Tài liệu Báo cáo khoa học: Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase Investigations of the putative catalytic glutamate–arginine pair and of residues responsible for substrate specificity docx

Functional studies of active-site mutants from Drosophila
melanogaster deoxyribonucleoside kinase
Investigations of the putative catalytic glutamate–arginine pair and
of residues responsible for substrate specificity
Louise Egeblad-Welin
1,2,*
, Yonathan Sonntag
1,*
, Hans Eklund
3
and Birgitte Munch-Petersen
1
1 Department of Science, Systems and Models, Roskilde University, Denmark
2 Department of Molecular Biosciences, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden
3 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden
Drosophila melanogaster deoxyribonucleoside kinase
(Dm-dNK) phosphorylates the four natural deoxyribo-
nucleosides, thymidine, deoxycytidine, deoxyadenosine
and deoxyguanosine, which is a crucial step in the bio-
synthesis of DNA precursors via the salvage pathway.
In addition, Dm-dNK phosphorylates a number of
important nucleoside analogue pro-drugs [1,2], making
it a potential candidate for use in suicide gene therapy.
Keywords
catalytic mechanism; deoxyribonucleoside
kinase; dTTP; enzyme kinetics; nucleoside
analogues
Correspondence
B. Munch-Petersen, Department of Science,
Systems and Models, Roskilde University,
Box 260, DK 4000 Roskilde, Denmark

Fax: +45 46743011
Tel: +45 46742418
E-mail:
L. Egeblad-Welin, Department of Molecular
Biosciences, Swedish University of
Agricultural Sciences, Box 575, Biomedical
Center, S-751 25 Uppsala, Sweden
Fax: +46 18536971
Tel. +46 184714192
E-mail:
*These authors contributed equally to this
work
(Received 2 November 2006, revised 4
January 2007, accepted 16 January 2007)
doi:10.1111/j.1742-4658.2007.05701.x
The catalytic reaction mechanism and binding of substrates was investi-
gated for the multisubstrate Drosophila melanogaster deoxyribonucleoside
kinase. Mutation of E52 to D, Q and H plus mutations of R105 to K and
H were performed to investigate the proposed catalytic reaction mech-
anism, in which E52 acts as an initiating base and R105 is thought to sta-
bilize the transition state of the reaction. Mutant enzymes (E52D, E52H
and R105H) showed a markedly decreased k
cat
, while the catalytic activity
of E52Q and R105K was abolished. The E52D mutant was crystallized
with its feedback inhibitor dTTP. The backbone conformation remained
unchanged, and coordination between D52 and the dTTP–Mg complex
was observed. The observed decrease in k
cat
for E52D was most likely due

to an increased distance between the catalytic carboxyl group and 5¢-OH of
deoxythymidine (dThd) or deoxycytidine (dCyd). Mutation of Q81 to N
and Y70 to W was carried out to investigate substrate binding. The muta-
tions primarily affected the K
m
values, whereas the k
cat
values were of the
same magnitude as for the wild-type. The Y70W mutation made the
enzyme lose activity towards purines and negative cooperativity towards
dThd and dCyd was observed. The Q81N mutation showed a 200- and
100-fold increase in K
m
, whereas k
cat
was decreased five- and twofold for
dThd and dCyd, respectively, supporting a role in substrate binding. These
observations give insight into the mechanisms of substrate binding and
catalysis, which is important for developing novel suicide genes and drugs
for use in gene therapy.
Abbreviations
ACV, 9-(2-hydroxyethoxymethyl)-guanine; AraA, 9-(b-
D-arabinofuranosyl)-adenine; AraC, 1-(b-D-arabinofuranosyl)-cytosine; AraT,
1-(b-
D-arabinofuranosyl)-thymine; BVDU, (E)-bromvinyl-2¢-deoxyuridine; CdA, 2-chloro-2¢-deoxyadenosine; dAdo, deoxyadenosine; dCK,
cytosolic deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine; Dm-dNK, Drosophila
melanogaster deoxyribonucleoside kinase; dThd, deoxythymidine; F-AraA, 2-flouro-9-(b-
D-arabinofuranosyl)-adenine; FdUrd, 5-flouro-2¢-
deoxyuridine; HSV1-TK, Herpes simplex virus Type 1 thymidine kinase; TK, thymidine kinase.
1542 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS

Sequence alignments and structural studies suggest
that Dm-dNK belongs to the family comprising deoxy-
guanosine kinase (dGK), deoxycytidine kinase (dCK),
thymidine kinase 2 (TK2) and herpes simplex virus
type 1 thymidine kinase (HSV1-TK) (Fig. 1) [3]. The
structures of Dm-dNK, human dGK and human dCK
were determined a few years ago [4,5]; although the
structure of HSV1-TK has been known for several
years, the first structures being solved in 1995 [6,7].
The structure of human thymidine kinase 1 was solved
recently, and was shown to belong to a structural fam-
ily of its own [8,9].
Fig. 1. Structural alignment of Dm-dNK, TK2, dGK, dCK and HSV1-TK. Mutated amino acids are marked by their numbers in the Dm-dNK
sequence. Alignment of Dm-dNK, TK2, dGK and dCK was carried out with
CLUSTALW ( Alignment of HSV1-TK
was carried out by structural comparison with the Dm-dNK structure in
O [26].
L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase
FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1543
Human deoxyribonucleoside kinases are targets for
the chemotherapeutic treatment of cancer and viral
diseases because they catalyse the addition of the first
phosphate group to the nucleoside analogue. This
primes the nucleoside for further phosphorylation to
the corresponding triphosphate nucleoside, converting
the pro-drug to the active cytotoxic drug. The nucleo-
side analogue can be incorporated into the DNA chain
and cause chain termination, induce apoptosis or inhi-
bit DNA polymerase [10,11]. Because of the high cata-
lytic rate and the broad substrate specificity, it has

been suggested that Dm-dNK may be a putative sui-
cide gene in gene therapy. In vivo experiments with
cancer cell lines showed increased sensitivity towards
nucleoside analogues [12] and a bystander effect was
observed [13,14].
The 3D structures of Dm-dNK, human dCK, human
dGK and HSV1-TK show a similar binding mode for
the substrates in the active site. Three key residues in
Dm-dNK, identified and proposed as being responsible
for substrate specificity [4], were mutagenized and the
mutant enzymes characterized for their ability to phos-
phorylate native deoxyribonucleosides and nucleoside
analogues [15]. These mutations of residues 84, 88 and
110 (Fig. 2) converted dNK substrate specificity from
predominantly pyrimidine into purine.
It has been suggested that the reaction mechanism
proposed for HSV1-TK [16] also applies to other deoxy-
ribonucleoside kinases [3]. It is believed that E52 acts
as a base in the deprotonation of 5¢-OH, while the
transition state is stabilized by the positively charged
R105 (Fig. 2). The pK
a
of E52 is probably influenced
by the proximity of R105 which is high enough to act
as a base in the initial catalysis step. In a structural
study of Dm-dNK in which the enzyme was cocrystal-
lized with both deoxythymidine (dThd) and dTTP sep-
arately, E52 formed a hydrogen bond with the 5¢-OH
group of dThd, whereas it was moved 6.5 A
˚

in the
dTTP complex and coordinated the Mg ion. R105 is
also affected; when dThd is bound R105 forms a
hydrogen bond with E52, thus stabilizing the position
and charge of E52, and when dTTP is bound it forms
a hydrogen bond with the a-phosphate of dTTP
instead [17]. Knowledge regarding the enzymatic reac-
tion mechanisms is central to the design of mutant
enzymes or nucleoside analogues for use in suicide
gene therapy.
We investigated the catalytic mechanism by muta-
ting E52 to D, Q and H. Provided that the pro-
posed reaction mechanism [3] holds true, a profound
effect on the catalytic rate should be evident from
these mutations. Binding of the substrates to the
enzyme should not be altered to the same extent, as
reflected by the lower impact on K
m
values. Q is
similar in size to E but cannot function as a base,
whereas D and H should be able to act as a base
but the differences in their pK
a
values and size may
affect their efficiency. Likewise, mutations of R105K
and R105H to other positively charged residues are
expected to influence catalytic rate rather than sub-
strate binding.
Two further active site residues responsible for
substrate binding were investigated, these being Y70

and Q81 (Fig. 2). These two amino acid residues are
conserved among Dm-dNK, TK2, dGK, dCK and
HSV1-TK (Fig. 1). Y70 which anchors the 3¢-OH of the
deoxyribose moiety of the nucleoside, together with
E172 (Fig. 2), was mutated to W. This mutation was
performed to see whether the larger side chain would
affect substrate specificity. Q81, which forms two hydro-
gen bonds with the base, was mutated to N in order to
see how the increased distance between the base and
substrate-binding amino acid affected the binding of
dThd and deoxycytidine (dCyd).
Results
We performed site-directed mutagenesis of four active
site residues of Dm-dNK. Residue E52 was mutated to
D, H and Q, residue Y70 to W, residue Q81 to N and
residue R105 to K and H. The kinetic properties of
the active site mutants are summarized in Table 1. All
mutants were characterized with dThd and dCyd,
Fig. 2. Binding of the substrate dThd at the active site of Dm-dNK
[17]. Hydrogen-bonding residues are shown. E52, Y70, Q81 and
R105 were mutated in this study. Residues V84, M88 and A110,
mutated in a previous study [15], are also included.
Active site mutation of deoxyribonucleoside kinase L. Egeblad-Welin et al.
1544 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS
Y70W was further characterized with three nucleoside
analogues: 1-(b-d-arabinofuranosyl)-cytosine (AraC),
1-(b-d-arabinofuranosyl)-thymine (AraT) and (E)-brom-
vinyl-2¢-deoxyuridine (BVDU).
Mutants of catalytic residues: E52 and R105
It is evident from the kinetic results that mutation of

E52 changes only the catalytic rate. For the E52D
mutant the K
m
value was approximately the same as
for the wild-type with dThd, whereas k
cat
was  20 000
times lower. This was also the scenario for the E52H
mutant exhibiting k
cat
 1100 times lower than the
wild-type. The E52Q mutant did not show any meas-
urable activity.
Mutation of R105 also showed a decreased catalytic
rate. When mutated to K, the activity was lost com-
pletely with both dThd and dCyd as substrates. The
R105H mutant showed a slightly increased K
m
value,
sevenfold higher with dThd, and k
cat
was decreased by
 2000-fold. For dCyd as a substrate K
m
was  50-fold
higher and k
cat
was 275-fold lower.
Table 1. Kinetics of dThd and dCyd phosphorylation for active-site mutants of Dm-dNK. AraC, AraT and BVDU were tested only with the
Y70W mutant. k

cat
values were determined using a calculated mass of 26 785 kDa. It is assumed that there is one active site per monomer.
Where cooperativity is observed, the Hill coefficient (n) is given. When k
cat
⁄ K
m
is compared with the wild-type, dThd and dCyd is set to
100%. Kinetic parameters were determined from three independent experiments, except where indicated by * or # , which were based on
one or two experiments, respectively. The results are given as mean ± SD. ND, not detected.
Enzyme Substrate K
m
or K
0.5
(lM) (n) V
max
(lmolÆmin
)1
Æmg
)1
) k
cat
(s
)1
) k
cat
⁄ K
m
(s
)1
ÆM

)1
)
Wild-type
a
dThd 1.2 29.5 14.2 1.2 · 10
7
(100%)
dCyd 2.3 34.2 16.2 7.0 · 10
6
(100%)
AraC
b
24.3 5.6 2.9 1.2 · 10
5
AraT 62 ± 7.4 10.4 ± 0.7 0.3 7 · 10
4
BVDU 2.2 ± 0.1 13.2 ± 1.7 5.9 2.6 · 10
6
E52D dThd 3.8 ± 0.6 0.00162 ± 0.00002 7.2 · 10
)4
1.9 · 10
2
(< 1%)
dCyd 3.7 ± 0.4 0.00160 ± 0.00018 7.1 · 10
)4
1.9 · 10
2
(< 1%)
E52H dThd* 3.7 0.02612 1.2 · 10
)2

3.2 · 10
3
(< 1%)
dCyd 5.8 ± 2.0 0.00259 ± 0.0006 1.2 · 10
)3
2.0 · 10
2
(< 1%)
E52Q dThd ND ND – –
dCyd ND ND – –
Y70W dThd# 251 ± 86
(n ¼ 0.6 ± 0.07)
5.2 ± 0.9 2.3 9.2 · 10
3
(< 1%)
dCyd 246 ± 34
(n ¼ 0.76 ± 0.002)
15.2 ± 0.3 6.8 2.8 · 10
4
(< 1%)
AraC# 1441 ± 463 4.6 ± 0.6 2.1 1.4 · 10
3
(< 1%)
AraT# 357 ± 43 2.3 ± 0.1 1.0 2.9 · 10
3
(< 1%)
BVDU# 4.9 ± 0.4 0.82 ± 0.07 0.4 7.5 · 10
4
(< 1%)
Q81N dThd 231 ± 24 12.1 ± 0.5 5.4 2.3 · 10

4
(< 1%)
dCyd 205 ± 15 14.7 ± 2.0 6.6 3.2 · 10
4
(< 1%)
R105H dThd 8.9 ± 1.5 0.015 ± 0.0008 6.7 · 10
)3
7.5 · 10
2
(< 1%)
dCyd 113 ± 24 0.124 ± 0.027 5.5 · 10
)2
4.9 · 10
2
(< 1%)
R105K dThd ND ND – –
dCyd ND ND – –
a
[2].
b
[15].
L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase
FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1545
Mutants of substrate-binding residues: Y70 and
Q81
Y70W showed an increase in K
m
values for both dThd
and dCyd, of approximately 200- and 100-fold,
respectively. The k

cat
values decreased by approxi-
mately fivefold with dThd and twofold with dCyd. An
interesting feature for this mutant is that it gained neg-
ative cooperativity with dThd and dCyd. The Y70W
mutant was further characterized using the nucleoside
analogues, AraC, AraT and BVDU. K
m
for AraC was
increased 59-fold, but this was less pronounced than
the increase of 106-fold for dCyd. V
max
was fairly
unchanged compared with the wild-type. The K
m
value
for BVDU was increased approximately twofold com-
pared that for the wild-type, which indicated a minor
change in the binding of BVDU. By contrast, k
cat
was
decreased approximately 15-fold. With AraT, K
m
increased approximately sixfold and k
cat
decreased
approximately fivefold compared with the wild-type.
Thus, for Y70W, the changes in K
m
with these ana-

logues were less pronounced than for dThd and dCyd.
Overall, changing Y70 to W had a greater impact on
catalytic efficiency (k
cat
⁄ K
m
) with the natural substrates
than with the analogues. Q81N had an increased K
m
value for dThd and dCyd, of approximately 200- and
100-fold, respectively. The k
cat
values were only
decreased approximately twofold for both substrates.
Phosphorylation of nucleosides and nucleoside
analogues
Wild-type Dm-dNK and all mutants, with the exception
of E52H, were tested in a phosphotransferase assay
with natural nucleosides [dThd, dCyd, deoxyadenosine
(dAdo) and deoxyguanosine (dGuo)] and some nucleo-
side analogues [AraC, 2-chloro-2¢-deoxyadenosine (CdA),
2-flouro-9-(b-d-arabinofuranosyl)-adenine (F-AraA) and
5-flouro-2¢-deoxyuridine (FdUrd)]. The mutant Y70W
was also investigated with some additional compounds
[dUrd, AraT, 9-(b-d-arabinofuranosyl)-adenine (AraA),
9-(2-hydroxyethoxymethyl)-guanine (ACV) and BVDU].
The catalytic mutants E52D, E52Q, R105H and R105K
did not show any detectable activity with either nucleo-
sides or nucleoside analogues (data not shown) in this
assay. Only the wild-type, Y70W and Q81N were active

(see Table 2).
The most striking result for Y70W was that it
became an almost entirely pyrimidine-specific kinase,
because phosphorylation of dAdo and dGuo was
almost abolished, compared with the wild-type. The
pyrimidine nucleoside analogues AraT, AraC, FdUrd
and BVDU were also phosphorylated by Y70W. The
purine analogue CdA was phosphorylated but less effi-
ciently compared with the wild-type, in accordance
with the lowered activity with purines for Y70W.
Mutant Q81N was slightly less efficient towards the
nucleosides compared with the wild-type. In particular,
phosphorylation of dGuo was reduced markedly.
Structure of Dm-dNK-E52D in complex with dTTP
The structure of one of the mutants, E52D, was solved
using X-ray crystallography at a resolution of 2.5 A
˚
in
complex with the feedback inhibitor dTTP. The R-fac-
tor and R
free
were 23.2 and 24.2%, respectively
(Table 3). Most of the protein could be found in
the electron-density map, with the exception of resi-
dues 1–11 and 210–230. The loop connecting a9 and
b5 (residues 195–200) was flexible and poor density
was observed. The electron density for the mutant resi-
due and the ligand was well defined. Structural super-
positioning of Dm-dNK-E52D–dTTP to the wild-type
Dm-dNK–dTTP (PDB ID: 1OE0) was performed and

showed an rmsd of 0.249 A
˚
2
over 358 Ca (for the
dimer). This indicates that the E52D mutation does
not change the overall structure of the enzyme,
because the folds are almost identical. D52 in this
structure has a similar position to E52 in Dm-dNK–
dTTP, i.e. removed from the active site and binding a
Table 2. Nucleoside and nucleoside analogue phosphorylation by
recombinant Dm-dNK mutant enzymes, using the phosphotransf-
erase assay. Relative levels of phosphorylation expressed in rela-
tion to percentage dThd phosphorylation of the wild-type. Relative
phosphorylation expressed as a relation of the percentage of dThd
phosphorylation of the corresponding mutant is given in paren-
theses. The substrate concentration is 100 l
M. Experiments were
repeated twice with the exception of Q81N, which was assayed
once. Results are given as mean ± SD. ND, not detected. NI, not
investigated.
Substrate ⁄
Enzyme WT Y70W Q81N
dThd (%) 100 78.4 ± 0.3 (100) 79.7 (100)
dCyd (%) 87.7 ± 4.7 84.5 ± 3.1 (108) 87.9 (110)
dAdo (%) 65.3 ± 2.3 2.5 ± 0.6 (3.2) 33.1 (41.5)
dGuo (%) 38.4 ± 4.6 < 1 (< 1) 2.1 (2.6)
dUrd (%) NI 56 ± 9 (71.4) NI
AraT (%) NI 22 ± 4 (28.1) NI
AraC (%) 52 ± 2 7 ± 2 (8.9) 1 (1.3)
AraA (%) NI < 1 (< 1) NI

F-AraA (%) 19 ± 6 ND 4 (5.0)
CdA (%) 120 ± 11 11.5 ± 0.5 (14.7) 96 (120)
ACV (%) NI ND NI
FdUrd (%) 48 ± 0 37 ± 8 (47.2) 23 (28.9)
BVDU (%)
a
54 19.5 ± 8.5 (24.9)
a
[15].
Active site mutation of deoxyribonucleoside kinase L. Egeblad-Welin et al.
1546 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS
Mg ion that coordinates the phosphates of dTTP
(Fig. 3).
Discussion
Mutation of residues in the active site was intended to:
(a) validate the proposed reaction mechanism [3] by
mutating the putative catalytic base (E52) and arginine
(R105), thought to stabilize the transition state and
holding E52 in position during catalysis; and (b)
investigate the amino acid residues involved in sub-
strate binding (Y70 and Q81). The steady-state kinetics
of Dm-dNK is compulsory ordered with formation of
a ternary complex [1,2]. Pre-steady-state measurements
indicate that either the catalytic step or a preceding
step is rate determining for the overall forward reac-
tion (R. Browne, G. Andersen, G. Le, B. Munch-
Petersen and C. Grubmeyer, unpublished results).
Therefore, and because ATP is saturating in our
experiments, when evaluating the impact of the muta-
tions from the kinetic data, a change in the K

m
value
can be interpreted as an effect on substrate binding,
and a change in k
cat
would reflect an effect on the
catalytic step.
E52 mutations
E52D
The point mutation E52D was investigated using both
kinetic and structural studies. Kinetic results with an
unchanged K
m
value indicated that the affinity for sub-
strates was unchanged, whereas catalytic activity was
altered because the k
cat
value had decreased dramatic-
ally. The structural study showed that the backbone
conformation of the enzyme was unchanged. Because
the chemical properties of Glu and Asp are very sim-
ilar, both having a carboxylic acid functional group,
the dramatically decreased catalytic rate is most likely
due to the increased distance between the catalytic
base and the 5¢-OH of either dThd or dCyd. These
results favour the reaction-mechanistic hypothesis’
emphasis on arginine and glutamate acting as a pair in
the phosphorylation.
E52H
Mutation of E52 to H resulted in a greatly reduced

k
cat
value, although the K
m
value was relatively
unchanged. With its imidazole ring, histidine can act
as both a proton donor and an acceptor in enzymatic
reactions, and it should therefore theoretically be able
to replace glutamate as a base, to some extent. How-
ever, this is not the case. One reason may be an altered
local conformation, because the normal hydrogen
bond network will be affected. Modelling mutation of
E52H into the structure of wild-type Dm-dNK with
dCyd bound (PDB ID: 1J90) reveals an increase in the
distance between the 5¢-OH group and histidine of
 1A
˚
relative to glutamate. This alone could explain
the reduced catalytic rate.
Table 3. Data collection and refinement statistics for the dNK-E52D
in complex with dTTP
Dm-dNK E52D
Space group P2
1
Cell dimensions (A
˚
)a¼ 33.5
b ¼ 119.5
c ¼ 68.9
b ¼ 92.42°

Content of asymmetric unit One dimer
Resolution range (A
˚
) 32.3–2.5
Completeness (%) 100.0 (100.0)
R
sym
(%) 9.1 (31.1)
I ⁄ rI 6.9 (2.3)
Redundancy 3.8 (3.8)
Number of unique reflections 18,797
Beamline I711
Wavelength (A
˚
) 1.087
Temperature (K) 100
R-factor (%) 23.2
R
free
(%) 24.2
rmsd
Bond length (A
˚
) 0.007
Bond angles (°) 0.912
Mean B-value (A
˚
2
) 37.1
Fig. 3. Structural alignment of wild-type Dm-dNK (blue) (PDB ID:

1OE0) and Dm -dNK-E52D (red) illustrating the binding of the feed-
back inhibitor dTTP and the position of Mg
2+
in the active site.
L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase
FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1547
E52Q
To further support to the hypothesis of the role of E52
as a proton abstractor it was also mutated to its amide –
glutamine. This mutant did not show any detectable
activity with either dThd or dCyd. The two amino acids
take up roughly the same volume, the position of the
side chain can be expected to occupy roughly the same
position and glutamine can participate in hydrogen
bonding. The result that E52Q did not show any activity
must therefore be a consequence of the reactivity of the
functional group and further support the hypothesis
that E52 acts as an initiating base in the reaction.
R105 mutations
R105H
R105 is thought to stabilize the transition state and
hold E52 in the correct position to initiate the catalytic
reaction. The transition state of this type of kinases is
considered to be close to trigonal bipyramidal geo-
metry of the phosphate to be transferred. Arg105 as
well as a Mg ion and arginines of the Lid-region sta-
bilize the negative phosphates of this state. The most
striking effect of mutation of arginine to histidine is an
almost 2000-fold decrease in the k
cat

value for dThd,
and a 270-fold decrease for dCyd. The K
m
value was
increased sevenfold for dThd and 49-fold for dCyd.
The large decrease in k
cat
value indicates that Arg
plays an important role in catalysis. Arginine adopts
two distinct conformations depending on whether a
substrate or an inhibitor is bound [17]. When mutated
to histidine, the residue is no longer able to make
the same hydrogen bonds in the different states. The
decrease in k
cat
and increase in K
m
may be due to the
bulky and rigid structure of histidine that can cause
steric hindrances for the substrate, wherefore correct
positioning and stabilization of the negative charge of
E52 will be less than optimal.
R105K
Surprisingly, mutation of R105 to K completely abol-
ishes the catalytic efficiency. A possible explanation for
this is that lysine is more flexible than Arg, and therefore
not able to position E52, and catalyse the reaction.
Mutations of substrate-interacting residues:
Y70W and Q81N
Y70W

When Y70 was mutated to W the kinetic results
showed that the K
m
values with dThd and dCyd were
dramatically increased, whereas the k
cat
values were
only slightly decreased. Thus, the mutation primarily
affects binding of the substrate. A similar point muta-
tion was made in HSV1-TK, namely Y101 to F. In
this study, the K
m
value for HSV1-TK-Y101F was
increased 12.5-fold, whereas the k
cat
value was twofold
lower [18]. The structure of HSV1-TK-WT was deter-
mined in complex with (North)-methanocarba-thymidine,
as was the structure of HSV1-TK-Y101F. A structural
superposition showed that there were no significant
changes in the polypeptide chain, except that the
hydrogen bond from Y101–3¢-OH was lost [18]. Based
on our results and the information from the structures
of HSV1-TK we suggest that the network of hydrogen
bonds is disrupted, and this gives rise to an increase in
K
m
. Also, the polarity is changed, and the increase in
the size of the side chain may create steric hindrance
for the substrate, making the base moiety of the sub-

strates bind in a nonoptimal conformation.
Another interesting feature concerning the Y70W
mutant is that it became almost entirely pyrimidine spe-
cific, which is also true for the nucleoside analogues.
This must be a consequence of the tryptophan creating
steric hindrance for the purines. The intention behind
the mutation of Y70 to W was to create an enzyme with
increased affinity towards ACV, because tryptophan
was thought to make a better fit for the acyclic ribose
moiety in the active site compared with the bulkier
ribose ring of naturally occurring nucleosides. However,
no activity with ACV was detected for Y70W. The pres-
ence of the bulky dGuo base in ACV may be the reason.
When the kinetic constants were determined for
Y70W with the analogues AraT, AraC and BVDU, it
was surprising that BVDU had a very low K
m
value
(4.9 lm,  50-fold lower than the K
m
value for dThd
and dCyd). The crystal structure of Dm-dNK with var-
ious substrates shows that there is a deep hydrophobic
pocket at the 5-position of the base. The bromovinyl
group may interact with the amino acids lining this
space. Binding of BVDU to Dm-dNK-Y70W compen-
sates for the loss of one hydrogen bond to 3¢-OH by a
tighter fitting of the bromovinyl group, thus restoring
the tight binding lost due to the mutation. At the same
time, it is possible that the positioning of 5¢-OH is chan-

ged, and this may be the reason for the low k
cat
value.
Q81N
The kinetic data for Q81N show a dramatic increase
in the K
m
values, whereas k
cat
is decreased slightly.
These results suggest that the tight anchoring of the
base (dThd or dCyd) is lost, thereby resulting in
poorer binding and higher K
m
values.
Active site mutation of deoxyribonucleoside kinase L. Egeblad-Welin et al.
1548 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS
The relative phosphorylation of dAdo and dGuo
also showed a significant decrease compared with the
wild-type, most likely because of poorer binding of the
purine substrates.
In a previous study of HSV1-TK, Q125 (equivalent to
Q81 in Dm-dNK) was point mutated to N. The struc-
tures of both wild-type HSV1-TK and HSV1-
TK-Q125N were solved in complex with dThd, and the
main difference between the two structures was that the
tight binding of dThd (wild-type) was replaced by a
single water-mediated hydrogen bond (Q125N). Their
kinetic data supported this, because the K
m

value for
dThd with HSV1-TK-Q125N was increased 50-fold and
the catalytic rate was not affected, seen in comparison
with the wild-type [19]. The results obtained in this study
together with the information from HSV1-TK suggest
that the increased distance between base and amino acid
is the main reason for the increase in K
m
values. The
point mutation probably does not alter the backbone
conformation of the Ca-atoms.
In conclusion, the kinetic and structural data presen-
ted here, emphasizing the role of R105 and E52 in the
catalytic mechanism, have gained further support.
Together with findings of substrate-binding interac-
tions via the Q81N and Y70W mutations, additional
knowledge about the structure–function relationship of
the ultra fast Dm-dNK has been obtained.
Experimental procedures
Materials
Glutathione–Sepharose, pGEX-2T vector, Escherichia coli
strain BL21(DE3)pLysS, thrombin, [methyl-
3
H]thymidine
(25 CiÆmmol
)1
), [5–
3
H]-deoxycytidine (24 CiÆmmol
)1

) and
[
32
P]ATP[cP] (3000 CiÆmmol
)1
) were purchased from Amer-
sham Biosciences ( Uppsala, Sweden). BVDU (14.3 CiÆmmol
)1
),
1-b-d-arabinofuranosyl thymine (2.89 CiÆmmol
)1
) and 1-b-d-
arabinofuranosyl cytosine (23.30 CiÆmmol
)1
) were from
Moravek Biochemicals Inc. (Brea, CA). Radiolabelled nucle-
osides were diluted with the nonradioactive compounds to
the appropriate concentrations. When present in the radiola-
belled deoxynucleosides, ethanol was evaporated before use.
Non-radioactive nucleosides were from Sigma. Materials for
cloning, PCR, DNA sequencing, assay and crystallization
were standard commercially available products.
Site-directed mutagenesis and expression
plasmid
Expression plasmid pGEX-2T-Dm-dNK has been described
previously [2]. All mutants were constructed using site-
directed mutagenesis on the plasmid pGEX-2T-Dm-dNK
with truncation for 20 terminal amino acids. The primers
used to create the point mutations, where the changed
nucleotides are in boldface and underlined, are as follows:

E52D-fwd:5¢-GCCTGCTGACCGA
CCCCGTCGAGAAG
TGGCGC-3¢. E52D-rev:5¢-GCGCCACTTCTCGACGGG
GTCGGTCAGCAGGC-3¢. E52H-fwd:5¢-GCCTGCTGAC
C
CACCCCGTCGAGAAGTGGCGC-3¢. E52H-rev:5¢-GC
GCCACTTCTCGACGGG
GTGGGTCAGCAGGC-3¢. E52Q-
fwd:5¢-GCCTGCTGACC
CAGCCCGTCGAGAAGTGG
CGC-3¢. E52Q-rev:5¢-GCGCCACTTCTCGACGGGCT
G
GGTCAGCAGGC-3¢. Y70W-fwd:5¢-CTGCTGGAGCT
GATGT
GGAAAGATCCCAAGAAG-3¢. Y70W-rev :5¢-CTT
CTTGGGATCTTT
CCACATCAGCTCCAGCAG-3¢. Q81N-
fwd:5¢-TGGGCCATGCCCTTT
AACAGTTATGTCACG
CTG-3¢. Q81N-rev:5¢-CAGCGTGACATAACT
GTTAAA
GGGCATGGCCCA-3¢. R105H-fwd:5¢-GCTAAAAATAA
TGGAGC
ACTCCATTTTTAGCGCTCGC-3¢ . R105H-
rev:5¢-GCGAGCGCTAAAAATGGAG
TGCTCCATTAT
TTTTAGC-3¢. R105K-fwd:5¢-GCTAAAAATAATGGAG
AAATCCATTTTTAGCGCTCGC-3¢. R105K-rev:5¢-GCG
AGCGCTAAAAATGGA
TTTCTCCATTATTTTTAGC-3¢

Sequence verification
Plasmids of the seven mutants were transformed into XL1-
Blue Supercompetent Cells. Plasmids were isolated and the
insert sequenced using the dye terminator method (ABI
PRISM 310), in order to verify that the point mutations
were introduced, and that no other mutations or frame-
shifts had occurred.
Expression and purification
The seven pGEX-2T Dm-dNKDC20 mutants were trans-
formed into E. coli BL21-competent cells. Recombinant
proteins were expressed and purified and thrombin was
cleaved as described previously [2].
All proteins were stored at )80 °C, and a cryoprotectant
solution was added to a final concentration of: 10% (v ⁄ v)
glycerol, 0.1% (v ⁄ v) Triton X-100, 5 mm MgCl
2
and 5 mm
dithiothreitol, with the exception of Dm-dNK E52D CD20;
it was stored in 30% glycerol.
The purity of the proteins was determined by
SDS ⁄ PAGE [20] and the protein concentrations were deter-
mined using Bradford reagent [21].
Enzyme assays
Deoxynucleoside kinase activities were determined by initial
velocity measurements based on four time samples (0, 4,
8 and 12 min) using the DE-81 filter paper assay with trit-
ium-labelled substrates as described previously [2].
The standard assay conditions were: 50 mm Tris ⁄ HCl
pH 7.5, 2.5 mm MgCl
2

,10mm dithiotreitol, 0.5 mm
L. Egeblad-Welin et al. Active site mutation of deoxyribonucleoside kinase
FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS 1549
CHAPS, 0.5 mgÆmL
)1
bovine serum albumin and 2.5 mm
ATP.
The relative phosphorylation of nucleosides and nucleo-
side analogues was determined using the phosphoryl transfer
assay. This was performed using [
32
P]ATP[cP]. The nucleo-
sides ⁄ analogues were added to a final concentration of
100 lm in a reaction mixture of 25 lL. The standard reaction
buffer contained 50 mm Tris–HCL pH 7.5, 2.5 mm MgCl
2
,
10 mm dithiothreitol, 0.5 mm CHAPS, 0.5 mm bovine serum
albumin, 100 lm nonradioactive labelled ATP, radioactively
labelled ATP, 50 ng enzyme per reaction. After incubation of
the reaction mixtures for 20 min at 37 °C, 1 lL was spotted
on a TLC sheet. The nucleotides were separated in a buffer
containing NH
4
OH, isobutyric acid and destilled H
2
Oina
ratio of 1:66:33 (v ⁄ v ⁄ v). Sheets were autoradiographed using
phosphorimaging plates.
The kinetic data were evaluated using nonlinear regres-

sion analysis and the Michaelis–Menten equation v ¼
V
max
Æ[S] ⁄ (K
m
+[S]) or the Hill equation v ¼ V
max
Æ[S]
n
⁄
(K
n
0:5
+[S]
n
) as described previously [22]. All kinetic data
were analysed using sigma plot.
Crystallization
Crystals of a C-terminally truncated (D20) recombinant
Dm-dNK mutant E52D were grown using the vapour diffu-
sion method by hanging drop geometry. The crystallization
solution was: 0.12 m NaAc pH 7.0, 0.1 m Mes pH 6.5 and
18% (w ⁄ v) monomethyl polyethylene glycol 2000. The
enzyme solution consisted of 5 mgÆmL
)1
mutant enzyme in
a1· NaCl ⁄ P
i
buffer with 5 mm dTTP, 5 mm Mg
2+

,5mm
dithiothreitol and 10% glycerol. The crystallization solution
was diluted 1:2 with water before 2 lL was mixed with
2 lL enzyme solution on a cover slip. The well solution
was covered with 250 lL Al’s Oil. It was left to equilibrate
against the crystallization solution at 15 °C. After approxi-
mately 3 days, small crystals appeared and after 5 days
larger singular crystals were obtained.
Data collection
The E52D–dTTP crystals were flash-frozen in liquid nitro-
gen. The cryoprotectant had the same composition as the
crystallization solution plus an added 20% (v ⁄ v) glycerol.
The data set was collected at MAXLab in Lund, Sweden,
on beam line I711 at a temperature of 100 K. The data
were indexed scaled and merged using mosflm [23] and
scala [24]. The crystal belonged to the monoclinic space
group P2
1
and had a solvent content of 48%.
Structure determination and refinement
The structure was solved by molecular replacement using
molrep [25] with the wild-type structure Dm-dNK-dTTP
(PDB ID: 1OE0) as the search model. The mutated residue
was replaced using o v. 9.0.7 ( />[26], and rigid body refinement was performed using
refmac5 [27]. Constrained refinement with a twofold non-
crystallographic symmetry was carried out using refmac5.
Water molecules, Mg
2+
and the ligand dTTP were added
using the program o [26]. Data collection and refinement

statistics are shown in Table 3. The coordinates have been
deposited with the PDB ID: 2jcs.
Acknowlegdements
This work was supported by grants from the Swedish
Research Council (to HE), the Swedish Cancer Foun-
dation (to HE), the Danish Research Council (to
BM-P) and the NOVO Nordisk foundation (to BM-P)
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