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INTRODUCTION |
Deoxyuridine nucleotides pose severe problems for cells. Although
necessary precursors for thymine nucleotide biosynthesis, deoxyuridine
nucleotides can interfere with DNA polymerase activity, either by
inhibiting polymerases or by causing them to misincorporate 2'-deoxyuridine 5'-triphosphate (dUTP) in place of 2'-deoxythymidine 5'-triphosphate (dTTP) (1, 2). Ubiquitous DNA glycosylase enzymes
remove uracil bases from DNA, which are otherwise formed by spontaneous
cytosine deamination. The resulting apyrimidinic sites are cleaved by
endonucleases, fragmenting cellular DNA (3).
At temperatures of 70-95 °C, rate constants for spontaneous
cytosine deamination (in denatured DNA or cytidine nucleotides) are
several orders of magnitude higher than those measured at more moderate
temperatures (4). Therefore hyperthermophilic archaea that grow at such
temperatures counter the mutagenic effects of cytosine deamination with
a variety of thermostable uracil-DNA glycosylase enzymes (5). As a
consequence of having efficient base excision repair mechanisms, these
organisms must avoid misincorporating deoxyuridine nucleotides into DNA
during replication by using highly discriminatory DNA polymerases and
by maintaining low dUTP levels in the cell (1, 6).
Eukaryotes, some bacteria and some archaea use
zinc-dependent cytidine or deoxycytidine deaminase enzymes
to produce uridine or deoxyuridine nucleotides. Yet many bacteria,
including Escherichia coli and Salmonella
typhimurium, produce most of their 2'-deoxyuridine 5'-monophosphate (dUMP) from 2'-deoxycytidine 5'-triphosphate (dCTP)
using two different enzymes. These bacteria use dCTP deaminase (DCD,1 EC 3.5.4.13) to
catalyze the nucleophilic substitution of a hydroxide ion for the
4-amino group of the cytosine base (7). A second enzyme, dUTP
diphosphatase (DUT, EC 3.6.1.23), catalyzes the hydrolysis of the dUTP
-phosphorus anhydride bond to produce pyrophosphate (diphosphate)
and dUMP, the obligatory precursor for thymidine nucleotides synthesis
(8) (Fig. 1). Due to the toxicity of dUTP produced by DCD, the DUT
enzyme is required to remove the accumulated product. Null mutations in
the E. coli dut gene are lethal unless suppressed
by null mutations in the dcd gene (9).
Despite catalyzing markedly different hydrolytic reactions, DUT and DCD
proteins are encoded by homologous genes (9, 10). dUTP diphosphatase
enzymes have been extensively studied, and four crystal structure
models have been published for DUT proteins from E. coli
(11), human (12), and two retroviruses (13, 14). However, only one DCD
enzyme from S. typhimurium has been partially purified and
characterized (15) and the dcd gene was subsequently
identified in E. coli (9).
DUT proteins share five conserved signature motifs with the DCD
proteins; four of these correspond to conserved uridine binding regions
in DUT. These conserved domains have been used to identify dUTP
diphosphatase homologs from diverse organisms (16) and to predict that
two paralogous genes in the hyperthermophilic archaeon
Methanococcus jannaschii encode DCD and DUT proteins (10).
The original annotation of the complete genome sequence from that
organism described both gene products as dCTP deaminases because of
their considerable sequence divergence from canonical DUT proteins
(17).
In this work, we describe the characterization of two paralogous genes
from M. jannaschii that are members of the
dut-dcd family. Both genes were cloned in
E. coli, and the heterologously expressed proteins were
purified and characterized. The protein encoded by locus MJ1102 was
shown to be a dUTP diphosphatase, similar to previously characterized
DUT homologs. Unexpectedly, the enzyme encoded by locus MJ0430 was
found to be a dCTP deaminase/diphosphatase (MjDCD-DUT). It has two
functions, catalyzing both the deamination of dCTP and release of
pyrophosphate to produce dUMP. It also acts as a dUTP diphosphatase
with a lower affinity for dUTP than for dCTP. Compared with the
S. typhimurium dCTP deaminase, MjDCD-DUT has a higher
affinity for dCTP and is a more active deaminase. The discovery of this
bifunctional enzyme ties together the apparently unrelated deaminase
and diphosphatase activities of DCD and DUT homologs and shows how
M. jannaschii circumvents the problem of accumulating dUTP
during thymidine biosynthesis.
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EXPERIMENTAL PROCEDURES |
Materials--
All reagents were purchased from Sigma unless
otherwise specified. Stock solutions of nucleotides were prepared in
deionized water. Nucleotide concentrations were determined in sodium
phosphate buffer (pH 7.2, 0.1 M NaCl) by UV spectroscopy
using established molar extinction coefficients for cytidine
nucleotides, uridine nucleotides, adenosine nucleotides, and guanosine
nucleotides (18).
Cloning and Expression of the MJ0430 and MJ1102 Proteins in
E. coli--
The M. jannaschii genes at loci MJ0430
(Swiss-Prot accession number Q57872) and MJ1102 (Swiss-Prot accession
number Q58520) were amplified by PCR from genomic DNA using
oligonucleotide primers synthesized by Invitrogen. The MJ0430 gene was
amplified using primers: Fwd,
(5'-GGTGGTCATATGATTCTTAGTGATAAAG-3') and Rev,
(5'-GATCGGATCCTTAATCTTTTTTATGATTGTC-3'). The MJ1102 gene was amplified
using primers: Fwd, (5'-GGTGGTCATATGGTGGTGAAATTAATG-3') and Rev,
(5'-GATCGGATCCTTATCTCCCTTTATAAATTC-3'). The forward primers introduced an NdeI restriction site at the 5'-end of the
amplified DNA whereas reverse primers introduced a BamHI
site at the 3'-end. PCR amplifications were performed as described
previously using a 50 °C annealing temperature for MJ0430 and a
55 °C annealing temperature for MJ1102 (19). PCR products were
purified using a QIAQuick spin column (Invitrogen) and then digested
with NdeI and BamHI restriction enzymes
(Invitrogen). Bacteriophage T4 DNA ligase (Invitrogen) was used to
ligate the MJ0430 and MJ1102 genes into compatible sites in plasmids
pET17b (Novagen) or pT7-7, respectively (20). DNA sequences were
verified by dye-terminator sequencing at the University of Iowa DNA
facility. The resulting recombinant plasmids were transformed into
E. coli BL21-CodonPlus(DE3)-RIL (Stratagene) cells.
Transformed E. coli cells were grown in Luria-Bertani medium
(200 ml; Difco) supplemented with 100 µg/ml ampicillin. Cultures were
shaken at 37 °C until they reached an absorbance at 600 nm of 0.8. Recombinant protein production was induced with 28 mM lactose. After an additional 2-h incubation with shaking at 37 °C,
the cells were harvested by centrifugation (4000 × g,
5 min) and frozen at
20 °C. Induction of the desired proteins was
confirmed by SDS-PAGE analysis of total cellular proteins.
Site-directed Mutagenesis--
To test the possible function of
conserved Asp135 and Glu145 residues of
MjDCD-DUT, Asp135 was replaced by Asn (D135N) and
Glu145 was replaced by Gln (E145Q). The
QuikChangeTM site-directed mutagenesis kit (Stratagene) was
used according to the manufacturer's instructions with template
pMJ0430 (MJ0430 in pET17b). The oligonucleotide primers (Invitrogen)
used were D135N-Fwd (5'-CTGCTGGATGGATTAACGCTGGATTTAAAGG-3'),
D135N-Rev (5'-CCTTTAAATCCAGCGTTAATCCATCATCCAGCAG-3'), E145Q-Fwd
(5'-GGAAAAATAACCTTGCAGATTGTTGCTTTCG-3'), and E145Q-Rev (5'-CGAAAGCAACAATCTGCAAGGTTATTTTTCC-3'). DNA sequences of the mutated genes were confirmed by dye-terminator sequencing at the University of Iowa DNA facility.
Purification of MjDCD-DUT--
Heterologously expressed
MjDCD-DUT protein was purified from soluble cell-free extract by heat
treatment and chromatographic methods. E. coli cells (13 g,
wet weight) were suspended in 25 ml of extraction buffer (20 mM Tris/HCl, 2 mM DL-dithiothreitol (DTT), pH 7.6) and sonicated in a 50-ml plastic tube using a microprobe tip driven by a W-385 Ultrasonic processor (Heat Systems-Ultrasonics, Inc.) operating at 50% power output. Samples cooled by ice water were
sonicated for 8 min with 3 s on/off intervals. Soluble cell-free extract was obtained after centrifugation at 20,000 × g for 15 min at 4 °C. The E. coli proteins
were denatured by heating the soluble cell-free extract at 70 °C for
15 min, followed by centrifugation at 20,000 × g for
10 min at 4 °C to remove the insoluble material. Heat-soluble cell
extract (16 ml) was applied to a Mono Q HR anion-exchange column
(1 × 8 cm; Amersham Biosciences) equilibrated with buffer A (20 mM Tris/HCl, pH 7.5). Pumps attached to the column were controlled by a Biologic HR workstation (Bio-Rad). Bound protein was
eluted with a 45-ml linear gradient from 0 to 1 M NaCl in buffer A at a flow rate of 1.0 ml/min. Fractions containing dCTP deaminase activity were pooled and concentrated in a
N2-pressurized stirred cell with a YM10 ultrafiltration
membrane (Millipore). Concentrated protein was applied to a Sephacryl
S-200 HR size exclusion column (1.6 × 60 cm; Amersham
Biosciences) equilibrated with buffer B (50 mM HEPES/NaOH,
0.15 M NaCl, 2 mM DTT, pH 7.2). MjDCD-DUT
protein was eluted at a flow rate of 0.5 ml/min and was collected in
1-ml fractions. Fractions containing dCTP deaminase activity were
pooled and concentrated in a N2-pressurized stirred cell
with a YM10 ultrafiltration membrane (Millipore) and then stored at
70 °C. Heterologously expressed MJ1102-encoded protein and the
MjDCD-DUT D135N and MjDCD-DUT E145Q variant proteins were purified by
the same procedure.
Protein purity was evaluated by silver diamine staining of proteins
separated by SDS-polyacrylamide gel electrophoresis (12% T, 4% C
acrylamide) using a Tris/glycine buffer system. Sizes of the denatured
proteins were determined by comparison to low molecular weight protein
standards (Bio-Rad). Protein concentrations were measured using the
Bradford total protein assay (Bio-Rad) with bovine serum albumin as the standard.
Analytical Size Exclusion Chromatography--
Size exclusion
chromatography was performed at room temperature on a Superose 12HR
column (1 × 30 cm; Amersham Biosciences). The column was
equilibrated in buffer B and run with a flow rate of 0.4 ml/min.
Protein standards used to calibrate the sizing column were horse spleen
apoferritin (440,000), potato
-amylase (200,000), yeast alcohol
dehydrogenase (150,000), bovine serum albumin (67,000), bovine
erythrocyte carbonic anhydrase (29,000), and horse heart cytochrome
c (12,400). Eluted protein was detected by their absorbance
at 280 nm and/or dCTP deaminase activity.
MALDI-TOF Mass Spectrometry of MjDCD-DUT Protein--
Purified
MjDCD-DUT protein was analyzed by MALDI-TOF mass spectrometry. A
saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid matrix was
prepared in 50% (v/v) acetonitrile with 0.1% trifluoroacetic acid.
Purified MjDCD-DUT (0.5 µl of a 2.2 mg/ml protein solution) was mixed
with an equal volume of matrix solution on a stainless steel target
(Kratos Analytical). Dried, crystalline samples were washed with 5 µl
of 0.1% trifluoroacetic acid and dried under a stream of
N2 gas. Samples were analyzed in a Kratos KOMPACT SEQ
MALDI-TOF instrument (Kratos Analytical) operated on the linear mode at
20 kV. For each sample, 50 spectra were collected by scanning across
the sample. Average masses were identified using the combined sample
set. Ions of sodium, matrix, bovine serum albumin, bovine pancreas
insulin chain B (oxidized), and horse heart cytochrome c
were used for mass calibration.
Measurement of dCTP Deaminase Activity--
dCTP deaminase
activity was measured using a continuous spectrophotometric assay (15).
In a thermostated quartz cuvette, the following reaction mixture (1 ml)
was equilibrated at 60 °C for 15 min: 50 mM TES/NaOH
buffer (pH 7.5), 0.1 M NaCl, 5 mM
MgCl2, 1 mM 2-mecaptoethanol (Fisher), and
enzyme (0.1-1 µg). The reaction was initiated by addition of dCTP to
a final concentration of 0.2 mM. The decrease in absorbance
as a result of deamination of cytosine compounds was measured at 284 nm
with an UV-VIS recording spectrophotometer UV-160A (Shimadzu). The
molar extinction coefficient difference between deoxycytidine and
deoxyuridine compounds is 3.8 × 103
M
1 cm
1 (15).
Measurement of dUTP Diphosphatase Activity--
Standard dUTP
diphosphatase activity assays included 50 mM TES/NaOH
buffer (pH 7.5), 0.1 M NaCl, 5 mM
MgCl2, 1 mM 2-mecaptoethanol, and enzyme in 200 µl. The reaction mixture was pre-incubated at 60 °C for 15 min
before the addition of dUTP. After 10 min of incubation, reaction was
terminated by the addition of 10 µl of 100 mM pH 8.0 EDTA
and cooled on ice. Pyrophosphate released from dUTP was measured using
an enzymatic assay in a pyrophosphate reagent kit (Sigma).
Alternatively, inorganic phosphate was produced from pyrophosphate by
including a thermostable coupling enzyme in the dUTP diphosphatase
assay. The coupling enzyme used was purified, heterologously expressed
GTP cyclohydrolase from M. jannaschii, which has
pyrophosphate phosphohydrolase activity, but has no effect on
pyrimidine nucleotides (21). Inorganic phosphate was then quantified by
the malachite green dye-enhanced phosphomolybdate assay (21).
Inorganic phosphate concentrations were calculated from a standard
curve using 0-8 nmol of KH2PO4 in 700 µl
deionized H2O. Absorbances from reactions incubated without
enzyme were subtracted from absorbances of the enzymatic reactions to
calculate activities.
Product Identification--
The nucleotide products of the
enzymatic reaction were analyzed by separation on a Mono Q HR
anion-exchange column (0.5 × 5 cm, Amersham Biosciences). Bound
nucleotides were eluted with a 25-ml linear gradient from 0 to 0.5 M NaCl in a 25 mM Tris/HCl buffer (pH 8.5) at a
flow rate of 0.5 ml/min. The nucleotides eluted were monitored at 254 nm. Nucleotide standards were analyzed on the column before and after
the sample runs for calibration. The nucleotides (and their retention
times) were dCMP (20.1 min), dUMP (21.5 min), dCDP (24.1 min), dUDP
(25.3 min), dCTP (26.9 min), and dUTP (27.9 min).
Measurement of the Regiospecificity of the O-P Bond Cleavage
Reaction--
To 35 µl of buffer containing 50 mM
TES/NaOH (pH 7.5), 5 mM MgCl2, and 1 mM mercaptoethanol were added 45 µl of
H218O (98 atom% 18O) and 5 µl of
a 2.2 mg/ml solution of the MjDCD-DUT protein. The sample was incubated
at 60 °C for 15 min before 10 µl of 0.1 M dCTP was
added. After an additional 15 min at 60 °C the sample was evaporated
to dryness with a stream of dry nitrogen gas, and the residue was
reacted at 100 °C with 20 µl of a TMS reagent composed of
trimethylchlorosilane, hexadimethyldisilazane, and pyridine (1:3:9,
v/v/v). After removal of the insoluble material by centrifugation,
electron impact-mass spectrometry (EI-MS) analysis of the soluble
material was performed using a VG-70-70EHF instrument (VG Analytical)
operating at 70 eV with a direct probe inlet (probe temperature
programmed from room temperature to 400 °C at 5 °C/s). The mass
range scanned was 50-750 m/z at 5 s/decade.
Heat Stability, pH Optimum, and Metal Effect on dCTP Deaminase
Activity of MjDCD-DUT--
To test the heat stability, purified
MjDCD-DUT was incubated for 10 min at temperatures from 60 to 100 °C
and then assayed for dCTP deaminase activity using the standard assay
at 60 °C. The effect of pH on dCTP deaminase activity of MjDCD-DUT
was studied using a three-component buffer system in the pH range
5.0-9.8 (22). Buffer mixture containing 75 mM bis-Tris, 38 mM HEPPS, and 38 mM CHES were adjusted to pH
5.5-9.5 using either NaOH or HCl. Effects of NaCl (Fisher), DTT, and
2-mecaptoethanol (Fisher) were tested in standard dCTP deamination assays.
Divalent cation dependence was examined using enzyme, TES buffer, and
dCTP freed of divalent cations by passage through a 7.5 mm × 5 cm
column of Chelex 100-Na+ (Bio-Rad) (21, 23). MjDCD-DUT
elution was monitored by its absorbance at 280 nm. Metal replacement
reactions included 5 mM concentrations of
MgCl2·6H2O (Fisher),
MnCl2·4H2O (Fisher),
CaCl2·6H2O (Fisher),
NiCl2·6H2O,
CoCl2·6H2O (J. T. Baker Inc.),
CuCl2·6H2O (J. T. Baker Inc.),
BaCl2·2H2O (Fisher), or
ZnSO4·7H2O.
Substrate Specificity and Inhibitory Effects of Other Nucleotides
on MjDCD-DUT Deaminase Activity--
The substrate specificity of
MjDCD-DUT was investigated using a spectroscopic assay. Nucleotide
substrates tested at 0.2 and 1 mM concentrations included
dCDP, dCMP, CTP, CDP, CMP, cytosine, deoxycytidine, ATP, dTTP, UTP, and
GTP. The products of each reaction were identified by the Mono Q HR
anion-exchange column (0.5 × 5 cm; Amersham Biosciences) as
described above.
Inhibitory effects of various nucleotides on MjDCD-DUT dCTP deaminase
activity were also examined. Enzyme activity was measured by
quantifying the production of pyrophosphate. These reactions included
0.2 mM dCTP and 1 mM concentrations of other nucleotides.
Kinetic Analysis of MjDCD-DUT Activities--
For both dCTP
deaminase and dUTP diphosphatase activities of MjDCD-DUT, initial
reaction rates were measured in standard assays at various
concentrations of dCTP (0-100 µM) or dUTP (0-800
µM). Reaction mixtures were incubated at 60 °C for 15 min, and then reactions were started by the addition of substrate. A
curve from the Michaelis-Menten equation was fit through the initial
rate data by nonlinear regression, using the Sigma Plot 2000 program (SPSS Science).
 |
RESULTS |
Identification, Expression, and Purification of
MjDCD-DUT--
dCTP deaminase/dUTP diphosphatase homologs have been
identified in the genomes of several archaea (10). Two homologs, MJ0430 and MJ1102, were found in the complete genome sequences of M. jannaschii (17). However, it was not known whether one gene product functions as a dUTP diphosphatase and the other as a dCTP deaminase. We cloned both genes and expressed both proteins in E. coli for purification and characterization.
The recombinant MjDCD-DUT (encoded by MJ0430) was purified through
heating, anion-exchange, and gel-filtration chromatography. Table
I shows the purification of 33 mg of
MjDCD-DUT from 13 g of E. coli cells. The specific
activity of purified MjDCD-DUT was 10.5 µmol/min/mg. Enzyme specific
activity increased 4.2-fold though the purification, which indicates
the MjDCD-DUT constitutes 24% of the total E. coli soluble
protein.
Analysis of the purified MjDCD-DUT by MALDI-TOF mass spectrometry
showed a molecular mass of 23,619 ± 94 Da, which is close to its
predicted 23,432 Da molecular mass from the genomic data. The denatured
protein migrated on SDS-polyacrylamide gel an apparent molecular mass
of 30,800 Da. From a Sepharose 12 HR size exclusion column, MjDCD-DUT
eluted with an apparent molecular mass of 136,000 Da. This elution
pattern suggests that native MjDCD-DUT protein may form a hexamer.
MjDCD-DUT Catalytic Activities and Reaction Products--
When
MjDCD-DUT was incubated with 0.2 mM dCTP at 60 °C, the
enzyme not only catalyzed the deamination of dCTP, but also catalyzed the release of pyrophosphate to form dUMP. MjDCD-DUT can also catalyze
the hydrolysis of dUTP to form dUMP and pyrophosphate. These results
show that MjDCD-DUT is bifunctional: it catalyzes two consecutive
reactions to form dUMP using dCTP as substrate. In contrast,
-proteobacteria require separate dCTP deaminase and dUTP
diphosphatase enzymes to catalyze the reactions (Fig. 1) (15).
Enzymes can catalyze pyrophosphate release by promoting the
nucleophilic substitution by water at either the
- or
-phosphorus atoms of nucleotide triphosphates (24). To test the regiospecificity of
MjDCD-DUT, the enzyme was incubated with dCTP in
H218O, and the reaction products were converted
to TMS derivatives. EI-MS analysis identified two compounds. The
first corresponded to the (TMS)4 derivative of
pyrophosphate, M+ = 466 m/z
and M+-15 = 451 m/z. Isotopic
clusters of these ions had the same relative intensities observed for
unlabeled pyrophosphate and showed no 18O incorporation.
The second identified compound consisted of the (TMS)4
derivative of dUMP M+-15 = 509 m/z. The isotopic cluster of this ion showed the
incorporation of two labeled oxygens each with ~50% 18O
enrichment. A fragment ion 299 m/z originating
from the phosphate of the dUMP derivative contained only a single
18O confirming that one of the oxygens resided on the
phosphate. The other oxygen was incorporated at the C-4 position of
uracil during deamination. These data suggest that the phosphate
anhydride cleavage proceeds by attack of activated water at the
-phosphate of 5'-nucleotide triphosphates.
Characterization of MjDCD-DUT Activity--
The standard dCTP
deaminase assay was used to characterize the purified MjDCD-DUT under
various reaction conditions. When incubated with 0.1 M
NaCl, the enzyme exhibited its maximum activity, compared with no NaCl
(70%), 0.05 M (70%), 0.15 M (90%), or 0.2 M NaCl (90%) added. No change of enzyme activity was
observed when MjDCD-DUT was assayed in 50 mM TES/NaOH (pH
7.5), 50 mM HEPES/NaOH (pH 7.5), or 50 mM
MOPS/NaOH (pH 7.5) buffer. Nor did addition of 1 mM
2-mercaptoethanol and 1 mM DTT affect the activity,
although 1 mM 2-mercaptoethanol was added in the standard assay.
Purified MjDCD-DUT is a thermostable enzyme. When heated at 90 °C
for 10 min, MjDCD-DUT retained over 70% of its activity, assayed at 60 °C. Assays of MjDCD-DUT dCTP deaminase activity at various pH showed
maximum activity at pH 7.5. As shown in Fig. 2, over the range from pH 6 to 9, the
enzyme retains more than 50% of its maximum activity.

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Fig. 2.
MjDCD-DUT dCTP deaminase activity at
different pH values. A three-component buffer system was used with
bis-Tris, HEPPS and CHES as described under "Experimental
Procedures." Maximum specific activity in this experiment was 10.5 µmol/min/mg.
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The substrate specificity of MjDCD-DUT was investigated using the
standard deamination assay with 1 mM concentrations of
other cytidine nucleotides. dCDP is a very poor substrate; MjDCD-DUT catalyzes its deamination and hydrolysis to form dUMP at only 4% of
the rate of dCTP deamination. None of the following nucleosides or
nucleotides was deaminated by MjDCD-DUT: dCMP, CTP, CDP, CMP, cytosine,
or deoxycytidine. Neither ATP, dTTP, UTP, or GTP were substrates for
MjDCD-DUT. Nor was pyrophosphate released in these assays. These
results indicate that MjDCD-DUT is highly specific for dCTP as its
substrate for deamination. The MjDCD-DUT activity requires metals.
Divalent cation requirement was examined by assaying metal-free
MjDCD-DUT. No activity was detected in reactions without added metals.
Maximum dCTP deamination activity was observed with the addition of
2-5 mM MgCl2 in the presence of 0.2 mM dCTP. No other divalent cations supported full activity
at the concentration of 5 mM (Table
II). Compared with its dCTP deaminase
activity, dUTP diphosphatase activity of MjDCD-DUT showed a similar
pattern of metal activation (Table II).
To test the inhibitory effects of other naturally occurring nucleotides
on MjDCD-DUT dCTP deaminase activity, 1 mM concentrations of dCDP, dCMP, CTP, CDP, CMP, ATP, GTP, UTP, or dTTP were added to
standard assays that included 0.2 mM dCTP. Enzyme activity was analyzed by measuring the production of pyrophosphate using the
Sigma pyrophosphate reagent. Among these nucleotides, only dTTP
inhibited enzyme activity. The relative activity was 58% compared with
reactions without added dTTP. Because dUTP is a substrate for MjDCD-DUT
diphosphatase activity, the inhibitory effects of dUTP were tested
using the deamination assay. Here, 1 mM dUTP inhibited the
activity by 80%.
To characterize the kinetic properties of MjDCD-DUT, initial rates for
both dCTP deaminase and dUTP diphosphatase activities were measured at
various substrate concentrations. The relevant substrate for both
reactions is likely a Mg2+-substrate complex. Therefore, in
these experiments, the Mg2+ concentration was fixed at 5 mM, more than a 5-fold excess over the substrate
concentrations. Both sets of activity data were fit to the
Michaelis-Menten first-order rate equation. At 60 °C, apparent
kinetic parameters of MjDCD-DUT were Km = 17.6 ± 2.6 µM, Vmax = 14.7 ± 0.87 µmol/min/mg for dCTP deamination, and Km = 263 ± 64 µM, Vmax = 23.9 ± 2.6 µmol/min/mg for dUTPase activity.
Because both dCTP and dUTP are substrates for MjDCD-DUT diphosphatase
activity, a competition assay was used to test the relationship of
these two activities (25). Substrate concentrations were chosen to give
similar activities with 100% dCTP (40 µM) or 100% dUTP
(150 µM). The proportion of each substrate in the
reactions was varied from 100% dCTP and 0% dUTP to 0% dCTP and 100%
dUTP. The combined activity for pyrophosphate production was quantified by measuring the phosphate produced when the reaction was coupled to a
thermostable pyrophosphate phosphohydrolase enzyme (21). The results
were consistent with a general model for reactions occurring at the
same catalytic site and competing with each other, indicating that dCTP
and dUTP bind at the same sites (data not shown) (25).
Sequence Analysis and Site-directed Mutation of MjDCD-DUT--
An
alignment of the MjDCD-DUT protein sequence with homologous DCD and DUT
sequences identified conserved amino acids in four motifs that were
previously described for dUTP diphosphatases (Fig.
3) (16). Crystal structure models of
bacterial and viral DUT proteins implicate one of these conserved
residues, corresponding to Asp135 of motif 3 of MjDCD-DUT,
in binding the 3'-OH group of the substrate's deoxyribose moiety
(11-13). The bacterial enzyme may also use the corresponding residue
to activate a water molecule for nucleophilic attack on the
substrate's
-phosphate group (11). The viral DUT protein could use
this aspartyl side chain to promote subunit interactions (14). To test
this residue's importance to catalysis, we used site-directed
mutagenesis to replace Asp135 with Asn (MjDCD-DUT D135N).
Purified MjDCD-DUT D135N had neither dCTP deaminase nor dUTP
diphosphatase activity. The altered protein had an apparent molecular
mass of 57,900 Da, measured by analytical gel-filtration
chromatography, compared with a mass of 136,000 Da for the wild-type
MjDCD-DUT. Thus the replacement of Asp135 may destabilize
the protein's quaternary structure.

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Fig. 3.
Sequence alignment of MjDCD-DUT (Swiss-Prot
accession Q57872) with homologs from Aquifex aeolicus
(AaDCD; Swiss-Prot accession O67539),
Mycobacterium tuberculosis (MtDCD;
Swiss-Prot accession O07247), E. coli
(EcDCD and EcDUT; Swiss-Prot
accession P28248 and P06968), Helicobacter pylori
(HpDCD; Swiss-Prot accession O25136), M. jannaschii (MjDUT; Swiss-Prot accession
Q58520), Pyrococcus furiosus (PfDUT;
GenBankTM accession AAL47572.1), archaeal virus SIRV (SIRV-DUT;
GenBankTM accession 666605.1), Homo sapiens
(HsDUT; Swiss-Prot accession P33316; positions
112-252). Boxed residues indicate five previously
described motifs shared by dUTP diphosphatases. Positions of
identically conserved residues are shown in white on
black and regions of similarity conserved residues are
boxed. Two arrows indicate the Asp135
and Glu145 residues replaced by directed mutagenesis. DUT
and DCD sequences were aligned separately using the ClustalW program
(39), and then the alignments were combined by profile alignment using
ClustalW.
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In the analogous bacterial cytidine deaminase enzymes, the side chain
of a conserved glutamate residue is proposed to act as a general
acid/base catalyst, facilitating release of the leaving -NH2 group after a nucleophilic hydroxide ion attacks the
cytosine base (26-28). Although MjDCD-DUT has no sequence similarity
to this zinc-dependent cytidine deaminase, MjDCD-DUT
homologs do share a conserved glutamate residue (Glu145).
Because this glutamate residue is conserved in all DCD homologs, but in
few DUT homologs, we used site-directed mutagenesis to replace
Glu145 with Gln (MjDCD-DUT E145Q). Purified MjDCD-DUT E145Q
protein had no dCTP deaminase activity, but retained 25% dUTP
diphosphatase activity relative to wild-type enzyme. The purified
MjDCD-DUT E145Q also has a higher apparent affinity for dUTP compared
with the wild-type MjDCD-DUT (Table III).
This residue is also conserved in the archaeal virus SIRV DUT enzyme
(described below) that lacks dCTP deaminase activity, therefore these
results suggest that the Glu145 residue is necessary, but
not sufficient, for dCTP deaminase activity.
Characterization of MJ1102-encoded Protein--
Purified
MJ1102-encoded protein showed a single band on a SDS-polyacrylamide gel
with an apparent molecular mass of 18,000 Da, compared with its
predicted molecular mass of 18,640 Da. From the Sepharose 12 HR size
exclusion column, the MJ1102-encoded protein eluted with an apparent
molecular mass of 48,200 Da, which suggests that the native protein may
form a trimer. When incubated at 60 °C, it catalyzed hydrolysis of
dUTP to dUMP and pyrophosphate. It had no detectable dCTP deaminase
activity. Therefore the MJ1102 gene encodes a dUTP diphosphatase.
Sequence Alignment of MjDUT-DCD Homologs--
Fig. 3 shows the
amino acid sequences of MjDCD-DUT and MJ1102 proteins aligned with
sequences from homologous DCD proteins and four previously
characterized DUT enzymes. All of the sequences share conserved
residues in five previously defined sequence motifs (10, 16). Crystal
structure models of homotrimeric DUT proteins show each active site is
comprised of residues from motifs 2, 3, 4, or 5 contributed by all
three subunits. We predict that the hexameric MjDCD-DUT protein adopts
a similar fold and active site geometry. Despite the previous
identification of a set of residues specific to DCD but not DUT
homologs (10), few of those amino acid positions are conserved in an
alignment containing more diverse sequences (Fig. 3). Instead, residues
in the carboxy-terminal region of the DCD homologs distinguish DCD from
DUT proteins and may act alongside the Glu145 residue to
confer deaminase activity on DCD enzymes.
 |
DISCUSSION |
Previously, two pathways were known to convert deoxycytidine
nucleotides to dUMP for thymidine biosynthesis. Eukaryotes and some
microorganisms dephosphorylate deoxycytidine nucleotides to form dCMP
and then deaminate the nucleotide monophosphate using a
zinc-dependent deaminase (15, 29). This pathway does not produce dUTP, so the cells need only sequester dCMP and dUMP from pyrimidine nucleotide monophosphate kinases to avoid futile cycles. On
the other hand, many bacteria use the DCD protein to deaminate dCTP and
the DUT protein to rapidly hydrolyze dUTP to form dUMP. These organisms
require DUT proteins with high affinity for dUTP to avoid accumulating
that toxic intermediate (Table III). M. jannaschii has
circumvented the problem of dUTP accumulation by directly coupling dCTP
deamination to the release of pyrophosphate using the MjDCD-DUT enzyme
described here. This strategy of sequestering dUTP is similar to the
use of multifunctional, substrate channeling enzyme complexes by
eukaryotes to catalyze the initial steps in pyrimidine biosynthesis
(30, 31).
Like the S. typhimurium DCD protein, MjDCD-DUT activity is
inhibited by dTTP (15). Although the mechanism of this inhibition is
not yet clear, dTTP concentrations may regulate the pathway by feedback
inhibition. While some DUT proteins are inhibited by dTTP, they
hydrolyze dTTP and dCTP with much lower efficiency than dUTP (12).
Therefore members of the DCD/DUT family effectively discriminate among
pyrimidine nucleotides.
Having a bifunctional enzyme that sequesters the dUTP intermediate does
not relieve M. jannaschii of the need to hydrolyze free dUTP
produced by spontaneous deamination and pyrimidine nucleotide kinase
activity. Therefore that organism also has a dedicated dUTP
diphosphatase. Although M. jannaschii DUT has a higher
affinity for dUTP than does MjDCD-DUT, its affinity is still much lower than that of the E. coli or human DUT enzymes (Table III).
If M. jannaschii DUT has similar kinetics in
vivo, then the cells may require relatively high concentrations of
DUT to maintain low dUTP levels during DNA replication.
Although the order of the hydrolytic reactions has yet to be
determined, MjDCD-DUT's ability to hydrolyze dUTP to dUMP and pyrophosphate, but not to deaminate dCMP to dUMP, indicates that the
reaction likely proceeds in the order of dCTP
dUTP
dUMP. Similar metal activation profiles for dCTP deaminase and dUTP diphosphatase activities, along with the results of a competition assay, suggest that deamination and pyrophosphate release occur at the
same binding site. The fact that MjDCD-DUT E145Q protein has no
deaminase activity but can still catalyze the release of pyrophosphate
supports a model of both enzymatic reactions occurring at the same
substrate binding site, but independently.
Both the affinity for dCTP and the turnover of MjDCD-DUT are much
higher than those of the homologous S. typhimurium dCTP deaminase (Table III). In contrast to the cooperative kinetics observed
for the S. typhimurium DCD enzyme, MjDCD-DUT kinetics show
no sigmoidal relationship between dCTP concentration and the rate of
deamination. On the other hand, both enzymes require divalent cation
metals and the effective substrate for the enzymes is likely a
Mg2+-dCTP complex. Unlike the cytidine deaminase (26) or
adenosine deaminase (32, 33), MjDCD-DUT does not use a zinc-activated hydroxide to attack C-4 of the cytosine ring. The analogous mechanism of hydroxide activation by MjDCD-DUT is not yet known; however, the
enzyme may use either an activated Mg2+-H2O
nucleophilic complex or the Glu145 side chain carboxylate
could serve as a general base, abstracting a proton from water. The
loss of deamination activity in the MjDCD-DUT E145Q variant protein
indicates that this residue is essential for deaminase activity. This
glutamate residue is conserved in all DCD homologs, but not in most DUT
homologs, except for crenarchaeal DUT proteins (Fig. 3). Therefore
Glu145 is necessary but may not be sufficient for deaminase
activity. In addition, we found that residues in motif 5 of DCD
proteins differ from those in DUT homologs. In DUT crystal structure
models, positions of this glycine-rich carboxy-terminal domain vary.
Only in substrate-bound human and viral DUT structures did this domain become ordered (in a closed enzyme conformation), positioning the
residues near the active site (12, 34). Additional studies will be
needed to determine the contribution of this domain to the MjDCD-DUT
enzyme's activities.
Unlike the bacterial dCTP deaminase, the MjDCD-DUT enzyme also has dUTP
diphosphatase activity. Crystal structure models of DUT show that each
of three active sites in the homotrimer is comprised of residues at the
interface of all three subunits. One conserved acidic residue,
equivalent to Asp135 of MjDCD-DUT, forms hydrogen bonds to
tightly bound water molecules in the active site. It has been suggested
that this residue is also involved in catalysis, activating water by
proton abstraction (35, 36). A structural model of equine infectious
anemia virus DUT shows that the analogous aspartate side chain forms a
hydrogen bond with a main chain amide group from an adjacent subunit,
stabilizing the protein's oligomeric structure (14). The variant
protein MjDCD-DUT D135N is unable to catalyze either the dCTP deaminase or the dUTP diphosphatase reaction. Because results from gel-filtration chromatography suggest that the altered protein does not adopt the same
quaternary structure as the wild-type enzyme, the specific role of
Asp135 remains to be determined.
A molecular phylogeny of the DUT and DCD homologs implies that dCTP
deaminase activity evolved from a dUTP diphosphatase after a single
gene duplication event (10). Although both dut and dcd genes have been vertically inherited in many organismal
lineages, phylogenies indicate numerous instances of horizontal gene
transfer, which may have been mediated by viral vectors (37). Yet these phylogenies did not explain the evolution of deaminase activity in DCD,
presumably from an ancestral gene encoding a dUTP diphosphatase. The
bifunctional activity of the MjDCD-DUT protein described here illustrates how a superfamily of enzymes sharing a common partial reaction has evolved into a suprafamily of enzymes that catalyze mechanistically distinct reactions (38).