From the
Center for Crystallographic Studies,
Department of Chemistry, University of Copenhagen, Universitetsparken 5,
DK-2100 Copenhagen Ø, Denmark and the
Department of Biochemistry, Lund University,
P.O. Box 124, S-221 00 Lund, Sweden
Received for publication, April 25, 2003 , and in revised form, May 16, 2003.
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ABSTRACT |
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INTRODUCTION |
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Deamination of cytosine compounds also takes place in the pyrimidine salvage pathways, wherein preformed cytosine and (deoxy)cytidine is converted into uracil and (deoxy)uridine, respectively. The first of these reactions is catalyzed by cytosine deaminase, an enzyme that only is present in bacteria and fungi. Cytidine deaminase that catalyzes the second reaction is, on the other hand, present in almost all organisms, including higher eukaryotes. Deamination of a cytosine ring implies an attack by a water molecule (or hydroxide ion) and subsequent expulsion of the amino group. Cytidine deaminase, dCMP deaminase, and yeast cytosine deaminase utilize zinc ions, whereas bacterial cytosine deaminase makes use of an iron ion for formation of the nucleophilic hydroxide ion used in the reaction. dCTP deaminase has been shown to not contain any metal ions.2 Therefore, dCTP deaminase and the highly similar DCD-DUT must operate with a different catalytic machinery from the other enzymes studied that deaminate cytosine compounds. Nevertheless, as for dUTP hydrolysis by dUTPases, dCTP deaminase and DCD-DUT require magnesium ions in order to be catalytically active. Studies of Salmonella typhimurium dCTP deaminase suggest that the true substrate of the reaction is the magnesium-dCTP complex (13).
We have undertaken to determine the structure of DCD-DUT from M. jannaschii to obtain information on the structural relationship of this enzyme and the homotrimeric dUTPases, of which there are crystal structures available from five different organisms (11, 12, 14, 15). Moreover, the DCD-DUT structure provides information about the mechanism for generation of the nucleophile in this type of deaminase. There is still no crystal structure available for any monofunctional dCTP deaminase.
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EXPERIMENTAL PROCEDURES |
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Data CollectionDiffraction data were collected under
cryogenic conditions (100 K) at beamline I711, MAX-lab, Lund university,
Sweden (17) on a MAR Research
CCD detector. One hour prior to data collection, the crystal was transferred
to mother liquor to which ethylene glycol had been added to 25% for cryo
protection. Auto indexing, data reduction, and scaling were performed with
programs from the HKL suite
(18). The crystals belong to
the cubic space group P213 (a = b = c =
111.1 Å), and two protein chains per asymmetric unit give a reasonable
Mathews coefficient of 2.44 Å3/Da, corresponding to 50%
solvent content.
Structure Determination and RefinementThe three-dimensional structure was determined using the method of single isomorphous replacement with anomalous scattering. The wavelength of the data collection (1.098 Å) was not ideal for obtaining an optimal anomalous signal from lead, but, nonetheless, the positions of two lead atoms were found with the program SOLVE (19), which may be attributed to the high redundancy and accuracy of the data. RESOLVE (20) was used for density modification and automatic tracing. The phases were extended from 2.5-Å resolution (lead derivative) to 1.88-Å resolution (native data) using the ARP-wARP program (21), and a free atom model was produced. From this model, 97% of the 356 amino acid residues contained in the final model could be automatically traced by ARP-wARP. After one step of refinement with REFMAC5 (22), the remaining amino acid residues of the model were manually built in O (23). Cycles of refinement with REFMAC5 and water picking with ARP-wARP (21) were performed. During refinement, non-crystallographic symmetry (NCS) restraints were applied to the two molecules in the asymmetric unit, water molecules related by NCS were detected with WATNCS (24), and these were also added to the NCS restraints. The quality of the model was checked with PROCHECK (25) and WHATIF (26) as refinement progressed. The structure factors and coordinates have been deposited in the Protein Data Bank with accession code 1OGH [PDB] .
Sequence AlignmentStructure-based sequence alignment (Fig. 4) was performed with the INDONESIA program package (alpha2.bmc.uu.se/~dennis/).3 The sequence alignment in the supplementary material (Fig. 5) was prepared with T-Coffee (27).
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RESULTS |
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The subunit in homotrimeric DCD-DUT is composed of 204 amino acid residues
with a molecular mass of 23.4 kDa. The asymmetric unit in the crystal contains
two subunits (chain A and B) forming two independent homotrimers by the
crystallographic 3-fold symmetry. The A chain is composed of residues A
1175 and the B chain of residues B 1181, respectively. Of the
356 amino acid residues, 18 were modeled with double conformations of their
side chains. Although there is clear electron-density for all amino acid
residues in the model that could be refined to low R-values, the
remaining C-terminal residues (A 176204 and B 182204) could not
to be localized in the electron-density map. The C-atoms (1175)
of the two chains can be superimposed with a root mean square deviation of
0.30 Å using default parameters in the program O
(23). The model also contains
400 water molecules, of which 192 are related by NCS. In the Ramachandran plot
there are no non-glycine residues in disfavored regions except for residues
Lys20 and Pro21, which form a cis-peptide bond.
This proline residue is strongly conserved among dCTP deaminase amino acid
sequences (Fig. 5, supplementary material).
Overall FoldThe subunit of DCD-DUT from M.
jannaschii is composed of 13 -strands (
1
13), 3
-helices (
1
3), and one 310-helix
(
1) (Fig. 2, a and
c). These are arranged in a structure of mainly
-structural character with one mixed
-sheet and three
anti-parallel
-sheets, of which two (S1 and S2) form a distorted
-barrel (Fig.
2a). The N terminus starts with one long
-helix
and goes further into
1, a
-strand in the first curled
anti-parallel
-sheet (S1) that also contains
7,
11, and
9. After one turn of 310-helix, the second anti-parallel
-sheet (S2) with
-strands
2,
13,
8, and
10
is initiated. These two
-sheets (S1 and S2) form the distorted
anti-parallel
-barrel. From
2, the protein chain continues into
3 and
4, which form an extruding anti-parallel
-arm (S3)
with a long loop connecting the two strands. Succeeding
4 is
5,
which is the central
-strand surrounded by
12 and
6 in the
mixed
-sheet S4. A long
-helix (
2) is inserted between
5 and
6in this
-sheet. After
6, the protein chain
contributes to
-sheet S1 and S2 with
7
11.
3 is
inserted between
8 and
9 of
-sheets S2 and S1, respectively.
From
11, the protein chain continues into
-sheet S4 with
12,
and, finally,
13 finishes
-sheet S2.
The subunits related by crystallographic 3-fold symmetry give rise to a
homotrimeric structure, the presumed active form of the enzyme
(Fig. 2b). The surface
perpendicular to the 3-fold axis has an equilaterally triangular shape with a
side of 40 Å. The thickness of the trimer along the 3-fold axis is
50 Å. The N-terminal residues of the three subunits are buried in the
interior of the trimer, whereas the C-terminal residues extrude in the solvent
region. The last five residues from chain B in the model form an additional
-strand on the
-arm S3 of the A-chain (A 5661) in the
crystal. These interactions are not part of the homotrimer contacts and may be
considered a crystal-packing artifact. The long loop between
3 and
4 intrudes into the next subunit forming an important intersubunit
interaction. An analysis of the interactions of two of the subunits in the
trimer with the Protein-Protein Interaction Server
(28) gives a value of 1677
Å2 for the interface-accessible surface area with residues
from eight different segments. 65 and 35% of the residues in the surface are
non-polar and polar, respectively. The analysis was performed including all
the NCS-related water molecules in the asymmetric unit, and, of these, 79 form
bridges between the subunits.
Structural Similarity to Homotrimeric
dUTPasesFig.
3a shows the superimposition of one subunit of DCD-DUT
from M. jannaschii and dUTPase from feline immunodeficiency virus
(12), respectively. This
dUTPase structure has been chosen as an illustration because it has an ordered
C terminus. The two crystal structures superimpose with a root mean square
deviation of 1.8 Å for 92 C atoms as determined using default
parameters in the program O
(23). Panels b and
d in Fig. 2 display
schematics of the same trimers in equivalent views. There are additional
structural features in the DCD-DUT structure, as reflected by its additional
71 amino acid residues. This is made clearer in the topology diagrams in
Fig. 3, b and
c as well as in the structure based sequence alignment in
Fig. 4 where the crystal
structures of five dUTPases of different origins have been superimposed with
the DCD-DUT structure. Structural elements, which are present in DCD-DUT but
not in dUTPase, are the anti-parallel
-arm S3 and
-helices
1 and
2. The N-terminal
1 lies on top of the homotrimer
as seen in the view of Fig.
2b. The
-arm S3, with
3 and
4 and their
interjacent loop, gives rise to additional trimerization interactions, and the
long
-helix
2 and
-strand
6 generate an extension of
DCD-DUT along the 3-fold axis of the trimer, as compared with dUTPases.
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Active SiteThere are several crystal structures in the Protein Data Bank of dUTPases in complex with dUMP, dUDP, or dUTP, which all map to the same position in the trimer as illustrated in Fig. 2d. Fig. 2e shows a close-up of the presumed active site of DCD-DUT with dUDP and a strontium ion from the superimposed equine infectious anemia virus dUTPase (PDB code 1DUC [PDB] ). This structure has been chosen because it is the only one where a metal ion is bound in the active site (15). Like dUTPases, DCD-DUT requires a divalent metal ion such as magnesium to be active. dUDP and the strontium ion can be contained in DCD-DUT without any clashes with the protein. The binding site for the pyrimidine ring is occupied by two well ordered water molecules (w359 and w361).
Residues from two of the subunits of the trimers contribute to the active site in DCD-DUT, as illustrated in Fig. 2e. Among these residues, three are conserved in dCTP deaminases but not in dUTPases, namely Arg122, Thr130, and Glu145 (Fig. 4) (Fig. 5, supplementary material). In the active site, residue Phe138 seems to serve the same role as the corresponding residue in dUTPases, which is predominantly a tyrosine residue. The ring of this residue stacks with the deoxyribose moiety of the substrate (Fig. 2e). In a few cases, for example in the mouse mammary tumor virus dUTPase, this residue is also a phenylalanine (29) and, in other dCTP deaminases, it is a tryptophan residue, which agrees with the stacking ability.
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DISCUSSION |
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The fifth conserved sequence motif in the dUTPases (motif 5), located in the C terminus, is not preserved in dCTP deaminases. However, the C-terminal region in dCTP deaminases contains a different amino acid sequence (residues 184190 in M. jannaschii DCD-DUT) (Fig. 5, supplementary material). These conserved residues could be involved in closing the active site as has been shown for dUTPases (912). The C terminus is indeed required for activity of DCD-DUT, as has been found for a C-terminally truncated form of the enzyme (2).
The trimer interface in DCD-DUT contains only a few invariant residues among dCTP deaminases (Asp49, Leu97, Thr130, and Glu145 in M. jannaschii DCD-DUT). Therefore, the nature of the interaction with 65% non-polar and 35% polar residues with 79 bridging water molecules for M. jannaschii DCD-DUT may not be typical for dCTP deaminases. A similar trend is observed in the dUTPases, wherein the character of the subunit interactions varies from exclusively hydrophobic (E. coli dUTPase) to alternating layers of positively and negatively charged residues that contain numerous water molecules (human dUTPase) (30).
dUTPases contain a proline residue that is considered to be a hinge, which
is important for correct bending of the C-terminal tail so that it reaches all
over the trimer to the correct active site
(Fig. 2d)
(31). No such curvature is
observed in the structure of M. jannaschii DCD-DUT, where the last
visible residues in the C terminus continue in a relatively straight line
(Fig. 2b). This may
not represent the conformation in solution, and, as mentioned previously, the
-strand formed by the last residues in the C terminus of protein chain B
may be regarded as a crystal-packing artifact. There are no indications that
the C terminus of DCD-DUT could not cross around the adjacent subunit of the
trimer and reach the active site farthest away, as is the case for dUTPases
(Fig. 2d). On the
other hand, the number of amino acid residues in the C terminus is even
sufficient for going in the other direction (counterclockwise in
Fig. 2b) and reaching
the third active site. The cis-peptide bond between Lys20
and Pro21 in DCD-DUT is placed at the end of
-strand
1
before the peptide chain enters the 310-helix
1. The bond is
placed on the protein surface and, though there is no obvious reason for its
presence, it may be involved in interactions with the C terminus upon
catalysis.
In motif 3, the aspartate residue corresponding to Asp135 is
strictly conserved in dUTPases and strongly conserved between the dCTP
deaminases (Fig. 5, supplementary material). Crystal structures of dUTPases
complexed with substrate analogues show that the carboxylate group of this
aspartate is hydrogen bonded to the 3-hydroxy group of deoxyribose. In the
dUTPase from E. coli in complex with dUDP, this carboxylate group
(Asp90) also interacts with a water molecule, appropriately
positioned for a nucleophilic in-line attack on the -phosphate. It has
therefore been suggested that this residue may act as a general base in dUTP
hydrolysis. Site-directed mutagenesis of the residue in other dUTPases has
consistently generated an inactive enzyme
(32,
33). In DCD-DUT,
Asp135 was recently mutated to an asparagine residue by Li et
al. (3), and this enzyme
had neither deaminase nor dUTPase activity. Whether the residue is essential
for catalysis or substrate binding or both remains to be answered, as an
aspartate or glutamate residue is also found at this position in
monofunctional dCTP deaminases.
A crucial role as general acid/base catalyst has been ascribed to a glutamic acid residue in cytidine deaminases, namely Glu104 in dimeric cytidine deaminase from E. coli (34) and Glu55 in tetrameric cytidine deaminase from Bacillus subtilis (35). The structures of these enzymes were determined using transition state analogues of cytidine with a hydroxyl group attached to a tetrahedral C4 atom. Assisted by a firmly bound zinc atom, the glutamate residue is supposed to be involved in both the generation of the nucleophilic hydroxide ion and the protonation of N3. Li and co-workers have mutated the strongly conserved Glu145 to glutamine in DCD-DUT (3). The mutant enzyme was devoid of deaminase activity but showed 25% residual dUTPase activity. Glu145 may have a role analogous to that of the residue in cytidine deaminases, because it is found in the active site close to the plausible position of the pyrimidine ring (Fig. 2e).
In cytidine deaminases, dCMP deaminases, and cytosine deaminases, a metal ion such as zinc or iron is used for water activation prior to the nucleophilic attack. dCTP deaminase and DCD-DUT do not contain any metal ions, as has been examined by energy dispersive x-ray fluorescence for dCTP deaminase from E. coli2 and seen in the crystal structure described here. Hence, formation of the nucleophile that performs the attack on the C4 atom of the pyrimidine ring must occur in a different way. The region of the active site in DCD-DUT, where the deamination reaction is assumed to take place, contains a network of hydrogen bonds involving well defined water molecules and the side chains of five amino acid residues, Ser118, Arg122, His128, Thr130, and Glu145 (Fig. 2f). All of these amino acid residues, except for His128, are invariant among dCTP deaminases (Fig. 5, supplementary material). His128 is substituted by an asparagine residue in some dCTP deaminases, a replacement that may preserve the hydrogen bonding abilities of the histidine residue. There is a very narrow pocket formed by motif 3 in homotrimeric dUTPases, where the O4 atom of uracil is hydrogen bonded to a water molecule that is held in place by hydrogen bonds to main chain atoms (11, 30). In DCD-DUT, the His128 side chain occupies the position equivalent to this water molecule, but, nevertheless, binding of uracil is possible (Fig. 2e).
The active site (Fig. 2f) contains three water molecules, i.e. w326, w350, and w359. Both w326 and w359 are hydrogen bonded to residues that are strictly conserved among the dCTP deaminases, which make them candidates as nucleophiles for the deamination reaction. w359 is hydrogen bonded to a positively charged side chain (Arg122), which could favor its tendency toward deprotonization and conversion to a hydroxyl group. However, it is also hydrogen bonded to Glu145, which counterbalances the positive charge. If Glu145 interacts with the pyrimidine ring, as in cytidine deaminase (34, 35), this could also favor the conversion of w359 to a nucleophilic hydroxyl ion. Binding of the substrate may perturb the dynamic hydrogen bonding network, altering the positions of the protons. The structure of the enzyme in complex with an inhibitor or product is required to resolve this issue and may give structural information on the role of the flexible C terminus.
The structure of DCD-DUT has confirmed that this bifunctional enzyme is not a fusion protein of a dCTP deaminase and a dUTPase. The evolutionary aspect of whether the bifunctional enzyme evolved before or after the dUTPases remains an open question.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains Figure 5, a sequence alignment prepared with T-Coffee, as
supplementary material.
¶ To whom correspondence should be addressed. Tel.: 45-35320282; Fax: 45-35320299; E-mail: sine{at}ccs.ki.ku.dk.
1 The abbreviations used are: DCD-DUT, bifunctional dCTP deaminase-dUTPase;
NCS, non-crystallographic symmetry.
2 J. Neuhard, unpublished results.
3 D. Madsen, P. Johansson, and G. J. Kleywegt, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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