and is the multiple from are for a of the data that was from the in a and data have been in the with and for the the the and the wild type The were J. Crystallogr. 1991; Google Scholar), E.A. PubMed Scopus Google Scholar), and Acta Crystallogr. D. Biol. Crystallogr. 1999; PubMed Scopus Google Scholar). were the in and a The are to the in The dUTPase structures identified the protein and the of active to provide an understanding of the catalytic mechanism (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar). This was to the that structures the relevant complexes with the the substrate dUTP was by the substrate of are used as of the G. Nat. Struct. Biol. 4: PubMed Scopus Google Scholar, W. J. A. Nature. PubMed Scopus Google with to and for and and and for and R. Science. PubMed Scopus Google Scholar). The of the atom in reactivity of the several enzymes W. A. J. PubMed Scopus Google Scholar), J. Biol. Chem. Full Text PDF PubMed Google Scholar), and D. W. D. PubMed Scopus Google were to arguing for reactivity with the that dUTPase is of the of no hydrolysis of the the of dUTPases for cleavage is in the reaction with the as kinetic and binding data for the dUTP and The in the hydrolysis reaction the determined is for Larsson G. Persson Persson R. Nyman P.O. PubMed Scopus Google Scholar). as suggested by an kinetic analysis (15Larsson G. Nyman P.O. Kvassman J.O. J. Biol. Chem. 1996; 271: 24010-24016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and are to be the The in the data for dUTP and the highly for in the dUTPase active the of hydrolysis by of on kinetic and binding the and the be in the as complexes and binding data for wild type and mutant E. coli dUTPase the of three to of and was and not from from Larsson G. Persson Persson R. Nyman P.O. PubMed Scopus Google in a the dUTPase catalytic (15Larsson G. Nyman P.O. Kvassman J.O. J. Biol. Chem. 1996; 271: 24010-24016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, D. A. S. A. 2003; PubMed Scopus Google Scholar), not in crystal (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 11Gonzalez A. Larsson G. Persson R. Cedergren-Zeppezauer E. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 767-774Crossref PubMed Scopus (28) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar, E.S. Larsson G. Nyman P.O. Z. Wilson K.S. Nature. PubMed Scopus Google Scholar, G.S. Stura E.A. G.S. Elder J.H. Stout C.D. 1996; PubMed Scopus Google Scholar), was binding in the complexes was by at or since of side chains has a on A. Larsson G. Persson R. Cedergren-Zeppezauer E. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 767-774Crossref PubMed Scopus (28) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, E.S. Larsson G. Nyman P.O. Z. Wilson K.S. Nature. PubMed Scopus Google or of C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, G.S. Stura E.A. G.S. Elder J.H. Stout C.D. 1996; PubMed Scopus Google Scholar). a was to the strictly conserved aspartate side the active into an residue. This was to the reaction and for a structural analysis of the in the of the Structure of substrate with to and of dUTP that is essential to hydrolysis of dUTPases are with conserved sequence motifs in (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar). for conserved dUTPase motifs in (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, A. Res. 2000; PubMed Scopus (39) Google Scholar). and active are conserved among dUTPases from and to (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar). the of the the of the and and was determined at with (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar), and contributes the to the The three active of the are in and conserved motifs from in a A by conserved from A the uracil and A conserved to the and is by conserved and of and and Therefore, active ultimately on of the this of binding is among in and complexes are shown for the moiety of the Due to the of the interactions in the three complexes determined in the and the was to the as in the wild type is the mutant and mutant the significant are that (i) is and and (ii) in the mutant the is in are The the catalysis-assisting Mg2+, not in and the triphosphate of the substrate in the enzyme active with a data Detailed analysis of the catalytically competent enzyme conformation of the enzymatic mechanism. three are the of the that water molecules by and of and The are by of the and the of from of the and and water molecules are and for and to be compared with the of the These a role in interactions the protein residues and the of and in the active and of are involved in residues of responsible for The of and by and provide an conformation for hydrogen-bonding The catalytic in the already side for binding of the uracil and the for significant by the dUTP and and of the for at the of the A the of the a for in-line nucleophile a water oxygen as in the in and A and to in A suggested to be a catalytic residue (12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google protein or water were to the the role of the water A with of in the this was for of may in hydrogen-bonding to is to the is to and water that and The may a role in the side side were to this catalytic However, protein atom is to as This suggested that side oxygen of with an enzyme The mutant was and to be in catalytic for the mutant was determined to be as compared with for wild a of of was the for mutant and wild type enzymes that substrate is the in The crystal of the mutant in with and was at resolution and is shown as on the wild type and The structures are used for of is for the catalytic water and that is from the mutant A of this is that with an with with in the A of atom for into the could provide for this is by the with in the wild type The enzymatic of the mutant it to the crystal of the mutant and this substrate and are highly to in the and of the and the the nucleophilic attack are is These with the kinetic that the water molecule the nucleophile Although the structures that identified have been determined for the complexes of wild type and mutant the catalytic of the mutant was in the reaction (dUTP Therefore, the residue plays a role in dUTP have been with this (i) of and Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google and (ii) to and The mutant is not in the role of dUTP as by in and structures in and the catalytic of the mutant enzyme is to of to the catalytic water molecule in the reaction. in the and complexes are that the nucleophile attack mechanism be in the catalytic water molecule to be in the A. Larsson G. Persson R. Cedergren-Zeppezauer E. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 767-774Crossref PubMed Scopus (28) Google as at a of to the is to as as to the as it is in the determined the a to the to out nucleophilic attack on the the as and the water has the the to the protein atom and in that protein with the of water a binding for that in of the for the dUTP hydrolysis reaction (dUTP dUMP This binding is already available in the of the of the catalytic and are to be for (dUTP and the the of in the may be as of a water molecule in such an reaction. of the pyrophosphate at the of the active is to as it numerous with the The may pyrophosphate since it is to to the pyrophosphate as compared with dUMP at no could be to in the the has been in the of the and and The from in and in may in pyrophosphate via with strictly conserved side of the pyrophosphate moiety the dUMP is by investigations out on dUTPase into substrate that not the pyrophosphate in the enzyme active G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar). and A and that the reaction product dUMP of the binding interactions with the substrate with to the and However, side chains interactions with the triphosphate moiety side chains and are to in the This may to the significant of dUMP binding to compared with and (15Larsson G. Nyman P.O. Kvassman J.O. J. Biol. Chem. 1996; 271: 24010-24016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, Larsson G. Persson Persson R. Nyman P.O. PubMed Scopus Google Scholar). binding of dUMP as compared with dUTP of product with substrate in the active substrate is available in the The structural analysis of the apoenzyme, as as and product complexes atomic interactions to the catalytic mechanism of The were based on structures and kinetic data determined with the substrate as as the substrate and the that the reaction pathway may be of with dUTP and a water molecule in the to the determined is already in the and is in the the binding of the substrate the is to that of the and the was shown to to to the enzyme in the reaction. is to that (i) a water molecule to and is with in the active of the wild type enzyme dUTP is and (ii) this water molecule is for in-line to the such an it be that this water as the nucleophile in dUTP The of the for the catalysis-assisting significant as role in catalytically competent of the substrate in the dUTPase active as by and in the and of the was established in a of on dUTPases from (15Larsson G. Nyman P.O. Kvassman J.O. J. Biol. Chem. 1996; 271: 24010-24016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, D. A. S. A. 2003; PubMed Scopus Google Scholar, J. E. A. Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). was that to enzymatic of by the of the active from the determined crystal structures the of the catalytically competent the of the nucleophile The data on the were a significant in this (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar). However, to the catalytic of the as as the of the as compared with the of the nucleophile attack could not be determined in the the critical of the α-phosphorus is from competent as determined in and The of to the three is not in structures the is as to J. 1999; PubMed Scopus Google Scholar, R. J. A. Struct. 2000; Full Text Full Text PDF Scopus Google Scholar, R. W. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Z. A. Z. G. PubMed Scopus Google Scholar). the dUTPase the of contributes to an conformation for of enzymatic reaction is by interactions and residues from as as several water molecules the active residues to with and are strictly conserved among dUTPase The and role of of of the and were already suggested based on determined crystal structures of E. and dUTPases (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar). However, as to a catalytically competent substrate the relevant of conserved residues to be in the of the from structures the role of and in and of the the and with by determined structures of dUTPases from (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar, 12Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Crossref PubMed Scopus (88) Google Scholar, 13Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure (Lond.). 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Prasad G.S. Stura E.A. Elder J.H. Stout C.D. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1100-1109Crossref PubMed Scopus (39) Google Scholar). However, the the and in This be to the by the in Due to the of Mg2+, the triphosphate a conformation that from the The of the side is for by the The of the in the structures the the and an oxygen in the to oxygen in the This may to activation of the α-phosphorus for the nucleophile attack and the in the The data provide of role of as the (10Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Crossref PubMed Scopus (81) Google Scholar), of the of this conserved residue in the was to of the of the The of and catalytic the of the for mechanistic the side of in of water molecules in the of the that has The a description of the catalytic pathway via of the nucleophile and of interactions responsible for the and the reaction. The key residue was shown to to substrate binding via to the of the A role was in the of the of the nucleophile this role could not have been the of the side of to the catalytic and is the structural data that the of the critical side is by an of and the of the conserved from the This is strictly in dUTPase as no was to the mutant is in catalytic and in the the the with of the the atom of the mutant for the role of to the catalytic water The this role identified a protein atom of The oxygen of a conserved residue with a binding for this water that is the substrate of the of has been in this side oxygen a water molecule that in the water network The side in to of the on the reaction The of data for hydrolysis are in of a mechanism with significant since the for the mechanism is highly an is PubMed Scopus Google Scholar, R. J. Chem. Scopus Google Scholar). for the reaction are in with this the the oxygen and the reaction is with an of based on the Acta Crystallogr. Google Scholar), a of is to be for the mechanism PubMed Scopus Google Scholar). The data of with the for in-line attack that be with a mechanism of significant and for and for in and for Persson for the expression and and at and for data dUTP of of the mammalian genome numerous mutagenic and carcinogenic modifications on a daily there are such as is to the integrity of DNA. dUTPase the incorporation of uracil into DNA by catalyzing the cleavage of dUTP into dUMP and However, the by dUTPase dUTP was a Although crystal structures of dUTPase been no was to provide an understanding of the catalytic mechanism. PDF