Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes.

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

murium, is obtained via a pathway where dCTP is deaminated by dCTP deaminase (EC 3.5.4.13) to yield ammonia and dUTP that subsequently is hydrolyzed by dUTPase to generate dUMP and pyrophosphate (1). In contrast, Gram-positive bacteria and eukaryotic organisms synthesize dTTP from dUMP obtained by deamination of dCMP by the zinc-containing enzyme dCMP deaminase (2). Recently, a bifunctional enzyme from the archaeon Methanocaldococcus jannaschii has been identified (3,4) that possesses both the dCTP deaminase and dUTPase activities in one polypeptide suggesting that at least in some Archaea, dCTP serves as a source for dUMP. The structure of this archaeal enzyme is now known and the subunit shares an overall fold with dUTPases as well as the organization of subunits in a trimer (5). In the present work we demonstrate that dCTP deaminase from E. coli is yet another member of this family of enzymes, even though significant differences are found in the C-terminal stretch that closes the active site upon catalysis.
One very interesting feature of the dCTP deaminase is that the deamination reaction proceeds without aid from a metal cofactor. Other nucleobase or nucleoside deaminases such as cytosine deaminase (6,7), cytidine deaminase (8), adenosine deaminase, (9) and adenine deaminase (10) all require a catalytic metal ion, Zn 2ϩ , Mn 2ϩ , Fe 2ϩ , which is either tightly bound in the active site or has to be added together with the substrate to the enzyme. Here, we report the identification of catalytically important amino acid residues of E. coli dCTP deaminase. Furthermore we present the first structure of a monofunctional dCTP deaminase in a distinct complex with the reaction product dUTP and Mg 2ϩ (dUTP⅐Mg 2ϩ ) and also the structures of a mutant enzyme (E138A) in complex with bound substrate dCTP and Mg 2ϩ (dCTP⅐Mg 2ϩ ) as well as the same mutant enzyme in complex with dUTP⅐Mg 2ϩ . The structures of these complexes of enzyme and nucleotides allow us to suggest a plausible mechanism for the deamination reaction in the absence of a metal cofactor, involving conserved amino acid residues. Finally, also based on these new structures of dCTP deaminase and the previously determined structures of dUTPase and the bifunctional dCTP deaminase-dUTPase we identify amino acid residues that determine the bi-or monofunctionality of dCTP deaminases with respect to dUTPase activity. ard procedures (11). The dcd (12) gene was obtained by PCR using the Pfu polymerase (Stratagene) according to the suppliers manual with chromosomal DNA from a E. coli K12 strain as a template and the deoxy-oligonucleotides; DCD-5: GAAAATCATATGCGTCTGTGTG-ACCG and DCD-3: GCGGATCCTTAGTCTTTATCGATTCGGC. Lettering in italics indicate the restriction sites incorporated by the primers in the PCR product; NdeI in DCD-5 and BamHI in DCD-3. The PCR product, comprising the reading frame of the dcd gene, and plasmid pET11a (Novagen) were digested with the restriction enzymes NdeI and BamHI, ligated, and the ligation mix was subsequently transformed into BL21(DE3) cells. This yielded a strain, SØ5352, harboring the plasmid pETDCD that allows for overexpression of the dcd gene upon induction with IPTG. Mutant alleles of the dcd gene encoding the R115A, E138A, and E138Q enzymes were constructed using the QuikChange method (Stratagene) with pET11DCD as the template and the complementary deoxyoligonucleotides 5R115A, TCCTCACTGGCG-GCTCTGGGGCTGATG; 3R115A, CATCAGCCCCAGAGCCGCCAGT-GAGGA; 5E138A, TGCATTGTGCTGGCGTTCTACAACTCC; 3E138A, GGAGTTGTAGAACGCCAGCACAATGCA; 5E138Q, TGCATTGTGCT-GCAGTTCTACAACTCC and 3E138Q, GGAGTTGTAGAACTGCAGCA-CAATGCA. The entire coding region for wild-type and mutant alleles of the dcd gene were verified by sequencing using an ABI PRISM 310 DNA Sequencer as recommended by the supplier (PerkinElmer Life Sciences).
Purification of E. coli Wild Type and Mutant dCTP Deaminases-An overnight culture of BL21(DE3) derivatives harboring a plasmid encoding either wild type (SØ5352) or mutant dCTP deaminases constructed as described above were grown in LB medium at 37°C supplemented with ampicillin (100 g ml Ϫ1 ). These overnight cultures were then used to inoculate 300 ml of the same media as above, and growth was continued at 37°C to an OD 600 of 1 when IPTG 1 was added to a final concentration of 0.3 mM. After three hours of induction the cells were harvested by centrifugation in a Sorvall centrifuge in SLA3000 rotor at 5000 rpm for 8 min. All subsequent steps were conducted at 4°C. The cell paste was suspended in 20 ml of 50 mM potassium phosphate, pH 6.8, and centrifugation was repeated as above. Subsequently, the cell paste was suspended in 5 ml of the same buffer, and the cells were disrupted using a Sonics Vibra-Cell ultrasonic processor. Cell debris was sedimented by centrifugation in a Sorvall centrifuge using an SS34 rotor at 14,000 rpm for 20 min. A freshly prepared 10% (w/w) streptomycin sulfate solution was added to the extract to a final concentration of 1% (w/w). The precipitate was cleared by centrifugation as above, and the supernatant was dialyzed against several changes of washing buffer. The dialyzed protein was loaded on to a DEAE 52 column (38 ml) equilibrated with 50 mM potassium phosphate, pH 6.8, which was mounted on a Gradifrac (Amersham Biosciences). The column was eluted with a gradient from 0 to 0.4 M KCl in 50 mM potassium phosphate, pH 6.8 of a total of 240 ml. The protein eluted at ϳ0.1 M KCl and fractions containing dCTP deaminase were diluted 3-fold with 50 mM potassium phosphate, pH 6.8, and a second run of the DEAE 52 column as above was performed. The fractions eluted from the column were pooled and made 60% saturated (40 g/100 ml) with ammonium sulfate. The precipitate was recovered by centrifugation as above and dissolved in 5 ml of 50 mM Hepes, 2 mM dithiothreitol, pH 6.8 and dialyzed against several changes of the same buffer. This protocol yielded 10 -15 mg of dCTP deaminase that migrated as a single protein band in SDS-PAGE (13) of ϳ20 kDa corresponding to the dCTP deaminase monomer with a purity of more than 95%. For crystallization the enzyme was stored at 4°C whereas for enzymological studies it was stored in aliquots of 100 l at Ϫ20°C where the enzyme was stable for several months. All mutant enzymes were purified and stored by the same procedure as above. Enzyme concentration was determined using a calculated ⑀ 280 ϭ 22,190 cm Ϫ1 M Ϫ1 giving an A 280 (0.1%) of 1.044.
Preparation of Selenomethionine-substituted E138A dCTP Deaminase-Selenomethionine substituted E138A enzyme was produced from BL21(DE3) harboring the plasmid encoding this mutant enzyme by regulation of the normal methionine biosynthesis as described by Van Duyne et al. (14). Shortly, 2.5 ml of an overnight culture grown in M9 minimal medium with the addition of 0.4% (w/v) glucose and 100 g/ml ampicillin was used to inoculate 200 ml of the same medium, and growth was continued at 37°C. At an OD 600 of 0.4, 6 ml of a sterile filtered aqueous solution of 20 mg D,L-selenomethionine, 20 mg L-lysine, 20 mg L-threonine, 20 mg L-phenylalanine, 10 mg L-leucine, 10 mg L-valine, and 10 mg L-proline was added. After 15 min, IPTG to a final concentration of 0.5 mM was added. After three hours of induction cells were harvested, and the selenomethionine-substituted E138A enzyme was purified as described above.
Enzyme Kinetics and Recording of CD Spectra-Assays were conducted at 37°C in 50 mM Hepes, 2 mM dithiothreitol, pH 6.8 by measuring the decrease in absorbance from the deamination of dCTP to dUTP at 284 nm (for dCTP concentrations in the range of 0 -0.2 mM) or 291 nm (for dCTP concentrations exceeding 0.2 mM) with the extinction coefficients of ⌬⑀ 284 ϭ 3,800 cm Ϫ1 M Ϫ1 or ⌬⑀ 291 ϭ 1,340 cm Ϫ1 M Ϫ1 , respectively. For dCTP concentrations above 1 mM cuvettes with a light path of 2 mm was used. The concentration of dCTP and potassium phosphate varied as described under "Results and Discussion." Magnesium chloride was added in an excess of 2 mM above dCTP concentration. CD spectra of wild-type and mutant dCTP deaminases were recorded at 25°C as 5 overlaid scans on a Jasco J-810 instrument with a light path of 2 mm. The samples contained ϳ0.1 mg ml Ϫ1 of protein in 50 mM potassium phosphate, 2 mM dithiothreitol, pH 6.8. The analysis of initial velocity data were performed using Equation 1 for cooperative substrate binding, v ϭ V͓S͔ n /͑S 0.5 n ϩ [S] n ͒ (Eq. 1) where v is the initial velocity, V is the maximal velocity, S represents substrate, S 0.5 is the concentration of S for half-maximal velocity and n is the Hill-coefficient. Crystallization-Solubility screening (15) and crystal screen I from Hampton Research (16) were used for initial crystallization screening using the hanging drop vapor diffusion technique. Small, irregular crystals of the E138A inactive mutant enzyme cocrystallized with dCTP and Mg 2ϩ were obtained in both polyethylene glycol and salt conditions in the solubility screen and large crystals with sharp edges appeared in crystal screen I condition 23 (30% (v/v) polyethylene glycol 400, 0.2 M magnesium chloride, 0.1 M Hepes, pH 7.5). The crystals used for structure determinations were grown at room temperature with a hanging drop of 2 l of 5-8 mg/ml enzyme, 5 mM nucleotide (dCTP or dUTP, respectively), and 20 mM magnesium chloride in 50 mM Hepes, pH 7.5 mixed with 2 l of mother liquor (28 -30% (v/v) polyethylene glycol 400, 0.2 M magnesium chloride, 0.1 M Hepes, pH 7.5) equilibrated over 1 ml of mother liquor. The crystals grew to a size of 0.2 ϫ 0.1 ϫ 0.05 mm in 4 days.
Diffraction Data Collection-Diffraction data for the E138A crystals in complex with dCTP⅐Mg 2ϩ and wild-type crystals in complex with dUTP⅐Mg 2ϩ were collected under cryogenic conditions (100 K) at beamline I711, MAX-lab, Lund University, Sweden (17) on a MAR Research CCD detector. A copper rotating anode from Rigaku (RU300) operating at 46 kV/70 mA equipped with a MAR345 image plate was used for diffraction data collection on cryocooled (120 K) crystals of E138A in complex with dUTP⅐Mg 2ϩ . The mother liquor was used as cryoprotectant for all three crystals. Auto indexing, data reduction, and scaling were performed with programs from the HKL suite (18). The crystals of E138A in complex with dCTP⅐Mg 2ϩ and wild-type enzyme in complex with dUTP⅐Mg 2ϩ belong to space group P2 1 , with six protein chains in the asymmetric unit having a Matthews coefficient of 2.18 Å 3 /Da, corresponding to ϳ44% solvent content. The crystals of E138A in complex with dUTP⅐Mg 2ϩ belong to space group C2, also with six protein chains in the asymmetric unit (Matthews coefficient 2.10 Å 3 /Da, solvent content ϳ41%). This crystal was nonmerohedrally twinned which made auto indexing of the data nontrivial and as consequence the quality of the data was not as good as for the other two data sets reported here.
Structure Determination and Refinement-The three-dimensional structure of E. coli dCTP deaminase in complex with dCTP⅐Mg 2ϩ was determined by a combination of molecular replacement and single wavelength anomalous dispersion methods. First, molecular replacement as implemented in AMoRe (19) was applied using a trimmed polyalanine trimer of the bifunctional dCTP deaminase-dUTPase from M. jannaschii (Protein Data Bank code 1OGH; chain A, residues 1-45 and 86 -164) as search model. Diffraction data in the 15-4.5 Å range from a crystal of E138A mutant enzyme not including selenomethionine protein were used (data not shown), and all translational searches were performed using the centered correlation function (20). A solution with high contrast of the correlation coefficient and R-value placed two trimers in the asymmetric unit. Introduction of correct amino acid side chains, refinement using REFMAC5 (21), and rounds of prime and switch in RESOLVE (22) utilizing the 6-fold NCS of the crystal did not improve the electron density maps significantly and large parts (ϳ35%) were missing. An anomalous difference electron density map with phases from the present model and diffraction data from the selenomethionine substituted protein revealed the positions of 24 selenium sites, four for each protein chain. The selenium sites were refined with SHARP (23), DM (24) was subsequently used for density modification and 166 of the 193 amino acids in one of the six protein chains could be automatically traced by ARP-wARP (25). The remaining residues were manually built using O (26) and the five other protein chains were generated by NCS. After one cycle of positional refinement with REF-MAC5 (21), dCTP, and a magnesium ion could be introduced in each of the active sites. Cycles of refinement with REFMAC5 using NCS restrains, manual rebuilding in O and water picking with ARP-wARP were performed. The structure of the wild-type enzyme in complex with dUTP⅐Mg 2ϩ was determined using the first model, free from water molecules as well as dCTP and Mg 2ϩ , as starting point since the crystals both belonged to the same crystal form. After rigid body refinement allowing the six protein chains in the asymmetric unit to move separately, clear electron density was seen for dUTP, Mg 2ϩ , and the side chain of residue Glu 138 and these were model built in O. Further rounds of refinement in REFMAC5, rebuilding in O and water picking using ARP-wARP were performed. Despite electron density maps of high quality, the R free value did not reach below 28%. Closer examination of the cumulative intensity distribution of the diffraction data suggested presence of merohedral twinning. Therefore a twinning operator ((h,k,l) 3 (h,k,-h,-l)) was introduced and refinement was recaptured using SHELXL (27) resulting in considerably improved refinement statistics. The crystals of E138A in complex with dUTP crystallized in another space group (C2), and the structure was determined using the trimer of the wild-type product complex free from ligands as search model resulting in one clear molecular replacement solution with the dCTP Deaminase from Escherichia coli program AMoRe (19). Rigid body refinement, addition of dUTP, Mg 2ϩ , and water, as well as positional refinement with REFMAC5 and model building was performed similarly to the wild-type structure in complex with dUTP⅐Mg 2ϩ . Details from structure refinement are found in Table  I. The quality of the models was checked with PROCHECK (28) and WHATIF (29) as refinement progressed. The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 1XS4, 1XS1, and 1XS6 for E138A in complex with dCTP⅐Mg 2ϩ , wild-type enzyme in complex with dUTP⅐Mg 2ϩ , and E138A in complex with dUTP⅐Mg 2ϩ , respectively.

RESULTS AND DISCUSSION
Inhibition of dCTP Deaminase by Inorganic Phosphate-A previous enzyme kinetic analysis of the dCTP deaminase from S. typhimurium revealed positive cooperativity for the binding of dCTP with Hill coefficients close to a value of 2 (30). However, during our studies of the enzyme from E. coli we discovered that this cooperativity was caused by the presence of phosphate in the assay buffer. Omission of phosphate in the assay incubations gave nearly hyperbolic saturation curves for dCTP binding to E. coli dCTP deaminase (Fig. 1). From Fig. 1A it can be seen that the inhibition by inorganic phosphate of the dCTP deaminase activity does not affect the maximal velocity of the enzyme, but only has an effect on the half-saturation with dCTP (S 0.5 ) and the cooperativity of dCTP binding. Apparently, phosphate is a competitive inhibitor of dCTP binding but at the same time phosphate induces an increase in cooperativity of dCTP binding. Fig. 1B shows a linear correlation between increasing fixed concentrations of phosphate present in the incubation and a resulting increase in S 0.5 for dCTP, as well as concomitant increases in the Hill coefficient for dCTP binding. The specific mechanism by which phosphate acts on dCTP deaminase is not yet understood.
Analysis of Mutant dCTP Deaminases-None of the mutant dCTP deaminases prepared displayed any detectable activity (less than 1000-fold the wild-type activity) indicating that both side chains Arg 115 and Glu 138 are obligatory for the deamination reaction. As it will be discussed below an important function in catalysis of both residues can be deduced from the structure of the enzyme. Glu 138 corresponds to Glu 145 in the bifunctional Archaean enzyme for which it was also shown that the E145Q enzyme had lost the deaminase activity (3). The structural integrity of the mutant enzymes was verified by comparing the CD spectra of wild-type and mutant enzymes. The CD spectra of wild-type enzyme and the R115A and E138A enzymes were virtually superimposable (data not shown).
Crystal Structure Determination-The structure of the mutant enzyme E138A in complex with dCTP⅐Mg 2ϩ was determined by a combination of molecular replacement, using the bifunctional dCTP deaminase-dUTPase from M. jannaschii as search model, and single wavelength anomalous dispersion using selenomethionine-substituted enzyme. The experimental phases obtained from the single wavelength anomalous dispersion experiment gave a high quality electron density map sufficient for automatic model building of a large part of the final model. The structure of the wild-type enzyme in complex with dUTP⅐Mg 2ϩ was determined by difference Fourier methods, and the structure of the mutant enzyme E138A in complex with dUTP⅐Mg 2ϩ was determined using the molecular replacement method. The statistics of the diffraction data, and the refinement for the three structures are summarized in Table I.
The subunit of E. coli dCTP deaminase is composed of 193 amino acid residues with a molecular mass of 21.2 kDa. The asymmetric unit in the crystals contains six subunits (chains A-F) that form two independent homotrimers (chains A-C and D-E, respectively), and has clear electron density for all amino acid residues. The C␣ atoms of the subunits may be superimposed with root mean square deviation values ranging from 0.128 Å (chains A and F, E138A⅐dCTP⅐Mg 2ϩ ) to 0.296 Å (chains A and C, E138A⅐dCTP⅐Mg 2ϩ ) using default parameters in the program O (26). In the Ramachandran plots there are no nonglycine residues in disfavored regions except for residues Asn20 and Pro21, which form a cis-peptide bond. In the three structures, every active site (three per trimer) binds a well defined dCTP⅐Mg 2ϩ or dUTP⅐Mg 2ϩ complex.
Overall Fold-The dCTP deaminase subunit is composed of 14 ␤-strands (␤1-14), three ␣-helices (␣1-3), and four 3 10helices (␥1-4) ( Fig. 2A). The ␤-strands form three antiparallel ␤-sheets: S1 (␤1, ␤8, ␤12, and ␤10), S2 (␤2, ␤3, ␤14, ␤9, and ␤11), and S3 (␤4 and ␤6), and one mixed ␤-sheet, S4 (␤5 and ␤13 from one subunit of the homotrimer and ␤7 from another subunit), illustrated with different shades of blue in Fig. 2A. S2 pack on top of S1 and in this manner a distorted ␤-barrel is created. The subunits assemble, forming a compact homotrimer related by a 3-fold rotational NCS with one nucleotide-Mg 2ϩ complex bound between pairs of subunits, resulting in three active sites per trimer as illustrated in Fig. 2B. The homotrimer has an equilateral (ϳ55 Å long side) triangular face perpendicular to the 3-fold axis. The height along the 3-fold axis is ϳ45 Å. An analysis of the interactions of two of the subunits (chains A and C, E138A⅐dCTP⅐Mg 2ϩ ) in the trimer with the Protein-Protein Interaction Server (31) gives a value of 2,006 Å 2 for the interface accessible surface area with residues from eight different segments of chain A and five segments from chain C. Of the residues in the surface, 64% are non-polar and 36% polar.
Active Site Interactions with the Nucleotide-Mg 2ϩ Complexes-The wild-type dCTP deaminase from E. coli was cocrystallized with the reaction product, dUTP, and the mutant enzyme E138A was cocrystallized with both the substrate, dCTP, and the product, dUTP. In all three crystal structures a magnesium ion is octahedrally coordinated to the ␣-, ␤-, and sites of the three structures described in this work. A close up on the nucleotides and Mg 2ϩ binding between two subunits in the trimer (chains A and C) for the three determined structures are shown in Fig. 3. Fig. 3B gives a schematic representation of the residues forming hydrogen bonds to the respective nucleotides and the Mg 2ϩ ion. The magnesium ion clearly serves to shield the negative charge of the phosphates of the nucleotide so that the C-terminal fold can close over the active site nucleotide-Mg 2ϩ complex (Fig. 4). Interactions with the triphosphate moiety of the nucleotide are mediated by Arg 126 and Ser 111 (␣-phosphate), Ser 112 , and Arg 110 (␤-phosphate) and from the C-terminal fold, Lys 178 and Tyr 171 (␥-phosphate).
Trp 131 is stacked with the deoxyribose ring in an equivalent way to the tyrosine or phenyl alanine residues found in dUTPase structures (32)(33)(34)(35) and the bifunctional dCTP deaminase-dUTPase from M. jannaschii (5,36). The enzyme's discrimination against CTP appears to be a result of the large, hydrophobic side chain of Trp 131 that leaves no room for a hydroxyl group at position 2 on the ribose ring. The side chain of Ile 135 makes hydrophobic interactions with the substrate, stacking with the pyrimidine ring. The corresponding residue in a dCTP deaminase amino acid sequence alignment (5) is always a hydrophobic residue with matching stacking properties (Ile, Val, Leu, or Trp). O 2 in dCTP and dUTP (Fig. 3, B, D, and F) forms hydrogen bonds to Gln 182 and the backbone amino group of Val 136 as well as to the invariant Arg 115 from the other subunit. The hydrogen bond network, including hydrogen bonds between Arg 115 and Gln 182 , locks the two subunits together on one side of the pyrimidine ring (Fig. 3A). The backbone carbonyl of Val 136 forms a hydrogen bond to N3 and in dCTP, also to N4 (Fig. 3D). OD2 from the carboxylate of Asp 128 and the backbone amino group of the same residue both form hydrogen bonds to O3* of the deoxyribose ring.
Structural Similarity-The structure of E. coli dCTP deaminase show the greatest similarity to the previously determined structure of the bifunctional dCTP deaminase-dUTPase from M. jannaschii (5,36), as demonstrated by the possibility to use parts of this structure as a search model and obtaining a correct molecular replacement solution. One subunit of E. coli dCTP deaminase (chain A, E138A⅐dCTP⅐Mg 2ϩ ) and the bifunctional dCTP deaminase-dUTPase from M. jannaschii (PDB code 1OGH; chain A) superimpose with a root mean square deviation of 1.30 Å for 140 C␣ atoms as determined using default parameters in the program O (26) (Fig. 5A). The amino acid sequence identity between the superimposed residues is 31% (Fig. 5B). The central distorted ␤-barrel is conserved between the structures and the main differences are additional secondary structure elements (␥2, ␤5, and ␣2) in E. coli dCTP deaminase, while there is a ␣-helix and a ␤-strand in the bifunctional enzyme (residues 71-93), which are not present in the E. coli enzyme (Fig. 5, A and B). Like the bifunctional enzyme, dCTP deaminase is also similar to homotrimeric dUTPases (Fig. 5, A and B) (5). 98 C␣ atoms from E. coli dUTPase (PDB code 1DUD) superimpose, as above, with chain A from E. coli dCTP deaminase (E138A⅐dCTP⅐Mg 2ϩ ) with a root mean square deviation of 1.80 Å (21% identity). The primary structure of E. coli dUTPase is 40 amino acids shorter than the dCTP deaminase and as a result there is no correspondence in the dUTPase to ␤4-6, ␥2, and ␣2. The N terminus shows a different fold in the dCTP deaminase compared with dUTPases.
The C-terminal Fold-The 23 C-terminal residues of the bifunctional dCTP deaminase-dUTPase from M. jannaschii are not present in any of the published crystal structures (5,36) and therefore this part of the structures cannot be compared. The C terminus of one subunit in trimeric dUTPases has on the other hand been shown to interact with the active site in the cleft between two other subunits, resulting in each active site being built from residues contributed by all three subunits (33,34,37,38). This is apparently not the case for E. coli dCTP deaminase, where only two subunits of the trimer contribute to the active site. It is evident from our results that the C-terminal arm in dCTP deaminase folds back on the same subunit from which it is derived and serves to close the active site (Fig.  4). Thereby the C terminus provides interactions with the ␥-phosphate of dCTP via Lys 178 and Tyr 171 and to the O 2 of the base moiety via Gln 182 (Fig. 3, B, D, and F). Furthermore, the side chain of the C-terminal residue Asp 193 forms a salt bridge with Arg 110 of the other subunit (Fig. 3A). Since the primary structure of the C-terminal arm of the bifunctional enzyme has higher sequence identity with the dCTP deaminase than with the dUTPases, the dCTP deaminase and the bifunctional enzyme are likely to have a similar structural arrangement of the C terminus. Thus a slightly different arrangement of the active site of the bifunctional enzyme with respect to dUTPase activity can be expected as residues obligatory to dUTP hydrolysis, but not binding of substrate have been identified to reside in the C-terminal arm of dUTPase (38).
Determinants of dUTPase Activity for the Mono-and Bifunctional dCTP Deaminase Family Members-From the recent structures of the Mycobacterium tuberculosis (35) and E. coli (39) dUTPases in complex with dUTP and analogs hereof, a detailed understanding of the role in catalysis of individual conserved amino acid residues is provided. The conformation of dUTP and the magnesium ion coordination is almost identical in the dCTP deaminase and these dUTPase structures. The comparison of E. coli dCTP deaminase and M. tuberculosis dUTPase shows a completely conserved aspartate residue (Asp 128 and Asp 83 , respectively) that in both enzymes interact with the 3Ј-OH of the bound nucleotide. This residue (Asp 83 ) also prepares a water molecule for nucleophilic attack on the ␣-phosphate of dUTP in the M. tuberculosis dUTPase and other dUTPases (Asp 90 in the E. coli dUTPase, Fig. 5B). A structural comparison identifies Arg 126 as a residue that is crucial for the monofunctionality of the E. coli dCTP deaminase, as a residue of smaller size is found in the equivalent position in the dUTPases. Arg 126 occupies the position of the nucleophilic water molecule found in dUTPase and forms a salt bridge with Asp 128 (Fig. 5C). This exclusion of a potential nucleophilic water molecule is likely to prevent the hydrolysis of the phosphate chain of the bound nucleotide. The bifunctional enzyme from M. jannaschii has a tryptophan residue (Trp 133 ) replacing Arg 126 (Fig. 5B) but this large side chain is in a different conformation turned 180 degrees compared with Arg 126 . As a FIG. 4. Surface representations of the active site in E. coli dCTP deaminase. Surface representation of the active site cleft between subunit A (yellow) and subunit C (dark blue) of wild-type dCTP deaminase in complex with dUTP (A) with and (B) without the Cterminal residues 170 -193 from subunit A (white). dUTP atoms (colored by atom) and magnesium ions (green) are displayed as space-filling spheres. The panel was prepared using Chimera (43) result this tryptophan side chain occupies the same space as Trp 106 in the E. coli dCTP deaminase (Fig. 5C).
Catalytic Mechanism-We suggest a mechanism of the reaction catalyzed by dCTP deaminase based on the new structural information (Fig. 6A) 1DUD; dark blue). B, structurebased sequence alignment of E. coli dCTP deaminase, the bifunctional dCTP deaminase-dUTPase from M. jannaschii, and E. coli dUTPase. The secondary structure elements displayed above the sequences with twisted rods for ␣-helices and arrows for ␤-strands are according to the E. coli dCTP deaminase. The numbering of the secondary elements is the same as in Fig. 2A.
Residues identical in all three amino acid sequences are shown in dark gray and boxes, and identical residues in two of the sequences are displayed in light gray boxes. Equal signs under the sequences represent amino acid residues with C␣ positions considered to be equal in all three structures when superimposing the structures using default parameters in the program O (26), whereas asterisks under the sequences represents superimposable C␣ atoms in E. coli dCTP deaminase and the bifunctional enzyme from M. jannaschii. Observe that, for the structure of the bifunctional dCTP deaminase-dUTPase and E. coli dUTPase the 23 and 17 C-terminal residues are missing, respectively, and these are added in this alignment for the sake of completeness. C, close up view of the active site of M. tuberculosis dUTPase in complex with ␣,␤-imido dUTP and Mg 2ϩ (dark blue; PDB code 1SIX) superimposed with E. coli dCTP deaminase in complex with dUTP⅐Mg 2ϩ (yellow) and the bifunctional dCTP deaminase-dUTPase from M. jannaschii (light blue; PDB code 1OGH). Broken lines indicate the salt-bridge formed between Asp 128 and Arg 126 of dCTP deaminase and the hydrogen bond between the activating residue Asp 83 of dUTPase and the proposed nucleophilic water molecule 336 as well as direction of the in-line nucleophilic attack on the ␣-phosphate. Panels A and C prepared with MOLSCRIPT (41) and Raster3D (42). dCTP Deaminase from Escherichia coli and O 2 of the pyrimidine ring. The active site of the product complex with E. coli dCTP deaminase and dUTP⅐Mg 2ϩ holds two well defined water molecules (Fig. 6B). One of these (S5 in chain A) serves as a hydrogen bond donor interacting with Glu 138 and the backbone carbonyl group of Ala 124 , and as a hydrogen bond acceptor for the hydroxyl group of Ser 111 from the adjacent subunit (chain C). Ser 111 is firmly held in place by hydrogen bonds to Arg 115 and both these amino acid residues are invariant among dCTP deaminase amino acid sequences. In the suggested mechanism A (Fig. 6A) the water molecule (S5) is activated by Glu 138 . A tetrahedral reaction intermediate is formed and ammonia is expelled upon extraction of a proton from a second water molecule (S251), which is bifurcatedly coordinated to Glu 138 and to the carbonyl group of Val 136 . The formed hydroxide ion is thereafter neutralized and Glu 138 returns to its negatively charged starting position. dUTP can leave the active site and a new dCTP and water molecule may enter. The proposed mechanism A is analogous to previously suggested mechanisms of zinc containing enzymes (40) in that no tautomerization of the pyrimidine takes place. In a previous reaction model (Fig. 6A, mechanism B) suggested initially for the bifunctional dCTP deaminase-dUTPase (36) based on modeling of nucleotides into the active site, it was also proposed that the invariant Glu 138 (Glu 145 in M. jannaschii dCTP deaminase-dUTPase) is involved in activation of the nucleophile. Furthermore, tautomerization of the pyrimidine ring was suggested. The distance to Arg 115 from this oxygen atom is 3.3 Å in the product complex, and hence this arginine residue could help stabilize the negatively charged O 2 formed upon tautomerization. Glu 145 of the bifunctional enzyme was proposed to donate the second proton directly. This we do not find plausible, since the distance from Glu 138 to O4 is much longer (ϳ4.6 Å) than from Ser 251 (2.4 Å) in the product complex. Therefore, for the alternative mechanism B water molecule (Ser 251 ) most likely donates this second proton to the ammonia-leaving group.
The ammonia molecule that is expelled during the reaction may be harbored in a hydrophobic pocket shielded with the side chains of residues Leu 107 , Ile 127 , and Leu 158 . These amino acid residues are situated on the opposite side of the pyrimidine ring to the attacking water molecule (S5). The mechanism of product release is not obvious, but it must involve opening of the lid assembled of the 20 C-terminal residues that closes over the substrate/product in the active site.