Title: The Flexible Motif V of Epstein-Barr Virus Deoxyuridine 5′-Triphosphate Pyrophosphatase Is Essential for Catalysis
Abstract: 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 Mg2+-dUTP complex in the active site. Point mutations inside motif V highlighted the essential role of lid residue Phe273. Magnesium appears to play a role mainly in substrate binding, since in absence of Mg2+, the Km of the enzyme is reduced, whereas the kcat is less affected. 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 Mg2+-dUTP complex in the active site. Point mutations inside motif V highlighted the essential role of lid residue Phe273. Magnesium appears to play a role mainly in substrate binding, since in absence of Mg2+, the Km of the enzyme is reduced, whereas the kcat is less affected. Epstein-Barr virus, a human γ-herpesvirus, is the causative agent of infectious mononucleosis and establishes a lifelong persistent infection in over 90% of the world's population. EBV 3The abbreviations used are:EBVEpstein-Barr virusSPRsurface plasmon resonanceWTwild typeBicineN,N-bis(2-hydroxyethyl)glycineDTTdithiothreitol.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. Epstein-Barr virus surface plasmon resonance wild type N,N-bis(2-hydroxyethyl)glycine dithiothreitol. One protein of the lytic cycle is deoxyuridine 5′-triphosphate pyrophosphatase, a ubiquitous enzyme catalyzing the cleavage of dUTP into dUMP and pyrophosphate (PPi). 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 (1Harkiolaki M. Dodson E.J. Bernier-Villamor V. Turkenburg J.P. González-Pacanowska D. Wilson K.S. Structure. 2004; 12: 41-53Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). 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 (2Cedergren-Zeppezauer E.S. Larsson G. Nyman P.O. Dauter Z. Wilson K.S. Nature. 1992; 355: 740-743Crossref PubMed Scopus (144) Google Scholar), Homo sapiens (3Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure. 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), equine infectious anemia virus (4Dauter 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), feline immunodeficiency virus (5Prasad G.S. Stura E.A. McRee D.E. Laco G.S. Hasselkus-Light C. Elder J.H. Stout C.D. Protein Sci. 1996; 5: 2429-2437Crossref PubMed Scopus (77) Google Scholar), Mason-Pfizer monkey virus (6Németh-Pongrácz V. Barabás O. Fuxreiter M. Simon I. Pichová I. Rumlová M. Zábranská H. Svergun D. Petoukhov M. Harmat V. Klement E. Hunyadi-Gulyás E. Medzihradszky K.F. Kónya E. Vértessy B.G. Nucleic Acids Res. 2007; 35: 495-505Crossref PubMed Scopus (40) Google Scholar), Mycobacterium tuberculosis (7Chan S. Segelke B. Lekin T. Krupka H. Cho U.S. Kim M.Y. So M. Kim C.Y. Naranjo C.M. Rogers Y.C. Park M.S. Waldo G.S. Pashkov I. Cascio D. Perry J.L. Sawaya M.R. J. Mol. Biol. 2004; 341: 503-517Crossref PubMed Scopus (76) Google Scholar), Plasmodium falciparum (8Whittingham J.L. Leal I. Nguyen C. Kasinathan G. Bell E. Jones A.F. Berry C. Benito A. Turkenburg J.P. Dodson E.J. Ruiz Perez L.M. Wilkinson A.J. Johansson N.G. Brun R. Gilbert I.H. Gonzalez Pacanowska D. Wilson K.S. Structure. 2005; 13: 329-338Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), Arabidopsis thaliana (9Bajaj M. Moriyama H. Acta Crystallogr Sect. F Struct Biol. Cryst. Commun. 2007; 63: 409-411Crossref PubMed Scopus (7) Google Scholar), and vaccinia virus (10Samal A. Schormann N. Cook W.J. DeLucas L.J. Chattopadhyay D. Acta Crystallogr. D Biol. Crystallogr. 2007; 63: 571-580Crossref PubMed Scopus (23) Google Scholar). The active site is formed by five conserved motifs that are distributed over the entire sequence (11McGeoch D.J. Nucleic Acids Res. 1990; 18: 4105-4110Crossref PubMed Scopus (191) Google Scholar) (Fig. 1A). Each subunit of the trimer contributes to the formation of each of the three active sites (2Cedergren-Zeppezauer E.S. Larsson G. Nyman P.O. Dauter Z. Wilson K.S. Nature. 1992; 355: 740-743Crossref PubMed Scopus (144) Google Scholar). Whereas the first four motifs are well ordered, motif V is most often disordered and is only observed in some structures after inhibitor binding such as in the feline immunodeficiency virus dUTPase structure (Protein Data Bank entry 1f7r) (12Prasad 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 the human dUTPase structure (Protein Data Bank entry 1q5h) (3Mol C.D. Harris J.M. McIntosh E.M. Tainer J.A. Structure. 1996; 4: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), both in complex with dUDP. Recently, several structures showing motif V and α,β-imido-dUTP in a conformation close to the situation during catalysis became available: one of the human enzyme (Protein Data Bank entry 3ehw) (13Varga B. Barabás O. Kovári J. Tóth J. Hunyadi-Gulyás E. Klement E. Medzihradszky K.F. Tölgyesi F. Fidy J. Vértessy B.G. FEBS Lett. 2007; 581: 4783-4788Crossref PubMed Scopus (54) Google Scholar) and one from M. tuberculosis (Protein Data Bank entry 2py4) (14Varga B. Barabás O. Takács E. Nagy N. Nagy P. Vértessy B.G. Biochem. Biophys. Res. Commun. 2008; 373: 8-13Crossref PubMed Scopus (58) Google Scholar). Together with kinetic information about the human enzyme (15Tóth J. Varga B. Kovács M. Málnási-Csizmadia A. Vértessy B.G. J. Biol. Chem. 2007; 282: 33572-33582Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), a model of enzymatic action became available: (i) rapid, probably diffusion-limited binding of the substrate; (ii) a substrate-induced structural change required for the formation of the catalytically competent conformation; (iii) the rate-limiting hydrolysis step; and (iv) a rapid release of the reaction products PPi and dUMP. Hydrolysis occurs through a nucleophilic in-line attack on the α-phosphate by a water molecule activated by an aspartic acid residue (16Barabás O. Pongrácz V. Kovári J. Wilmanns M. Vértessy B.G. J. Biol. Chem. 2004; 279: 42907-42915Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The third family contains monomeric dUTPases encoded by avian and mammalian herpesviruses, which include important human pathogens, such as Epstein-Barr virus and herpes simplex virus. They have limited sequence homology to the trimeric enzymes; the five motifs forming the active site are present but reshuffled and spread out over a single polypeptide that is twice as long as the sequence of the subunit in trimeric enzymes. Working on the enzyme from EBV, we reported the first crystal structures of a monomeric dUTPase determined in complex with the product dUMP or the non-hydrolyzable substrate analogue α,β-imido-dUTP (17Tarbouriech N. Buisson M. Seigneurin J.M. Cusack S. Burmeister W.P. Structure. 2005; 13: 1299-1310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) (Fig. 1B). Like most dUTPase structures, those of the EBV enzyme showed four of the five conserved sequence motifs (Fig. 1A), with motif V invisible due to its flexibility, and revealed an active site that is extremely similar to those of trimeric dUTPases. EBV dUTPase furthermore exhibited similar kinetic parameters (17Tarbouriech N. Buisson M. Seigneurin J.M. Cusack S. Burmeister W.P. Structure. 2005; 13: 1299-1310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Bergman A.C. Nyman P.O. Larsson G. FEBS Lett. 1998; 441: 327-330Crossref PubMed Scopus (22) Google Scholar). This therefore implies that the conclusions of studies of the enzymatic mechanism for trimeric dUTPases are also valid for the EBV enzyme and vice versa. In the previously published EBV dUTPase structures (17Tarbouriech N. Buisson M. Seigneurin J.M. Cusack S. Burmeister W.P. Structure. 2005; 13: 1299-1310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), the N and C termini of the ordered structure were linked by a disulfide bridge between Cys4 and Cys246. We therefore wished to check the influence of this bridge on activity and its possible regulatory role by studying mutant C4S. EBV dUTPase structures (17Tarbouriech N. Buisson M. Seigneurin J.M. Cusack S. Burmeister W.P. Structure. 2005; 13: 1299-1310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) revealed that residues 114–133 linking the two domains of monomeric dUTPase and containing part of motif I show different structures in the α,β-imido-dUTP-bound form (Fig. 1B) and in the dUMP-bound form (Fig. 1C). This leads indirectly to a non-productive conformation of catalytic residue Asp76 of motif III (Fig. 1B). Motif I is always well ordered in trimeric dUTPases. We hypothesized first that the clustering of negative charges of two aspartic acid residues (catalytic residues Asp76 and Asp131 of the linker region) and the γ-phosphate of the inhibitor leads to destabilization of this part of the structure. When dUTPase structures showing motif V in a productive conformation became available (13Varga B. Barabás O. Kovári J. Tóth J. Hunyadi-Gulyás E. Klement E. Medzihradszky K.F. Tölgyesi F. Fidy J. Vértessy B.G. FEBS Lett. 2007; 581: 4783-4788Crossref PubMed Scopus (54) Google Scholar, 14Varga B. Barabás O. Takács E. Nagy N. Nagy P. Vértessy B.G. Biochem. Biophys. Res. Commun. 2008; 373: 8-13Crossref PubMed Scopus (58) Google Scholar), they showed an interaction of the residue corresponding to Asp131 with the conserved arginine residue of motif V. On the other hand, Asp131 is only partially conserved in sequence alignments. These revealed two classes of dUTPases, the first containing EBV dUTPase with a conserved aspartic acid residue in a position corresponding to Asp131 and the second group containing human and E. coli enzymes with a conserved aspartic acid residue at the position corresponding to residue 78 in EBV (Fig. 1A). We decided to study three single mutants of Asp131 (D131S, D131N, and D131E) along with a double mutant (D131S/G78D), making the EBV enzyme more similar to the second group. Finally, we report the structure of a dUTPase mutant with motif V deleted (ΔV), constructed in order to facilitate binding studies and crystallographic studies in view of potential antiviral drug design. This mutant showed the presence of intact Mg2+-dUTP in its active site, posing the question of why this mutant is completely inactive although most of the catalytic machinery is in place. In order to understand the precise mechanism of action of motif V, we decided to study two conserved residues of motif V, Arg268 and Phe273, since it has been shown that they interact with the bound substrate analogue α,β-imido-dUTP (13Varga B. Barabás O. Kovári J. Tóth J. Hunyadi-Gulyás E. Klement E. Medzihradszky K.F. Tölgyesi F. Fidy J. Vértessy B.G. FEBS Lett. 2007; 581: 4783-4788Crossref PubMed Scopus (54) Google Scholar, 14Varga B. Barabás O. Takács E. Nagy N. Nagy P. Vértessy B.G. Biochem. Biophys. Res. Commun. 2008; 373: 8-13Crossref PubMed Scopus (58) Google Scholar). Since binding of an Mg2+-substrate complex appeared possible without catalysis, we wanted to further characterize the role of Mg2+. Previous studies carried out on E. coli dUTPase (16Barabás O. Pongrácz V. Kovári J. Wilmanns M. Vértessy B.G. J. Biol. Chem. 2004; 279: 42907-42915Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Larsson G. Nyman P.O. Kvassman J.O. J. Biol. Chem. 1996; 271: 24010-24016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 20Vertessy B.G. Larsson G. Persson T. Bergman A.C. Persson R. Nyman P.O. FEBS Lett. 1998; 421: 83-88Crossref PubMed Scopus (43) Google Scholar) indicated an important role of Mg2+ for catalytic activity, by promoting enzyme-substrate complex stabilization (21Mustafi D. Bekesi A. Vertessy B.G. Makinen M.W. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 5670-5675Crossref PubMed Scopus (36) Google Scholar). Various dUTPase structures in the presence of α,β-imido-dUTP show the metal ion coordinating all three phosphate groups. Mutagenesis was carried out according to the manufacturer's instructions, using the QuikChange II kit (Stratagene) on EBV wild type (WT) dUTPase coding sequence cloned into pPROEx Htb plasmid (17Tarbouriech N. Buisson M. Seigneurin J.M. Cusack S. Burmeister W.P. Structure. 2005; 13: 1299-1310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Proteins were expressed in E. coli BL21 (DE3) strain and purified on Ni2+-nitrilotriacetic acid resin, as described previously (17Tarbouriech N. Buisson M. Seigneurin J.M. Cusack S. Burmeister W.P. Structure. 2005; 13: 1299-1310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). ΔV mutant is a 257-residue construct with the last 22 C-terminal residues removed. Screening for crystallization conditions was carried out with a PixSys4200 Cartesian robot (high-throughput crystallization laboratory at EMBL Grenoble) in 100 + 100-nl drops. Hits were reproduced manually in 1 + 1-μl drops. Protein solutions (about 1.5 mg ml−1) used for crystallization trials contained 10 mm dUTP in 250 mm NaCl, 10 mm MgCl2, 20 mm imidazole, and 20 mm Hepes, pH 7.5. Mutants D131S/G78D and D131N were crystallized using as precipitant 0.3 m ammonium sulfate, 25% polyethylene glycol 3350, 0.1 m Hepes, pH 7; C4S was crystallized using 150 mm malic acid, pH 7.5, 25% PEG 3350. The ΔV mutant was crystallized using 0.1 m Tris, pH 8.5, 20% polyethylene glycol 3350, 200 mm Li2SO4. Single crystals were harvested from the drop, dipped into paraffin oil from the panjelly kit (Jena Biosciences), and frozen directly at 100 K in a nitrogen gas stream (Oxford Cryosystems). Diffraction data (Table 1) were collected at the European Synchrotron Radiation Facility (Grenoble, France). Data were integrated using MOSFLM (22Leslie A.W.G. Int. CCP4/ESF-EACMB Newsletter Protein Crystallogr.1992Google Scholar) and further processed using the CCP4 package through ccp4i (23Potterton E. Briggs P. Turkenburg M. Dodson E. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 1131-1137Crossref PubMed Scopus (1070) Google Scholar). Structures were solved by molecular replacement using MOLREP (24Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4152) Google Scholar). Structures were refined with REFMAC (25Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar), and models were built using COOT (26Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23384) Google Scholar). Illustrations were made with PyMol (27DeLano W.L. The Pymol Molecular Graphics System. DeLano Scientific, LLC, San Carlos, CA2002Google Scholar).TABLE 1Data collection and refinement statisticsMutantΔV dUTPΔV dUMPD131ND131S/G78DC4SProtein Data Bank entry2we32we12we22we0Data collection statisticsESRF beamlineID14_eh4ID23_eh2ID23_eh1ID23_eh1ID23_eh2Space groupI4P212121P212121P212121P212121Cell parameters a/b/c (Å)aAt 100 K.103.2/103.2/47.754.6/56.5/80.555.6/56.9/81.155.7/57.0/81.256.0/56.8/81.4Resolution (Å)51.6-2.046.6-1.4546.6-1.845.9-1.546.3-2.0Completeness (%)99.5 (99.9)bValues for the highest resolution bin are given in parentheses.100 (99.8)98.1 (99.9)99.3 (99.7)99.7 (99.3)Multiplicity3.8 (3.8)4.1 (4.1)3.5 (3.4)3.8 (3.9)6.9 (6.9)〈I/σ(I)〉7.2 (1.7)4.4 (1.7)6.6 (2.2)8.4 (4.5)5.0 (2.2)Rsym0.064 (0.431)0.083 (0.371)0.083 (0.348)0.048 (0.166)0.105 (0.339)RefinementAtomic B modelIsotropicAnisotropicIsotropicAnisotropicIsotropicNo. of reflections1619444150227913957116536Rcryst0.212 (0.275)0.193 (0.215)0.196 (0.244)0.195 (0.213)0.189 (0.242)Rfree0.272 (0.333)0.221 (0.253)0.247 (0.318)0.224 (0.252)0.249 (0.366)r.m.s. deviations from ideal bond lengths (Å)0.0150.0080.0160.0090.017r.m.s. deviations from ideal bond angles (degrees)1.71.21.61.31.7Ramachandran plotcFrom PROCHECK (35). most favorable/additional/ generously allowed/disallowed (%)89.9/9.5/0.5/093.2/6.8/0/091.1/8.9/0/093.8/5.2/1.0/094.1/5.4/0.5/0ModelModeled residues240248245248247dUMP1111Mg2+-dUTP1Water molecules85258225299173SO42−21311Malic acid1a At 100 K.b Values for the highest resolution bin are given in parentheses.c From PROCHECK (35Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab Reducing conditions were obtained by adding 3 mm DTT to the protein in buffer for 1 h at room temperature. 1 h prior to gel electrophoresis, free thiol groups were blocked with 10 mm N-ethylmaleimide. Loading buffer did not contain β-mercaptoethanol for non-reducing conditions; otherwise it contained 10 mm β-mercaptoethanol. The pH drop due to proton release during dUTP hydrolysis was followed using cresol red as a pH indicator by monitoring absorbance at 573 nm. The reaction was carried out at 25 °C at pH 7.8 in quartz cells (Hellma), 1-cm path, using a Varian Cary 50 Bio spectrophotometer. The mixture contained 25 μm dUTP, 25 μm cresol red, 150 mm KCl, 1 mm MgCl2, 0.5 mm Bicine-NaOH, pH 7.8. The reaction was initiated by adding dUTPase (in 5 mm Hepes, pH 7.5, 250 mm NaCl, 10 mm MgCl2) to a final concentration of 31 nm. Kinetic values for Km and kcat were determined by linearization of the progress curve using the integrated Michaelis-Menten equation (19Larsson G. Nyman P.O. Kvassman J.O. J. Biol. Chem. 1996; 271: 24010-24016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Less active mutants were studied using the EnzChek pyrophosphate assay kit (Invitrogen), allowing photometric detection of PPi released during dUTP hydrolysis. It consists of a cleavage of PPi to phosphate by pyrophosphatase and a detection of inorganic phosphate by the conversion of 2-amino-6-mercapto-7-methylpurineribonucleoside to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine with an absorption maximum at 360 nm by purine ribonucleoside phosphorylase. Reactions were carried out at 25 °C in 96-well plates in volumes of 250 μl with variable dUTP and protein concentrations. The buffer contained 50 mm Tris, pH 7.5, 1 mm MgCl2, 0.05 mm NaN3. Absorbance was continuously monitored at 355 nm using a Thermo Labsystems iEMS Reader MF plate reader. Enzyme concentrations were chosen so that the initial velocity of the reaction with the dUTP substrate was at least 10 times slower than the initial velocity of the chromogenic reaction using the same concentration of a PPi standard. Kinetic constants were determined using Lineweaver-Burk plots (supplemental Fig. 1). WT dUTPase concentrations were determined using an ϵ280 = 30,160 m−1 cm−1. To assess the importance of Mg2+ for dUTPase activity, the "cresol red" assay was carried out after removing Mg2+. WT protein solution and reaction mixture without cresol red (25 μm dUTP, 150 mm KCl, 0.5 mm Bicine-NaOH, pH 7.8) were passed onto a column containing 1 ml of Chelex 100 resin (Sigma) to eliminate metal cations. 25 μm cresol red was then added, and the assay was performed as described above. The remaining Mg2+ in the assay was quantified after dilution by flame atomic absorption spectrometry (PerkinElmer Life Sciences) at the Grenoble University Hospital. Samples and standards were prepared in a solution of 1 g/liter lanthanum oxide, and concentrations were obtained using external calibration. The Mg2+ concentration in the Chelex-treated assay was below the detection limit of 0.08 μm. The absence of a lasting effect of Mg2+ depletion was checked by the addition of 1 mm Mg2+ to the mixture after Chelex treatment. Kinetic constants were the same as without the treatment (data not shown). Surface plasmon resonance (SPR) measurements were performed with a BIAcore T100 (Biacore AB) operated with BIAcore T100 evaluation Software 1.1. All measurements were performed at 25 °C, at 10 μl/min using a running buffer composed of 20 mm Hepes, pH 7.5, 150 mm KCl, 1 mm MgCl2. The carboxymethylated dextran layer of a CM5 sensor chip (Biacore AB) was activated by a 7-min pulse of a 1:1 mixture of freshly prepared 50 mm N-hydroxysuccinimide and 200 mm N-ethyl-N′-(dimethylaminopropyl)-carbodiimide. Recombinant human CSF1 (colony-stimulating factor-1), produced according to a modified protocol (28Halenbeck R. Kawasaki E. Wrin J. Koths K. Bio-Technologie. 1989; 7: 710-715Google Scholar), and streptavidin (Sigma) in 10 mm acetate buffer, pH 4, WT dUTPase, and truncated C-terminal mutant V in 20 mm Hepes, pH 7.5, 250 mm NaCl, 10 mm MgCl2 were bound to the activated surfaces during several pulses. Remaining N-hydroxysuccinimide ester groups were blocked by a 7-min injection of 1.0 m ethanolamine hydrochloride, pH 8.5. CSF1- and streptavidin-modified surfaces were used as reference for the WT dUTPase (7000 resonance units)- and for the ΔV mutant (8000 resonance units)-modified surfaces, respectively. Curves obtained on the reference surface are deduced from the curves recorded on the recognition surfaces, allowing elimination of refractive index changes due to buffer effects. Substrate dUTP and inhibitor α,β-imido-dUTP diluted in running buffer were injected at concentrations ranging from 1 to 1000 μm. No regeneration procedure was required. The absence of mass transport effects was checked on each surface by separately running one injection of α,β-imido-dUTP (5 μm) for 120 s at different flow rates ranging from 10 to 70 μl/min. It is possible to overlay the obtained curves, confirming the kinetic control of the experiments (not shown). KD values are obtained by fitting the steady-state response versus the concentration according to a 1:1 interaction model using BIAcore T100 evaluation software version 1.1. The presence in solution of the disulfide bridge between residues 4 and 246 could be verified by SDS-PAGE under non-reducing conditions in the presence of the blocking agent N-ethylmaleimide compared with a C4S mutant (Fig. 2) or with the WT protein under reducing conditions. The disulfide-linked form migrates faster than the reduced protein and represents the major fraction of dUTPase. Incubation of dUTPase under reducing conditions followed by the same analysis on non-reducing PAGE showed an increase of the reduced form in solution, although most of the protein remains oxidized. The enzymatic activity of the native enzyme, the one partially reduced with DTT, and the C4S mutant were virtually identical (Table 2) excluding a regulatory role of the disulfide bridge. The structure of the C4S mutant was almost identical to that of WT dUTPase with dUMP. The remaining disulfide bridge partner Cys246 stayed in place; only Ser4 became disordered, and Pro5 adopted a different conformation.TABLE 2Enzymatic activity assaysAssayKmkcatkcat/Kmμms−1m−1 s−1WT (1 mm Mg2+)Cresol red0.8 ± 0.2aErrors are based on 2–5 independent determinations of the kinetic constants and a minimal error of 10%.1.4 ± 0.11.8 × 106Enzcheck≪5bThe minimum concentrations (5 μm) of dUTP used in the test did not allow us to determine a value of Km for the WT enzyme, confirming that the Km is ≪5 μm.1.2 ± 0.2Without Mg2+ (<0.08 μm)Cresol red6.0 ± 0.91.2 ± 0.20.2 × 106WT with DTTCresol red0.71 ± 0.051.6 ± 0.12.3 × 106C4SCresol red0.6 ± 0.21.7 ± 0.22.8 × 106R268AEnzcheck8 ± 40.06 ± 0.010.008 × 106F273AEnzcheckNMcNM, not measurable.NMNMD131SEnzcheck5.2 ± 0.50.04 ± 0.010.008 × 106D131EEnzcheckNMNMNMD131NEnzcheck7 ± 20.04 ± 0.010.006 × 106D131S/G78DEnzcheck9 ± 20.18 ± 0.020.02 × 106a Errors are based on 2–5 independent determinations of the kinetic constants and a minimal error of 10%.b The minimum concentrations (5 μm) of dUTP used in the test did not allow us to determine a value of Km for the WT enzyme, confirming that the Km is ≪5 μm.c NM, not measurable. Open table in a new tab We speculated earlier (17Tarbouriech N. Buisson M. Seigneurin J.M. Cusack S. Burmeister W.P. Structure. 2005; 13: 1299-1310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) that the disorder of the loop containing motif I in the α,β-imido-dUTP-bound structure might be due to a clustering of negative charges contributed by Asp76, Asp131, and the γ-phosphate group of α,β-imido-dUTP (Fig. 1B) observed so far only in the EBV dUTPase structure. Site-directed mutagenesis of the apparently EBV-specific residue Asp131 to serine, the residue present in the E. coli enzyme, or to asparagine or glutamic acid led to a large loss in catalytic activity (Table 2). This activity was undetectable in the cresol red assay and could only be measured in the more sensitive EnzChek-based assay. With the EnzChek assay, WT kcat could be estimated as 1.2 s−1, allowing the values determined with this assay to be validated, although the Km was too low to be determined, since substrate concentrations had to be above 5 μm. Mutants D131S and D131N showed a kcat of 0.04 s−1 and Km value of 5 or 7 μm, respectively, whereas activity of the D131E mutant remained undetectable. A double mutation D131S/G78D, which will be discussed below, showed some recovery of enzymatic activity, with a 5-fold increase of its kcat to 0.18 s−1 compared with the single mutants and a doubled Km. Crystallization of these mutants in the presence of dUTP was attempted using a robotic screen of 576 crystallization conditions. Only for mutant D131N and the double mutant D131S/G78D was it possible to obtain crystals that turned out to be isomorphous, with the WT crystals containing dUMP. Structural changes concern only the mutated residues (Fig. 3) and the degree of disorder of the flexible region 114–133, which varies between structures (not shown). Using the same conditions as for the WT enzyme in the presence of dUTP, after 2 days, crystallization trials of ΔV mutant yielded crystals belonging to tetragonal space group I4 (Table 1) containing unhydrolyzed dUTP in the active site. After 2–3 weeks, orthorhombic dUTPase crystals appeared and were isomorphous with the WT-dUMP complex crystals described previously (17Tarbouriech N. Buisson M. Seigneurin J.M. Cusack S. Burmeister W.P. Structure. 2005; 13: 1299-1310Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). These crystals diffracted X-rays to 1.5 Å resolution and showed a bound dUMP molecule in the active site, obviously arising from slow spontaneous or enzymatic hydrolysis of dUTP during the 2–3-week period needed for crystallization and a protein structure virtually identical to the one of the WT protein-dUMP complex (not shown). The tetragonal crystals diffracted X-rays to 2 Å resolution and showed a bound Mg2+-dUTP complex (Fig. 4, A, C, and D) and a largely disordered linker region (residues 114–133), where residues 115–128 are invisible (Fig. 1C). Still, the catalytic residue Asp76 is co