Tài liệu Báo cáo khoa học: Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP binding to an inactive form of the enzyme ppt

Regulation of dCTP deaminase from Escherichia coli by
nonallosteric dTTP binding to an inactive form of the
enzyme
Eva Johansson
1,2
, Majbritt Thymark
1
, Julie H. Bynck
1
, Mathias Fanø
3
, Sine Larsen
1,2
and Martin Willemoe
¨
s
3
1 Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Denmark
2 European Synchrotron Radiation Facility, Grenoble, France
3 Department of Molecular Biology, University of Copenhagen, Denmark
Synthesis of dTMP by thymidylate synthase proceeds
by the reductive methylation of dUMP, which is
obtained via one of two parallel pathways. One path-
way, considered to be a minor supplier of dTTP [1–3],
involves the reduction of UDP (UTP) by the action of
ribonucleotide reductase. Subsequently, dUDP is phos-
phorylated to dUTP and cleaved to dUMP. The main
supply of dUMP, however, involves the deamination
Keywords
deoxynucleotide metabolism; dUTP; enzyme
regulation; hysteresis; deamination

Correspondence
E. Johansson, Diabetes Protein Engineering,
Novo Nordisk A ⁄ S, Novo Nordisk Park,
DK-2760 Ma
˚
løv, Denmark
Fax: +45 4444 4256
Tel: +45 4442 1189
E-mail:
or
M. Willemoe
¨
s, Department of Molecular
Biology, University of Copenhagen, Ole
Maaløes vej 5, DK-2200 Copenhagen N,
Denmark
Fax: +45 3532 2128
Tel: +45 3532 2030
E-mail:
Database
The atomic coordinates and structure fac-
tors have been deposited in the Protein
Data Bank with the PDB ID codes 2j4q
(E138:dTTP) and 2j4 h (H121A:dCTP) and
can be accessed at
(Received 12 April 2007, revised 12 June
2007, accepted 18 June 2007)
doi:10.1111/j.1742-4658.2007.05945.x
The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP
by the structurally closely related dUTPase. This pathway provides

70–80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP
deaminase is regulated by dTTP, which increases the substrate concentra-
tion for half-maximal activity and the cooperativity of dCTP saturation.
Likewise, increasing concentrations of dCTP increase the cooperativity of
dTTP inhibition. Previous structural studies showed that the complexes of
inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type
enzyme with dUTP bound were all highly similar and characterized by hav-
ing an ordered C-terminal. When comparing with a new structure in which
dTTP is bound to the active site of E138A, the region between Val120 and
His125 was found to be in a new conformation. This and the previous con-
formation were mutually exclusive within the trimer. Also, the dCTP com-
plex of the inactive H121A was found to have residues 120–125 in this new
conformation, indicating that it renders the enzyme inactive. The C-ter-
minal fold was found to be disordered for both new complexes. We suggest
that the cooperative kinetics are imposed by a dTTP-dependent lag of
product formation observed in presteady-state kinetics. This lag may be
derived from a slow equilibration between an inactive and an active confor-
mation of dCTP deaminase represented by the dTTP complex and the
dUTP ⁄ dCTP complex, respectively. The dCTP deaminase then resembles a
simple concerted system subjected to effector binding, but without the use
of an allosteric site.
Abbreviations
E138A, mutant dCTP deaminase with a Glu138 to Ala substitution; H121A, mutant dCTP deaminase with a His121 to Ala substitution;
V122G, mutant dCTP deaminase with a Val122 to Gly substitution.
4188 FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS
of a deoxycytidine nucleotide [1–3]. In eukaryotes and
most of the well-studied Gram-positive bacteria (e.g.
Bacillus subtilis) a dCMP deaminase supplies dUMP
directly by deamination of dCMP [4–6]. dCMP deami-
nase is structurally related to cytosine- and cytidine

deaminases [7], which are all metallo enzymes [8–10].
In other prokaryotes, dUMP is derived mainly from
dCTP. In Escherichia coli [11] and Salmonella enterica
serovar Typhimurium [3,12] a dCTP deaminase produ-
ces dUTP, which is subsequently cleaved by dUTPase
to dUMP. In the archaeon Methanocaldococcus janna-
schii, a bifunctional dCTP deaminase:dUTPase has
been identified. This enzyme produces dUMP directly
from dCTP by catalysing both the deamination and
the triphosphate cleavage reaction within the same act-
ive site [13,14].
Monofunctional and bifunctional dCTP deaminases
are both structurally closely related to trimeric
dUTPases. They belong to the same superfamily and
form trimers of identical subunits [15–18]. In accord-
ance with the ‘branch point’ position in deoxynucleo-
tide metabolism and in particular in dTTP synthesis,
both the dCMP- and the dCTP deaminases are
inhibited by dTTP [5,12]. For dCMP deaminase,
dTTP regulation occurs by binding of the inhibitor
to an allosteric site in competition with the activator
dCTP [7]. For dCTP deaminase, the mechanism of
dTTP regulation is not understood. Only dCTP
deaminases can bind dTTP, whereas the closely
related dUTPase has very low affinity for this nuc-
leotide in a concentration range of several orders of
magnitude above physiological levels [19,20]. This
selectivity against dTTP (and dCTP) in the dUTPase
active site is obviously to avoid the breakdown of
important deoxyribonucleotides while facilitating the

extremely important removal of the toxic intermedi-
ate, dUTP [21–23].
Kinetic analysis of dCTP deaminase from S. ent-
erica serovar Typhimurium showed competitive
inhibition of dCTP binding by dTTP. However, the
presence of dTTP in the assay incubation also
increased the apparent cooperativity of dCTP bind-
ing, which indicates that the mechanism of dTTP
inhibition is not caused only by a trivial competition
between substrate and inhibitor for binding to the
same site [12]. We have previously determined the
structures of wild-type dCTP deaminase in complex
with dUTP and the inactive E138A mutant protein
in complex with dUTP and dCTP. In all cases, we
observed an ordered C-terminal that was closed over
the active site, but in a different conformation to
that observed for dUTPase. For both the mono- and
bifunctional dCTP deaminases, as we show here, and
the dUTPase [24], the C-terminal fold is important
for the formation of a catalytically competent com-
plex by closing the active site, but not for binding
of the substrates. In this study, we present results
from structural and mechanistic studies on dTTP
inhibition of E. coli dCTP deaminase. Coordinated
closure of the active site and rearrangement of the
main chain and side chains in the active site appear
as key players in a slow transformation from an
inactive to an active enzyme. dTTP inhibition may
then be achieved by stabilizing the inactive form of
presumably both the mono- and bifunctional dCTP

deaminases.
Results
Structure analysis
The E138A E. coli dCTP deaminase variant in com-
plex with dTTP crystallized in space group P6
3
22. The
structure was determined using the molecular replace-
ment technique with wild-type E. coli dCTP deaminase
in complex with dUTP as a search model that was pre-
viously crystallized in space group P2
1
[18] (Protein
Data Bank code 1XS1). The two different crystal
forms were obtained under similar conditions using
PEG400 as precipitant. The structure forms a homo-
trimer that exists in two copies in the E138A:dTTP
structure. The two copies are designated A and B,
originating from the A and B chains in the structure.
dTTP binds at the site of the protein shown previously
to bind the nucleotides dUTP and dCTP in wild-type
and the E138A variant [18]. The nucleotide-binding
site is positioned between two of the subunits giving
rise to three active sites per trimer. In the previously
determined structures of the enzyme, the C-terminal
amino acid residues from one of these subunits are
folded to form a lid over the active site which interacts
with the bound nucleoside triphosphate. In the struc-
ture in which dTTP is bound, the C-terminal amino
acid residues are disordered and not visible in the elec-

tron-density map (Fig. 1). Furthermore, the c-phos-
phate of dTTP is not visible in the electron-density
map and a magnesium ion is only seen bound to the
phosphates of dTTP in one of the two subunits. Large
movement of a helix 2 (residues 55–65) [18] is also
observed. If the C-terminal residues had been folded
over the active site, as shown in previous structures of
E. coli dCTP deaminase, these residues would have
coincided with a helix 2 in this new position (Fig. 1B).
A loop containing active-site residues in the interior of
the enzyme (residues 120–125) is also totally different
compared with previously determined E. coli dCTP
E. Johansson et al. dTTP inhibition of dCTP deaminase
FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS 4189
deaminase structures (Figs 1B, 2 and 3). Interestingly,
the crystal structure of the other inactive mutant
enzyme H121A in complex with dCTP was very
similar to the structure of E138A in complex with
dTTP. In the H121A complex we also observed a dis-
ordered C-terminus and rearrangement of active-site
residues 120–125 that was almost identical to the
E138A complex (Fig. 2D,E)). Wild-type E. coli dCTP
deaminase in complex with dTTP crystallized in the
same form and under similar conditions as for the
E138A:dTTP and H121A:dCTP complexes. However,
resolution of the diffraction data for the wild-type
enzyme in complex with dTTP was poor (3.5 A
˚
). As a
result, details of the active site of the wild-type:dTTP

complex were not as informative as for the E138A
variant, although the active-site structure of the wild-
type complex was reminiscent of this.
Enzyme kinetics and equilibrium binding
Figure 4A shows the results from a steady-state kinetic
analysis of dTTP inhibition of dCTP deaminase by
varying dCTP in the absence or presence of 100 lm
dTTP. As found previously for the enzyme from
S. enterica serovar Typhimurium [12], the cooperativity
of dCTP saturation increased in the presence of dTTP.
The Hill coefficient, n, increased from ~ 1.5 to 3, the
apparent half-saturation constant, S
0.5
, increased 2.5-
fold and k
cat
remained the same as in the absence of
dTTP. When dTTP was varied in the presence of a
constant saturating or unsaturating concentration of
dCTP, inhibition was cooperative and the dTTP con-
centration for 50% inhibition, I
0.5
, and the correspond-
ing Hill coefficient increased with the increase in dCTP
concentration (Fig. 4B).
A
B
Fig. 1. Comparison of dCTP deaminase
structures. (A) Superposition of the E138A
trimer in complex with dCTP (grey) and dTTP

(yellow, cyan and magenta). (B) Stereo-
view of a superposition of one of the sub-
units of the E138A variant of E. coli dCTP
deaminase in complex with dTTP (yellow;
chain B), dUTP (cyan; Protein Data Bank
entry 1XS4, chain A), or dCTP (magenta;
Protein Data Bank entry 1XS6, chain A). The
nucleotides are shown in ball and stick rep-
resentations and the magnesium ions as
spheres. The N-terminus (N) and the extent
to which the C-termini were resolved in the
dTTP complex (C1) and the dCTP ⁄ dUTP
complexes (C2) are indicated. The solid
arrow points to the region of a helix 2 and
b strand 5 that moved towards the active
site in the absence of an ordered C-terminal
fold. The dotted arrow points to the region
in the active site constituted by residues
120–125 that deviated in position between
the dCTP ⁄ dUTP complexes and the dTTP
complex. The figure was created using
PYMOL (DeLano Scientific, San Carlos, CA).
dTTP inhibition of dCTP deaminase E. Johansson et al.
4190 FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS
Analysis of the presteady-state kinetic behaviour of
dCTP deaminase using rapid quench-flow experiments
showed a lag in the progress of product formation
(Fig. 4C). This lag, which indicates slow activation of
the enzyme upon substrate binding prior to the forma-
tion of a catalytic complex, increased in the presence

AC
D
E
B
Fig. 2. Electron-density maps and close up stereoview of residues 120–124, 138 and nucleotides in the active site of E. coli dCTP deami-
nase and mutant enzymes. Electron-density maps for the (A) E138A dTTP complex and (B) H121A dCTP complex where the blue mesh rep-
resents the 2F
o
) F
c
map contoured at 1 r and the green mesh represents the F
o
) F
c
electron density map contoured at 3 r. (C)
Superposition of the structures of E138A in complex with dTTP (yellow; chain B), and the wild-type enzyme in complex with dUTP (cyan;
Protein Data Bank entry 1XS1, chain A). Wat5 is the proposed catalytic water molecule. (D) Superposition of the structures of H121A in
complex with dCTP (magenta; chain B), and the wild-type enzyme in complex with dUTP (cyan; Protein Data Bank entry 1XS1, chain A).
Wat5 is the proposed catalytic water molecule. (E) Superposition of the structures of E138A in complex with dTTP (yellow; chain B) and
H121A in complex with dCTP (magenta; chain B).The figures were created using
PYMOL (DeLano Scientific).
E. Johansson et al. dTTP inhibition of dCTP deaminase
FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS 4191
of dTTP. Significant estimates of the initial velocity,
V
ini
, could not be obtained when fitting Eqn (4) to the
data. A fixed value of V
ini
to < 0.1 times the steady-

state velocity, V
ss
, greatly increased the errors of the
calculated constants in Eqn (4). Therefore, V
ini
was
fixed at 0 when performing the calculations. The late
data points obtained in the absence of dTTP showed a
deviation from linearity caused by beginning substrate
depletion (Fig. 4C) and were omitted from the calcula-
tions. Unfortunately, we were not able to perform
presteady-state experiments at subsaturating substrate
concentrations to fully characterize the kinetics of the
slow transition from inactive to active enzyme [25].
Attempts to do so were hampered by the experimental
requirement for high enzyme concentrations both in
terms of estimating the true free-ligand concentration
in the experiments and by rapid substrate depletion
resulting in an underestimation of V
ss
.
dTTP binding to dCTP deaminase was also
investigated by equilibrium binding. This revealed a
hyperbolic binding curve (Fig. 4D) with a stochiometry
of 1 : 1 of dTTP bound per subunit of dCTP
deaminase.
Mutational analysis of amino acid residues
involved in dTTP regulation of dCTP deaminase
The design of the mutant enzymes H121A and V122G
was inspired by the results from analysis of crystal

structures as discussed later. Both mutant enzymes
were produced in similar amounts as wild-type enzyme
and could be purified by the same procedure as for
wild-type enzyme. However, none of the mutant
enzymes displayed detectable activity.
Discussion
As mentioned, we have previously published the struc-
tures of wild-type dCTP deaminase in complex with
dUTP and the inactive mutant protein E138A in com-
plex with dUTP and dCTP [18]. In E138A the sugges-
ted catalytic base, Glu138, is replaced by alanyl.
Comparison between structures of the complexes of
wild-type and mutant dCTP deaminase revealed that
the E138A complexes provide a good model for the
interaction between dCTP deaminase and bound
ligand. The interactions with bound nucleotide are
Fig. 3. Close-up stereoview of the centre of
the homotrimer of E. coli dCTP deaminase
with focus on residues Val122 and Thr123.
(A) Superposition of E138A with dTTP
bound and wild-type enzyme with dUTP
bound suggested to represent the inactive
and active conformers of dCTP deaminase,
respectively. E138A in complex with dTTP is
shown in yellow and the wild-type enzyme
in complex with dUTP in cyan (Protein Data
Bank entry 1XS1, chain A). (B) Superposition
of the same region as above of the inactive
H121A in complex with dCTP shown in
magenta compared with the wild-type

enzyme in complex with dUTP in cyan (Pro-
tein Data Bank entry 1XS1, chain A). The
superposition demonstrates the likely struc-
tural incompatibility between the two con-
formers due to a clash of the side chains of
Val122 and Thr123 as indicate by the
arrows. The figure was created using
PYMOL
(DeLano Scientific).
dTTP inhibition of dCTP deaminase E. Johansson et al.
4192 FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS
similar with only small changes in the arrangement of
water molecules around the 4 position of the pyrimid-
ine ring [18]. In this study, we compared the structures
of dCTP deaminase, represented by E138A, in complex
with all three nucleotides that bind to the enzyme.
A superposition of the trimer of E138A with the
nucleotides dCTP or dTTP bound is shown in Fig. 1A.
Whereas the previously determined structures of
E138A in complex with dCTP or dUTP overall are
virtually identical [18], the new dTTP complex revealed
a disordered C-terminus. This difference between the
two types of complex is more easily reconciled in the
comparison of a single subunit of E138A in complex
with dUTP, dCTP or dTTP (Fig. 1B). In the dTTP
complex, the entrance to the active site had partly col-
lapsed caused by a movement of the lip formed by
a helix 2 and b strand 5 [18]. Apparently, movement of
the active site lip prevented binding of the C-terminal
residues over the active site, or the absence of the

C-terminal residues caused the movement of the lip
(Fig. 1B). In addition, the C
a
chain between amino
acid residues 120 and 125 (Fig. 2C) was rearranged in
the dTTP complex to accommodate the 5-methyl
group of the thymine moiety. As a result, the Ala124
carbonyl was moved from the 4-oxo ⁄ 4-amino group of
the bound nucleotide and the side chain of His121 was
flipped to a position where in the wild-type enzyme it
would intersect Glu138 and C4 of the pyrimidine ring
(Fig. 2C). In addition, the nucleophilic water molecule
[18], wat5, appeared in the dTTP complex to be
expelled by the His121 side chain from its position in
the dCTP(dUTP) complex between Glu138 and
the Ala124 carbonyl (Fig. 2C). Also, in the dTTP com-
plex the side chains of Thr123 and Val122 had moved
to new positions. The significance of this last observa-
tion is that due to the proximity of residues 120–125
from each subunit in the centre of the trimer, the side
chains of Val122 of one subunit and Thr123 of the
neighbouring subunit are likely to clash unless each
subunit is in the same conformation (Fig. 3A,B). As a
consequence, Thr123 and Val122 may mediate a
concerted switch between the dCTP(dUTP)-binding
conformer and the dTTP-binding conformer of dCTP
deaminase. We were not able to identify structural
changes in the main chain of the subunit, or in the
interaction of subunits within the trimer that linked
the conformation of residues 120–125 to the position

of the active site lip and closure of the C-terminal end
over the active site. However, it is reasonable to expect
these two events to be associated but the structural
change that mediates the communication between the
two regions appears to be very subtle.
Based on the observations described above, mutant
alleles encoding the enzymes H121A and V122G were
constructed to analyse the roles of His121 and Val122
in catalysis and regulation of dCTP deaminase.
Removal of the imidazole ring in H121A was anticipa-
ted to relieve or reduce inhibition by dTTP by prevent-
ing expulsion of the water molecule, wat5, as described
above (Fig. 2C). Replacing the Val122 side chain in
V122G aimed to relieve the suggested concerted struc-
tural transition of the trimer and perhaps reduce the
inhibition by dTTP. As mentioned in the results, the
AB
C
D
Fig. 4. Initial rate and presteady-state kinetics of dTTP inhibition
and dTTP binding to dCTP deaminase. Assays were performed as
described in Experimental procedures. (A) The concentration of
dCTP varied as indicated in the absence (closed circles) or pres-
ence (open circles) of 100 l
M dTTP. The kinetic constants calcula-
ted using Eqn (1) were (closed circles) k
cat
¼ 1.24 ± 0.09 s
)1
,

S
0.5
¼ 66 ± 9 lM, n ¼ 1.5 ± 0.3 and (open circles) k
cat
¼
1.20 ± 0.05 s
)1
, S
0.5
¼ 168 ± 8 lM, n ¼ 3.3 ± 0.4. (B) The dTTP
concentration varied as indicated in the presence of (open circles)
100 l
M dCTP and (closed circles) 500 lM dCTP. Kinetic constants
calculated using eqn (2) were (open circles) I
0.5
¼ 53 ± 6 lM and
n ¼ 1.31 ± 0.15 and (closed circles) I
0.5
¼ 826 ± 89 lM and n ¼
1.7 ± 0.3. (C) Presteady-state kinetics of dCTP deaminase. Experi-
ments were performed as described in Experimental procedures
with enzyme in the absence (closed circles) or presence (open cir-
cles) of dTTP. The kinetic parameters were calculated using Eqns
(4–6). The calculated constants were (closed circles) rate
ss
¼
0.79 ± 0.06 s
)1
with s ¼ 0.49 ± 0.13 s (k ¼ 2.0 s
)1

) and (open cir-
cles) rate
ss
¼ 0.16 ± 0.02 s
)1
with s ¼ 2.3 ± 0.5 s (k ¼ 0.43 s
)1
).
For comparison the straight lines represent the calculated steady-
state rate in the absence of a lag. (D) dTTP binding to dCTP
deaminase. Binding experiments were performed as described in
Experimental procedures. The nucleotide concentration varied as
indicated. The binding constants calculated using Eqn (3) were
N
max
¼ 1.01 ± 0.02 and K
d
¼ 35 ± 3 lM.
E. Johansson et al. dTTP inhibition of dCTP deaminase
FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS 4193
mutant proteins were both inactive and unfortunately
no suitable crystals for the structural analysis of
V122G could be obtained. However, the conformation
of the main chain in the region of residues 120–125 in
the H121A:dCTP complex was found to strongly devi-
ate from that of wild-type enzyme in complex with
dUTP and almost superimpose with the same region in
the structure of the E138A:dTTP complex (Fig. 2E,D).
In addition, the H121A:dCTP complex, like the
E138A:dTTP complex, had a disordered C-terminal

fold, which again indicates a connection between the
position of residues 120–125 and folding of the C-ter-
minal.
We anticipate that the E138A:dTTP complex resem-
bles the binding of dTTP to wild-type enzyme, as
also expected from the crystallographic analysis of
the wild-type:dTTP described. Therefore, the lack of
activity of H121A and the structural similarity between
the H121A:dCTP and the E138A:dTTP complexes
(Fig. 2E) suggest a mechanism for dTTP inhibition
that not only acts by physical blocking of the active
site, but also through a concerted change to an inac-
tive conformation of the active sites in the trimer. The
observation that the complexes of H121A:dCTP and
E138A:dTTP are also very similar in terms of the posi-
tion of Val122 and Thr123 (Fig. 3) supports such a
mechanism. Interestingly, there are no indications as
to why the inhibited ⁄ inactive conformation should
exclude the binding of dCTP (or dUTP). This import-
ant observation is discussed below.
Obviously, there is competition between dCTP and
dTTP for binding to the active site, as revealed by the
crystal structures of the various complexes. Also, results
from kinetic experiments point to a competitive mech-
anism for dTTP inhibition; an increase in S
0.5
for dCTP
in the presence of dTTP (Fig. 4A) and an increase in
I
0.5

for dTTP with increasing dCTP concentrations
(Fig. 4B). From the equilibrium binding experiment
presented in Fig. 4D it can be seen that dTTP binds to
only one type of site with no cooperativity.
The lag observed in presteady-state kinetics shown
in Fig. 4C is a clear indication that the mechanism of
regulation of dCTP deaminase is not a simple rapid
equilibrium mechanism. The observed increase in
cooperativity of dTTP inhibition at increasing dCTP
concentrations (Fig. 4B), but complete absence of
cooperativity in equilibrium binding of dTTP
(Fig. 4D), indicates that the cooperativity effect of
dTTP inhibition is a kinetic phenomenon. Given the
right circumstances, a lag in the progress of product
formation is known to produce what is termed kinetic
cooperativity and several enzyme systems have been
shown to possess such properties [25–28]. The k for
activation of dCTP deaminase is of the same order of
magnitude as the k
cat
(Fig. 4A,), a condition that qual-
ifies for causing kinetic cooperativity, and very import-
ant, the lag is increased in the presence of dTTP. The
increase in cooperativity of dCTP saturation in the
presence of dTTP may therefore be explained by a
mechanism in which dTTP stabilizes an inactive form
that dominates the population of free enzyme, recall
that V
ini
(or rate

ini
) is likely to be less than V
ss
(or
rate
ss
) by an order of magnitude. Upon binding of
dCTP the proceeding structural changes in the active
site and proper folding of the C-terminus may contrib-
ute to the lag observed in presteady-state kinetics
(Fig. 4C).
Finally, it should be pointed out that each of the
two species-specific, but dominant, pathways for
dUMP synthesis described above are very similar from
a regulatory point of view. dCMP deaminase is activa-
ted by dCTP and inhibited by dTTP and both nucleo-
tides act on the enzyme by binding to an allosteric site
to alter the cooperativity of dCMP binding [5,29,30].
The activity of dCTP deaminase depends on the con-
centration of dCTP and is inhibited by dTTP. Our
results suggest that regulation of dCTP deaminase is
not by a conventional allosteric mechanism, but appar-
ently utilizes the property of the enzyme to exist in
two conformations and that dTTP stabilizes the inac-
tive form by binding to the active site. In this way,
dCTP deaminase can use one nucleotide-binding site
to gain a pseudo-allosteric mechanism of regulation
that generates the apparently attractive feature of an
increase in both S
0.5

and the sigmoidity of the satura-
tion curve for dCTP in response to the binding of
dTTP to the enzyme.
Experimental procedures
Materials
All buffers, nucleotides and salts were obtained from Sig-
ma-Aldrich (Darmstadt, Germany). Radioactive nucleotides
were obtained as ammonium salts from Amersham Bio-
sciences (Hillerød, Denmark). TLC was performed with
poly(ethylene-imine)-coated cellulose plates from Merck
(Darmstadt, Germany).
Molecular biology and protein methods
Construction of mutant alleles of the dcd gene encoding the
dCTP deaminases H121A and V122G was achieved by
performing the QuikChange method (Stratagene, La Jolla,
CA) using the oligo-deoxynucleotides, where underlined
letters indicate the site of mutagenesis: H121A5–3,
dTTP inhibition of dCTP deaminase E. Johansson et al.
4194 FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS
GGGCTGATGGTGGCCGTCACCGCGCAC; H121A3–5,
GTGCGCGGTGACG
GCCACCATCAGCCC; V122G5–3,
GATGGTGCACG
GCACCGCGCACC; V122G3-5, GGT
GCGCGGTG
CCGTGCACCATC. The plasmid pETDCD
described previously [18] was used as a template for muta-
genesis. The pETDCD plasmid contains the reading frame
of the E. coli dcd gene under control of the late T7 promo-
ter in the vector pET11a (Novagen, Darmstadt, Germany).

All mutations were verified by sequencing of the entire dcd
reading frame on an ABI PRISM 310 sequencer according
to the supplier’s manual. Wild-type and mutant protein was
produced and purified as described previously [18].
Enzyme kinetics and equilibrium binding
experiments
Initial velocities were obtained at 37 °C using TLC and
subsequent liquid scintillation counting to first separate and
then quantify [5-
3
H] dUTP produced from [5-
3
H] dCTP, as
described in detail previously [14]. Data were recorded over
5 min at two enzyme concentrations (50–100 nm) and the
assay incubations contained in addition to varying concen-
trations of the nucleotides dCTP and dTTP, as shown
under results, 50 mm Hepes, pH 6.8, 2 mm MgCl
2
and
2mm dithiothreitol. Presteady-state experiments were per-
formed at 37 ° C using a KinTek RQF-3 rapid quench flow
instrument by mixing dCTP deaminase (20 lm) in the pres-
ence or absence of 100 lm dTTP and 150 lm [5-
3
H] dCTP
in 50 mm Hepes, pH 6.8, 2 mm MgCl
2
and 2 mm dithio-
threitol at time 0 and quenching the reaction with 3 m

formic acid at the time points given under results. Subse-
quently, the samples representing each time point were sub-
jected to TLC and analysed for the distribution of
radioactivity in spots of [5-
3
H] dCTP and [5-
3
H] dUTP as
above for steady-state kinetic samples.
In equilibrium binding experiments, the incubations con-
tained dCTP deaminase (50–100 lm), 50 mm Hepes,
pH 6.8, 2 m m MgCl
2
and between 0 and 320 lm [methyl-
3
H]
dTTP. Free nucleotide was separated from bound using
Amicon Ultrafree-MC 30.000 NMWL centrifugal filter
devices, as described previously [31,32]. Samples represent-
ing free and total radioactive nucleotide were washed by
TLC in 1 m acetic acid, cut out and quantified by liquid
scintillation as above for samples from kinetic experiments.
Data from presteady-state and steady-state kinetic and
equilibrium binding experiments were analysed using the
computer program ultrafit from biosoft (v. 3.0). The
equations used were: the Hill equation, Eqn (1), for sigmoid
saturation curves
rate ¼ k
cat
½S

n
=ðS
n
0:5
þ½S
n
Þð1Þ
where rate is the initial turnover of the enzyme with a
maximum of k
cat
, S
0.5
is the concentration of substrate S at
half-maximal saturation of the enzyme and n is the Hill
coefficient. Equation (2) was used for sigmoid inhibition
rate
inh
¼ rate I
n
0:5
=ðI
n
0:5
þ½I
n
Þð2Þ
where rate
inh
is the initial rate corresponding to the presence
of a given concentration of inhibitor I and I

0.5
is the concen-
tration of inhibitor for half-maximal inhibition. Equation (3)
was used for hyperbolic binding of ligands to the enzyme
N ¼ N
max
½L=ðK
d
þ½LÞ ð3Þ
where N is the degree of binding with the dissociation con-
stant K
d
of ligand L to the enzyme with a maximal number
of binding sites N
max
. Equations (4–6) were used to analyse
the data recorded for presteady-state kinetics
P ¼ V
ss
t ÀðV
ss
À V
ini
Þð1 À e
Àt=s
Þs ð4Þ
rate
ss
¼ V
ss

=½Enzymeð5Þ
rate
ini
¼ V
ini
=½Enzymeð6Þ
where P is the product and V
ini
and V
ss
are the initial and
steady-state velocities (rate
ini
and rate
ss
are the corresponding
Table 1. Diffraction data and refinement statistics. Values within
parentheses are data for the highest resolution shell. R
merge
¼
S|I–<I>|⁄SI, where I is observed intensity and <I> is aver-
age intensity obtained from multiple observations of symmetry
related reflections. R
factor
¼ S
work
||F
obs
| ) k|F
calc

|| ⁄S
work
F
obs
. R
free
¼
S
test
||F
obs
| ) k|F
calc
|| ⁄S
test
F
obs
, where F
obs
and F
calc
are observed
and calculated structure factors, respectively, k is the scale factor,
and the sums are over all reflections in the working set and test
set, respectively. rmsd, root mean square deviation.
Diffraction data statistics
Protein:ligand E138A:dTTP H121A:dCTP
Space group P6
3
22 P6

3
22
Wavelength (A
˚
) 1.046 1.046
Resolution (A
˚
) 50–2.6 (2.74–2.6) 50–2.7 (2.85–2.7)
R
sym
(%) 13.5 (51.5) 12.3 (47.2)
<I> ⁄ r(I) 21.9 (2.6) 4.9 (1.3)
Completeness (%) 92.7 (70.0) 94.2 (75.1)
Multiplicity 12.2 (5.5) 10.0 (5.4)
No. reflections 147198 111034
No. unique reflections 12085 11131
Refinement statistics
No. reflections (total) 12020 10539
No. reflections (working
set)
11438 9976
No. reflections (test set) 582 563
No. atoms 2702 2706
Resolution (A
˚
) 30–2.6 (2.67–2.60) 30–2.7 (2.77–2.70)
R
factor
(%) 24.7 (30.1) 22.8 (31.7)
R

free
(%) 30.7 (32.0) 27.2 (31.4)
Average B
factor
(A
˚
2
)29 29
Bond length rms
from ideal (A
˚
)
0.016 0.014
Bond angle rmsd
from ideal (deg)
1.7 1.6
E. Johansson et al. dTTP inhibition of dCTP deaminase
FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS 4195
rates) prior to and after the transition of the enzyme to a
more active form, respectively, t is the time and s is the lag-
time. The rate constant, k, for the activation of the enzyme is
obtained as 1 ⁄ s.
Crystallization
Crystals were grown in hanging drops as described previ-
ously [18] using the vapour-diffusion technique with hang-
ing drops. Protein solutions contained 3.7 or 5.1 mgÆmL
)1
protein for H121A and E138A mutant enzymes, respect-
ively, as well as 5 mm dCTP (H121A) or dTTP (E138A)
and 20 mm magnesium chloride in 50 mm Hepes, pH 6.8.

This solution was mixed in equal amounts with the mother
liquor (2 lL+2lL) that consisted of 34% poly(ethylene
glycol 400), 0.2 m magnesium chloride and 0.1 m Hepes,
pH 7.5 and the drop was equilibrated over 1 mL of mother
liquor at room temperature. Long (> 1 mm) needle-formed
crystals appeared after one week.
Diffraction data collection
Diffraction data were collected on cryo-cooled crystals
(100 K) at beam-line I911-2 at MAX-LAB (Lund, Sweden)
using a MarMosaic 225 CCD detector from MAR
Research. Auto-indexing and integration of the data were
performed with mosflm [33] and scala [34] was used for
scaling. All the crystals belonged to space group P6
3
22
with cell dimensions a ¼ b ¼ 61.6, c ¼ 244.8 A
˚
(E138A +
dTTP) and diffracted X-rays well. However, the long c-axis
prevented collection of high-resolution data.
Structure determination and refinement
The structure of the E138A mutant enzyme cocrystallized
with dTTP was determined with the molecular replace-
ment technique using the program amore [35]. The chain
A of wild-type dCTP deaminase in complex with dUTP
(Protein Data Bank entry 1XS1) stripped from ligands
and water molecules, was used as a search model. The
correct solution contained two molecules in the asymmet-
ric unit that each forms a separate trimeric structure as a
result of the crystal symmetry. The initial difference elec-

tron-density maps revealed density for the nucleotide
which was built using the program o [36]. However, the
final model only showed electron density for a magnesium
ion in one of the molecules of the asymmetric unit (chain
B) and there was no electron density visible for the
c-phosphate of dTTP. Therefore, the structure was
modelled with dTDP in the active sites. There was no
electron density for the C-terminal 20 amino acid residues
that were omitted from the model. Initially, the stretch of
amino acid residues from 121 to 125 was also unclear and
was excluded from the model. Cycles of refinement using
noncrystallographic symmetry restraints with refmac5 [37]
and model building with o [36] were performed and now
enabled model building of the 121–125 stretch and a new
position of a helix 2, containing residues 55–65 in one of
the molecules in the asymmetric unit (chain B). Further-
more, the model contains residues 1–174 in chain A, resi-
dues 1–171 in chain B and three water molecules in each
chain. The structure of the H121A mutant enzyme cocrys-
tallized with dCTP was determined using difference Fou-
rier techniques with the model of the E138A protein
crystallized in the same space group (P6
3
22). Refinement
and model building proceeded as for E138A cocrystallized
with dTTP. The final model includes residues 1–171 in
chain A, residues 1–174 in chain B and two magnesium
ions, both coordinated to the modelled dCDP, because
there was no electron density for the c-phosphate of
dCTP. Data and refinement statistics are shown in

Table 1. The quality of the models was checked with pro-
check [38] and whatif [39]. The Ramachandran plot has
91.2% of the residues in the most favoured regions. There
are no residues in the disallowed regions and 0.7% of the
residues are in the generously allowed regions (correspond-
ing to His121 in both chains) for the E138A mutant struc-
ture cocrystallized with dTTP. For the H121A structure,
92.3% of the residues are in the most favoured regions
and Ala121 and Val122 form a cis-peptide that puts them
in the generously allowed and disallowed regions, respect-
ively, of the Ramachandran plot.
Acknowledgements
We are grateful for the beam time provided at MAX-
LAB (Lund, Sweden). This study was supported by
the Danish Natural Science Council through a grant
and a contribution to DANSYNC. We acknowledge
the support by the European Community – Research
Infrastructure Action under the FP6 programme
‘Structuring the European Research Area’.
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