Probing the active site of the deoxynucleotide N-hydrolase Rcl encoded by the rat gene c6orf108

Article abstract of DOI:10.1074/jbc.M110.181594

Rcl is a potential anti-angiogenic therapeutic target that hydrolyzes the N-glycosidic bond of 2′-deoxyribonucleoside 5′- monophosphate, yielding 2-deoxyribose 5-phosphate and the corresponding base. Its recently elucidated solution structure provided the

Full text of DOI:10.1074/jbc.M110.181594

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 53, pp. 41806–41814, December 31, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
Probing the Active Site of the Deoxynucleotide N-Hydrolase
□
S
*
Rcl Encoded by the Rat Gene c6orf108
Received for publication, September 6, 2010, and in revised form, October 15, 2010 Published, JBC Papers in Press, October 20, 2010, DOI 10.1074/jbc.M110.181594
‡§2 ¶ʈ ‡§ ‡§3
‡
1
Christelle Dupouy , Chi Zhang , Andr e´ Padilla , Sylvie Pochet , and Pierre Alexandre Kaminski
‡
§
¶
From the Institut Pasteur, Unit e´ de Chimie et Biocatalyse, and CNRS, URA2128, 28 rue du Dr Roux, 75724 Paris Cedex 15, CNRS,
UMR5048, Centre de Biochimie Structurale, F34090 Montpellier, and INSERM, U554, F34090 Montpellier and
ʈ
Universit e´ Montpellier 1 et 2, IFR3, 29 rue de Navacelles, F34090 Montpellier, France
Rcl is a potential anti-angiogenic therapeutic target that hy-
drolyzes the N-glycosidic bond of 2ꢀ-deoxyribonucleoside 5ꢀ-
monophosphate, yielding 2-deoxyribose 5-phosphate and the
corresponding base. Its recently elucidated solution structure
provided the first insight into the molecular basis for the sub-
strate recognition. To facilitate the development of potent and
specific inhibitors of Rcl, the active site was probed by site-
directed mutagenesis and by the use of substrate analogs. The
nucleobase shows weak interactions with the protein, and the
causal role of Rcl in breast tumorigenesis was suggested as the
up-regulation of Rcl correlates with the tumor grade (4).
Thus, Rcl is a potential therapeutic target. However, its func-
tion remains to be determined.
We have shown that Rcl is a 2Ј-deoxynucleoside 5Ј-phos-
phate N-hydrolase that had not been previously described (9).
Rcl hydrolyzes 2Ј-deoxyribonucleoside 5Ј-monophosphate
4
(dNMP) to form a free nucleobase moiety (N) and 2-deoxyri-
bose 5-phosphate. 2-Deoxyribose 5-phosphate may be con-
deoxyribose binding pocket includes the catalytic triad Tyr-13, verted to 2-deoxyribose, a downstream mediator of thymidine
Asp-69, and Glu-93 and the phosphate binding site Ser-87 and
Ser-117. The phosphomimetic mutation of Ser-17 to Glu pre-
vents substrate binding and, thus, abolishes the activity of Rcl.
The synthetic ligand-based analysis of the Rcl binding site
shows that substitutions at positions 2 and 6 of the nucleobase
as well as large heterocycles are well tolerated. The phosphate
group at position 5 of the (deoxy)ribose moiety is the critical
binding determinant. This study provides the roadmap for the
design of small molecules inhibitors with pharmacological
properties.
phosphorylase (also known as the angiogenic factor endothe-
lial cell growth factor 1, ECGF1) that regulates tumor angio-
genesis and progression (10). 2-Deoxyribose is also a substrate
for glycolysis through its conversion to glyceraldehyde
3-phosphate, which may indirectly increase the development
and malignancy of cancer cell overexpressing Rcl.
Rcl belongs to the nucleoside 2-deoxyribosyltransferase
(NDT) family (EC 2.4.2.6; pfam 05014). NDT catalyzes the
reversible transfer of the deoxyribosyl moiety from a de-
oxynucleoside donor to an acceptor nucleobase (11). On the
contrary, Rcl is a hydrolase (or a transferase using only water
molecule as an acceptor) (9). Both NDT and Rcl are specific
for 2Ј-deoxyribose-containing substrates, whereas the corre-
sponding riboside analogs are inhibitors. Their amino acid
sequences display an overall low level of identity but share the
catalytic triad composed of a tyrosine, an aspartate, and a glu-
tamate, suggesting a similar mode of action (9, 12).
Rcl is a c-Myc target (1) that becomes tumorigenic when
the metabolic environment of the cell is permissive (2). Its
participation in tumorigenesis is supported by several pieces
of evidence as follows. rcl is up-regulated in several cancers
such as human prostate, breast cancers, and chronic lympho-
cytic leukemia (3–5). rcl is repressed in response to histone
deacetylase inhibitors, anticancer agents that induce tumor
cell death, differentiation, and/or cell cycle arrest (6). rcl is
over-expressed in response to chronic administration of the
synthetic glucocorticoid methylprednisolone or estrogen di-
ethylstilbestrol. Altogether these data indicate that Rcl has a
The structure of Rcl in solution was recently described (13,
14). Rcl is a symmetric homodimer; each monomer is com-
posed of five buried ␤-strands alternating with five ␣-helices.
The global architecture of the active site in Rcl resembles that
of known NDTs. It is mainly composed of the N-terminal re-
gion from one monomer and a few residues from second
role in cell growth and/or cell proliferation (7, 8). Moreover, a monomer. The overall orientation of the nucleotide in Rcl
appeared to be similar to that of the bound nucleoside in
NDTs. It is composed of three main parts that recognize each
*
This work was supported by the Institut Pasteur (Direction des applica-
tions de la recherche et des relations industrielles) and CNRS.
S
moiety of the ligand; that is, the nucleobase, the deoxyribose,
and the phosphate group (specific for Rcl).
□
The on-line version of this article (available at http://www.jbc.org) con-
tains supplemental data and a figure.
1
The nucleobase shows weak interactions with the protein
and is largely accessible to solvent. The deoxyribose binding
pocket is central and includes the catalytic triad (Tyr-13, Asp-
Funded by a CNRS post-doctoral fellowship. Present address: IBMM, UMR
5
3
247, CNRS-Universit e´ Montpellier 1 et 2 cc 1704, Place Eug e` ne Bataillon
4095 Montpellier Cedex 05, France.
2
3
Present address: INSERM, U845, Centre de Recherche “Croissance et Sig-
nalisation,” Equipe “PRL/GH Pathophysiology,” 156 rue Vaugirard, Paris,
F-75015, France.
To whom correspondence should be addressed: Institut Pasteur, Unit e´ de
Chimie et Biocatalyse, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France.
Tel.: 33-1-40-61-30-52; Fax: 33-1-45-68-84-04; E-mail: pierre-alexandre.
kaminski@pasteur.fr.
6
9, and Glu-93). The phosphate binding site is reminiscent of
4
The abbreviations used are: dNMP, 2Ј-deoxyribonucleoside 5Ј-monophos-
phate; NDT, nucleoside 2-deoxyribosyltransferase; ITC, isothermal titra-
tion calorimetry.
4
1806 JOURNAL OF BIOLOGICAL CHEMISTRY
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Rcl Active Site
other phosphate binding proteins (15), and hydrogen bonds
with Ser-17, Arg-19, Ser-87, and Ser-117 were suggested.
Here, we report the analysis of the contribution of the dif-
ferent amino acids participating at the active site. The binding
determinants of the active site were probed with various syn-
thetic nucleotide analogs. These studies defined chemical
groups that are important for molecular recognition and will
guide the rational design of inhibitors. Furthermore, a phos-
phomimetic mutation at serine 17 abolishes Rcl activity, sug-
gesting that phosphorylation of Ser-17 plays an important
role in Rcl regulation.
TABLE 1
Oligonucleotides used to generate the Rcl variants
Y13Aϩ GGCTCCATGCTCCGTGGCCTTCTGCGGGAGC
Y13AϪ GCTCCCGCAGAAGGCCACGGAGCATGGAGCC
Y13Fϩ CCATGCTCCGTGTTCTTCTGCGGGAGCATC
Y13FϪ GATGCTCCCGCAGAAGAACACGGAGCATGG
S17Aϩ CCGTGTACTTCTGCGGGGCCATCCGCGGCGGGCGCG
S17AϪ CGCGCCCGCCGCGGATGGCCCCGCAGAAGTACACGG
S17Eϩ GTGTACTTCTGCGGGGAAATCCGCGGCGGGCGCG
S17EϪ CGCGCCCGCCGCGGATTTCCCCGCAGAAGTACAC
R19Aϩ TTCTGCGGGAGCATCGCCGGCGGGCGCGAGGAC
R19AϪ GTCCTCGCGCCCGCCGGCGATGCTCCCGCAGAAG
H45Aϩ GGTGCTCACTGAGGCCGTGGCTGATGCTGAG
H45AϪ CTCAGCATCAGCCACGGCCTCAGTGAGCACC
S87Aϩ GGAAGTGACACAGCCAGCCTTGGGTGTTGGC
S87AϪ GCCAACACCCAAGGCTGGCTGTGTCACTTCC
S87Dϩ GGAAGTGACACAGCCAGACTTGGGTGTTGGC
S87DϪ GCCAACACCCAAGTCTGGCTGTGTCACTTCC
E93Qϩ GGGTGTTGGCTATCAACTGGGCCGGG
EXPERIMENTAL PROCEDURES
Chemicals—2Ј-Deoxyguanosine 5Ј-monophosphate, adeno-
sine 3Ј,5Ј-cyclic-monophosphate, adenosine 5Ј-monosulfate,
E93QϪ CCCGGCCCAGTTGATAGCCAACACCC
S117Aϩ GTCTGGCCGAGTGCTTGCCGCCATGATCCGCGG
S117AϪ CCGCGGATCATGGCGGCAAGCACTCGGCCAGAC
S117Nϩ GTCTGGCCGAGTGCTTAACGCCATGATCCGCGG
S117NϪ CCGCGGATCATGGCGTTAAGCACTCGGCCAGAC
S158A GCGGATCCTCAGGCACTTGGGTGACTGGCGGAAGCCGTCTTCTG
S158E CCCAAGCTTTCAGGCACTTGGGTGACTCTCGGAAGCCGTCTTCTGAGG
8
-bromoadenosine 5Ј-monophosphate, and xanthosine 5Ј-
monophosphate were purchased from Sigma. 2-Fluoroad-
enine-9-␤-D-arabinofuranoside 5Ј-monophosphate (Fludaraꢁ)
2
was from Schering. N -Ethyl-2Ј-deoxyguanosine 5Ј-mono-
7
phosphate, N -methyl-2Ј-deoxyguanosine 5Ј-monophos-
uct was purified by reversed phase HPLC (10–20% A in B) to
give, after repeated freeze-drying, compound 1 in a 65% yield.
Adenosine-5Ј-carboxylic acid (2) was synthesized via oxida-
tion of the 5Ј-hydroxyl group of 2Ј,3Ј-O-isopropylidene aden-
osine as reported (17). Isopropylidene was then removed by
phate, glyoxal-2Ј-deoxyguanosine 5Ј-monophosphate, and
6
O -methyl-2Ј-deoxyguanosine 5Ј-monophosphate were from
Jena Bioscience GmbH. 5-Aminoimidazole-4-carboxamide-1-
␤
-D-ribofuranoside 5Ј-monophosphate and acyclovir mono-
phosphate were from Carbosynth. Adenosine 5Ј-monophos-
phorothioate was from BioLog.
treatment with a solution of HCOOH/H O (70:30) at 35 °C
2
for 12 h. Purification by reversed phase HPLC (5–30% A in B)
General Synthetic Methods—Reagents and anhydrous sol-
vents were obtained from commercial suppliers and used
without further purification. Flash chromatography was per-
formed using silica gel 60 (Merck). Preparative HPLC were
carried out on a PerkinElmer Life Sciences system (200 Pump)
with a C18 reverse phase column (Kromasil, 5 ␮m 100 Å,
afforded compound 2 in a 85% yield.
7
7
N -Deaza-adenosine 5Ј-monophosphate (N -deaza-AMP)
was prepared from commercial 7-deaza-adenosine (tuberci-
din). First, the 2Ј and 3Ј hydroxyl groups were protected by an
isopropylidene acetal group with 2,2-dimethoxypropane and
catalytic amounts of p-toluene sulfonic acid as previously de-
scribed (18). Then, 7-deaza-2Ј,3Ј-O-isopropylidene-adenosine
was 5Ј-phosphorylated as for compound 1, and the cyano-
ethyl and isopropylidene groups were removed as described
above. Purification by reversed phase HPLC (5–15% A in B)
1
50 ϫ 4.5 mm) using a flow rate of 5.5 ml/min and a linear
gradient of acetonitrile (A) in 10 mM triethylammonium ace-
1
13
31
tate buffer (B) at pH 7 over 20 min. H, C, and P NMR
spectra were recorded on a Bruker Avance 400 spectrometer
operating at 400.13, 100.62, and 161.62 MHz, respectively.
High resolution mass spectra were recorded on a Waters Q-
TOF micro MS instrument using a mobile phase of acetoni-
trile/water with 0.1% formic acid. Structure of synthesized
compounds was confirmed by NMR and mass spectrometry.
7
9
afforded N -deaza-AMP. N -Deaza-dGMP was a gift of Pro-
fessor Ioan Lascu (Universit e´ de Bordeaux-2, Institut de Bio-
chimie et G e´ n e´ tique Cellulaires.
Construction of the Rcl Mutants—RclE93A, Rcl D69A, and
Rcl D69N were previously described in Ghiorghi et al. (9) and
Yang et al. (13) respectively. Oligonucleotides ϩ (Y13Aϩ,
Y13Fϩ, S17Aϩ, S17Eϩ, R19Aϩ, H45Aϩ, S87Aϩ, S87Dϩ,
E93Qϩ, S117Aϩ, S117Nϩ) and T7term, oligonucleotides Ϫ
2
Ј,3Ј-O-Isopropylidene adenosine 5Ј-monophosphate (1)
was synthesized via phosphorylation of commercial 2Ј,3Ј-O-
isopropylidene adenosine according to reported procedures
(
16). The product (50 mg, 0.16 mmol) was coevaporated three
(Y13AϪ, Y13FϪ, S17AϪ, S17EϪ, R19AϪ, H45AϪ, S87AϪ,
times with dry pyridine, then a 1 M solution of 2-cyanoeth-
ylphosphate in pyridine (320 ␮l, 0.32 mmol) was added, and
the mixture was coevaporated twice with dry pyridine. The
residue was dissolved in dry pyridine (1.6 ml) and N,NЈ-dicy-
clohexylcarbodiimide (198 mg, 0.96 mmol) was added at
room temperature. The mixture was kept stirring for 24 h at
room temperature. Water was added to the mixture, in-
solubles were filtered, and the solvent was removed under
vacuum. The cyanoethyl group was removed by treatment
with a 1% solution of NaMeO in methanol at room tempera-
ture for 2 h. The mixture was neutralized by the addition of a
S87DϪ, E93QϪ, S117AϪ, S117NϪ) and T7prom (Table 1)
were used in two separate PCR reactions using plasmid
pET28aRcl as DNA template. The parameters used 1 cycle of
5 min at 95 °C, 25 cycles of 30 s at 95 °C, 30 s at 53 °C, and 30 s
at 72 °C, and 1 cycle of 10 min at 72 °C. The annealing tem-
perature was dependent on the pairs of oligonucleotides used.
Oligonucleotides T7prom and T7term were used in a second
PCR using aliquots of the first one using the same parameters
as above with the exception of an annealing temperature of
61 °C.
The amplified DNA fragments were purified by using the
QIAquick PCR purification kit (Qiagen) and then digested
ϩ
resin Dowex H , filtered, and concentrated. The crude prod-
DECEMBER 31, 2010•VOLUME 285•NUMBER 53
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Rcl Active Site
with NdeI and BamHI enzymes over 2 h at 37 °C and repuri-
fied. Each PCR product was ligated with plasmid pET28a that
had been digested with the same restriction enzymes. The
ligation mixtures were used to transform strain DH5␣. Plas-
mids with the correct sequence were used to transform strain
Bli5.
The construction of the RclS158A and RclS158E mutants
was done by using T7prom and oligonucleotides S158A or
S158E, respectively. Each pair of oligonucleotides was used in
a PCR reaction, and the amplified products were digested
with NdeI and BamHI and with NdeI and HindIII, respec-
tively, purified, and cloned into plasmid pET28a that had been
digested with the same enzymes.
Overexpression and Purification of the N-terminal His-
tagged Rcls—The different pET28aRcl plasmids were used to
transform strain Bli5. Culture conditions and induction were
performed as described by Ghiorghi et al. (9). Frozen cells
resuspended in 40 ml of extraction/wash buffer (50 mM
Na HPO , NaH PO , 300 mM NaCl pH 7.0) were broken by
FIGURE 1. Schematic representation of the active site of Rcl.
2
4
2
4
using a French press at 14,000 p.s.i. The lysate was centri-
fuged at 25,000 ϫ g for 30 min at 4 °C. The supernatant was
loaded on a column containing 6 ml of TALON (BD Bio-
science) resin that had been previously equilibrated with the
same buffer. After washing, Rcl was eluted with 150 mM imid-
azole. Fractions containing Rcl were pooled and dialyzed
against sodium phosphate buffer (50 mM Na HPO ,
2
4
NaH PO , pH 6.0). The purity was checked by SDS-PAGE
2
4
electrophoresis and by measuring the specific activity. Puri-
fied His-tagged Rcls in 50 mM sodium phosphate buffer, pH
6
.0, were stored at Ϫ20 °C.
Kinetic Measurements—The enzyme activity was deter-
mined spectrophotometrically by incubating the enzyme with
dGMP and by following the production of 2-deoxyribose
5
-phosphate (one of the reaction products) as described pre-
viously (9). The initial velocity of the reaction was measured
either at a variable concentration of dGMP both in the ab-
sence and presence of inhibitors or at a fixed concentration of
dGMP and variable concentrations of inhibitors, allowing the
investigation of the nature of the inhibition.
Isothermal Titration Calorimetry (ITC)—ITC was per-
formed in a MicroCal VP-ITC calorimeter at 25 °C. Protein
samples were prepared as indicated above. After thermal
equilibration, titrant additions were made at 600-s intervals to
the 1.41-ml protein samples by adding 5-␮l aliquots of 10 mM
GMP to protein samples ([RCLwt] ϭ 257 ␮M, [Y13F] ϭ 259
FIGURE 2. 600 MHz one-dimensional NMR spectra of E93Q, S17E, S117A,
and Y13F Rcl mutants. The shifted methyl resonances chemical shifts of
Rcl wild type are indicated by the dotted lines.
␮
M, [D69N] ϭ 440 ␮M, [E93Q] ϭ 250 ␮M, [S11A] ϭ 400 ␮M,
deoxynucleoside 5Ј-monophosphate were chosen for mu-
and [S17E] ϭ 507 ␮M) in 25 mM Na HPO , NaH PO , pH 6.0, tagenesis. The residues Tyr-13, Ser-17, Arg-19, His-45,
2
4
2
4
2
5 mM NaCl, 2 mM ␤-mercaptoethanol.
Asp-69 (previously described in Yang et al. (13)), Trp-72, Ser-
The heat of dilution obtained from injecting the ligand into 87, Glu-93, and Ser-117 were replaced either by an alanine or
buffer was subtracted before fitting. Heat effects were inte-
by the most conservative amino acid. One-dimensional NMR
grated with ITC Origine 7.0 software, and a best fit was found spectra of Rcl and of the different mutants were recorded to
using a single binding-site model.
control their structure (Fig. 2). The shifted methyl resonances
of the Rcl mutants are very similar to those observed in Rcl
wild type, indicating that the core domains of the proteins
make similar residue-residue interactions. Furthermore, small
angle x-ray scattering experiments indicate that Rcl mutants
display the same overall shape and dimeric form as the wild
type (supplemental data and supplemental figure). As dGMP
RESULTS
Catalytic Activities of Rcl Mutants—Based on the NMR
structure of Rcl complex with GMP, the amino acids that
participate at the active site (Fig. 1) and are potentially in-
volved in the catalytic mechanism of the hydrolysis of 2Ј-
4
1808 JOURNAL OF BIOLOGICAL CHEMISTRY
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Rcl Active Site
was the best substrate of the native enzyme, it was chosen to
measure the activity of the mutants. The results are summa-
rized in Table 2. Mutants Y13A, D69A, S87A, and E93A did
not abolish the catalysis but caused a large decrease in en-
zyme activity (Ն98%). The side chains of Ser-17, Arg-19, and
His-45 did not significantly influence the catalytic activity.
Mutant W72F has a reduced catalytic activity due to an in-
crease of K
for dGMP, indicating that it may be involved in
m
the base stacking but is not essential for catalytic activity. Mu-
TABLE 2
tants E93Q and S117A showed no detectable activity. k val-
cat
Kinetic parameters of wild-type and Rcl mutants with dGMP as
substrate
ues showed less than a 2-fold variation if Asp-69 and Ser-87
were excepted (4- and 5-fold). In contrast, the K values var-
m
V
and K were obtained from double reciprocal plots of initial velocity
max
m
measurements. At least five different concentrations of dGMP were used. ND, not ied from 50 ␮M to 10 mM.
detectable.
Binding of GMP—To further characterize the mutants and
in particular those without any detectable activity (E93Q and
S117A), the dissociation constant of Rcl and mutants with
k
Km
kcat/K
cat
sϪ1
m
Ϫ1
␮M
M
Ϫ1ꢂs
WT
0.0298
0.0429
0.0267
0.0273
0.0168
0.0412
0.0033
0.0066
0.0309
0.0042
0.0495
ND
48
621
Y13A
Y13F
S17A
R19A
H45A
D69A
D69N
W72F
S87A
E93A
E93Q
S117A
10,000
4,000
74
4.5
GMP was determined by ITC (Table 3). The K value of Rcl
d
6.3
with GMP was 10.6 Ϯ 0.4 ␮M (Fig. 3), similar to that de-
scribed by Doddapaneni et al. (14). The Y13F mutant shows a
K value comparable with Rcl wild type, whereas their K
545
569
885
10.7
7.4
50
109
260
890
171
435
8,500
ND
ND
d
m
values for dGMP differ by a factor of 80. Asp-69 contributes
180
9.1
marginally to the binding of GMP as the K value was only
d
4.3
reduced by a factor of three as compared with the wild-type
ND
ND
enzyme. For both mutants E93Q and S117A the K value was
ND
d
enhanced by a factor of 14 and 30, respectively. These low K
d
TABLE 3
values indicate that these two amino acids are the major con-
tributors to the GMP binding.
Dissociation constants K of Rcl and of different mutants
d
All energies reported as kcal/mol. ND, not detectable.
The GMP binding is enthalpy-driven with ⌬H values from
Kd
⌬H
kcalꢂmol
⌬S
kcalꢂmol
⌬G
kcalꢂmol
Ϫ1
7
to 8 kcalꢂmol for the Rcl wild-type, Y13F, and D69N mu-
Ϫ1
Ϫ1
Ϫ1
Ϫ1
␮
M
tants. There is a loss in ⌬H of ϳ3–4 kcalꢂmol for both
S117A and E93Q mutants, consistent with their weaker bind-
ing interactions. In the latter, the positive ⌬S value indicates
an increased degree of freedom in the complex.
RCL WT
Y13F
10.6 Ϯ 0.4
11.0 Ϯ 0.5
33.3 Ϯ 1.3
143.7 Ϯ 8.6
363.8 Ϯ 14.6
ND
Ϫ8.11 Ϯ 0.05
Ϫ7.96 Ϯ 0.05
Ϫ6.96 Ϯ 0.06
Ϫ4.84 Ϯ 0.18
Ϫ4.61 Ϯ 0.18
ND
Ϫ4.44
Ϫ4.00
Ϫ2.86
ϩ1.33
ϩ0.29
ND
Ϫ6.79
Ϫ6.78
Ϫ6.11
Ϫ5.24
Ϫ4.70
ND
D69N
E93Q
S117A
S17E
FIGURE 3. ITC for Rcl binding to GMP. A, Rcl wild type is shown. The protein concentration in the calorimeter cell was 257 ␮M, and the titrant was 10 mM.
B, Rcl S117A is shown. The protein concentration was 400 ␮M, and the GMP was 10 mM. Both experiments were carried out in 25 mM phosphate sodium, pH
6
.0, 25 mM NaCl, 2 mM ␤-mercaptoethanol at 25 °C.
DECEMBER 31, 2010•VOLUME 285•NUMBER 53
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Rcl Active Site
FIGURE 4. Conservation of Rcl amino-acids sequences in eucaryotes. The catalytic triad YDE is indicated in bold characters. The phosphorylated serine
residues in the human Rcl are shadowed and underlined.
TABLE 4
modate several substitutions at positions 2, 6, and 7 and also
7
Kinetic parameters of wild-type and Rcl mutated at putative
phosphorylated site
ND, not detectable.
large heterocycles. The enhanced k value with N -methyl-
cat
dGMP is explained by the protonation of the nucleobase, cre-
ating a permanent positive charge and increasing the C1Ј-N9
bond cleavage reactivity.
k
Km
kcat/K
cat
sϪ1
m
Ϫ1
␮M
M
Ϫ1ꢂs
Rcl Inhibition by Nucleotides with Various Nucleobases—
GMP and 6-methylthio-GMP were previously reported as
WT
0.0297
0.0273
ND
48
74
ND
48
59
621
545
ND
385
313
S17A
S17E
S158A
S158E
inhibitors of the hydrolysis of dGMP by Rcl with K values of
i
0.0184
0.0171
20 and 10 ␮M, respectively. Four other naturally ribonucleoti-
des of the de novo purine nucleotides pathway, AMP, IMP,
xanthosine 5Ј-monophosphate, and 5-aminoimidazole-4-car-
boxamide 1-␤-D-ribofuranosyl 5Ј-monophosphate, display
similar inhibitory constants around 50 ␮M (Table 6).
Effect of Replacement of the Phosphorylated Serine Residues
of Rcl by Alanine or Glutamate (Phosphomimetic)—Phosphor-
ylated peptides corresponding to human Rcl were identified
by mass spectrometry in several proteomic studies (19–23).
Three phosphorylated peptides on three different serine resi-
dues (indicated in bold in Fig. 4) were identified. Two of these,
serine Ser-17 and Ser-158, are conserved in the rat Rcl. Re-
placement of the two serines by alanine did not affect either
the affinity for dGMP or the catalytic efficiency. A similar ob-
servation was made with the S158E change. On the contrary,
9
As N -deaza-dGMP is not a substrate for Rcl, it was tested
7
as a potential inhibitor in addition to N -deaza-AMP. The
7
absence of inhibition by N -deaza-AMP (Table 6) indicates
7
that N may participate in the binding. The absence of inhibi-
9
tion by N -deaza-dGMP (not shown) may be explained either
by the presence of a proton at position 7, which again indi-
7
cates that N may participate in the binding, or by the short-
9
the S17E change has a profound effect on the enzyme activity, ening (of about 10%) of the C1Ј-C9 bond in N -deaza-dGMP
which was no longer detectable (Table 4). In addition, no
GMP binds to the S17E mutant as determined by microcalo-
rimetry (Table 3). This suggests that phosphorylation of
Ser-17 negatively regulates the activity of Rcl.
versus the C1Ј-N9 bond. In both cases the determining factor
may be ionization and/or electron densities.
Substituted derivatives at position 8 of the nucleobase are
not allowed, as illustrated by 8-Br-AMP and previously by
8-oxo-dGMP (9). According to the structure, any substituent
at position 8 would point toward the phosphate group, lead-
ing to steric clashes.
Modified Deoxyribonucleotides as Substrates—As guanine
showed little interaction with Rcl, different purines modified
by endogenous oxidants, alkylating agents, or endogenous
aldehydes (24), found in cancerous cells as DNA adducts,
were tested. We previously showed that 8-oxo-dGMP was
neither a substrate nor an inhibitor of Rcl (9). As an example
Rcl Inhibition by Nucleotides with Modified Sugar—As pre-
viously shown, substitution of ribose by an acyclic chain di-
minishes the K by a factor of three, indicating that the 2Ј and
i
2
6
of alkylated dNMPs, N -ethyl-dGMP, O -methyl-dGMP, and
3Ј OH groups of the ribose contribute to the binding but are
not essential. The addition of an isopropylidene group cross-
ing at the 2Ј and 3Ј positions of the ribose abolishes the
inhibition.
7
2
N -methyl-dGMP were chosen in addition to 1,N -glyoxal
adduct (Table 5). k values varied only by a factor of 2 at the
cat
7
exception of N -methyl-dGMP (factor 70). K values were
m
7
also very close except for glyoxal-dGMP (factor 3) and N -
The orientation of the OH group at position 2Ј of the deox-
yribose is important because the chemotherapeutic agent 2-F
methyl-dGMP (factor 7). It is concluded that Rcl may accom-
4
1810 JOURNAL OF BIOLOGICAL CHEMISTRY
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Rcl Active Site
TABLE 5
Modified purine deoxyribonucleotides as Rcl substrates
ara-AMP, used for the treatment of chronic lymphocytic leu-
kemia, is neither a substrate nor an inhibitor of the reaction.
cidating its cellular role. Its solution structure was recently
solved and has provided insights into the molecular basis for
This may not be due to the presence of the fluor atom at posi- substrate recognition (13, 14). Tyr-13, Glu-93, and His-45 are
tion 2 of the nucleobase as Rcl accepts substitution at position located in the vicinity of the 3Ј-OH of the ribose. The side
2
2
as demonstrated with N -ethyl-dGMP (Table 7). The pres-
chains of Asp-69 and Trp-72 are in close contact with the 2Ј
position. A positively charged pocket formed by Ser-87, Ser-
ence of the phosphate group is also important because cAMP
is neither a substrate nor an inhibitor of Rcl activity and be-
cause phosphate cannot undergo substitution with a
carboxylate.
1
17Ј, Ser-17, and Arg-19 may constitute the phosphate bind-
ing site. Mutagenesis of these amino acids to alanine or to
conservative amino acids clearly shows that Glu-93 and Ser-
Sulfate is considered to be a good phosphate mimic as it is
isosteric and has a similar charge distribution and a net nega-
tive charge at physiological pH. However, adenosine 5Ј-mo-
nosulfate has no inhibitory effect. This result may be ex-
1
17 are the main contributors to substrate binding and to ca-
talysis. The differences of K values between variants Rcl
d
E93Q and Rcl S117A for GMP suggest that phosphate binding
is primordial.
plained either by the differences of pK between the sulfate
a
The low catalytic activity of Rcl with different 2Ј-de-
oxyribonucleoside 5Ј-monophosphates as substrates led us
to investigate other ligands. DNA damage plays a major
role in mutagenesis, carcinogenesis, and aging. DNA is
subject to modifications by various endogenous and exoge-
nous chemicals, and modified guanine, resulting from oxi-
dation or alkylation processes, is frequently found in can-
cerous cells (26, 27). These DNA lesions are subject to a
network of DNA repair mechanisms that generally excise
the modified base (28). However, in some cases the modi-
fied base can be recycled as illustrated by 8-oxo-2Ј-deox-
yguanosine and 8-oxo-guanine (29). We hypothesized that
and the phosphate group or by the difference of size between
sulfur and phosphorus. Substitution of the ␣ oxygen of AMP
by sulfur (5Ј-AMPS) results in a diminution of the K by a fac-
i
tor of 3, which might be due to their difference of pK : 6.8
a
(
AMP) versus 5.3 (5Ј-AMPS).
DISCUSSION
The anti-apoptotic protein Rcl was described as an enzyme
with unprecedented activity. Rcl is a hydrolase that catalyzes
the cleavage of the N-glycosidic bond of dNMP yielding 2-de-
oxyribose 5-phosphate and the corresponding nucleobase. Rcl
may be considered as another catabolic enzyme that cleaves
dNMP, contributing to the maintenance of a balanced pool of modified dNMPs could be present during this cycle and
nucleotides in addition to 5Ј-nucleotidases (25) but with a
that Rcl could be involved in their catabolism. The pres-
different activity. We hypothesize that understanding Rcl sub- ence of an ethyl or a glyoxal group at position 2 or of a
strate specificity and catalytic mechanism should help in elu-
methyl at position 6 of the guanine ring has no significant
DECEMBER 31, 2010•VOLUME 285•NUMBER 53
JOURNAL OF BIOLOGICAL CHEMISTRY 41811

Rcl Active Site
TABLE 6
TABLE 7
Ribonucleosides 5ꢀ-monophosphate as potential ligands;
modifications on the nucleobase
Ribonucleosides 5ꢀ-monophosphate as potential ligands;
modifications on the sugar part
NI, no inhibition; XMP, xanthosine 5Ј-monophosphate; ZMP, 5-aminoimidazole-
NI, no inhibition; 2-F-ara-AMP, fludarabine; 5Ј-AMS, adenosine 5Ј-monosulfate;
4
-carboxamide 1-␤-D-ribofuranosyl 5Ј-monophosphate.
5Ј-AMPS, adenosine 5Ј-monophosphorothioate.
influence on the affinity and on the catalytic efficiency. The
addition of a methyl group at position 7 results in the for-
mation of a reactive cation that enhances depurination of
the 2Ј-deoxyribonucleotide. Thus, none of the modified
deoxyribonucleoside 5Ј-monophosphates tested is a spe-
cific substrate of Rcl. The question of the role of Rcl in the
maintenance of a balanced pool of nucleotides or in the
catabolism of anomalous deoxynucleotides remains
unanswered.
ous nucleobases lead to similar conclusions. As observed by
Holguin and Cardinaud (30) with purine as substrates for
trans-N-deoxyribosylase, substitutions at positions 2 and 6 are
of minor importance, whereas the imidazole moiety is less
tolerant. The position 8 does not accept substituents for steric
reasons.
Nevertheless, the fact that they are all substrates, even if
they have different affinities, confirms that the active site is
open, making relatively smooth interactions with the nucleo-
The recognition of the sugar part displays some similitude
with NDT but also important differences. Although not abso-
lutely required, the OH groups at positions 2Ј and 3Ј of the
base. The K values for Rcl inhibition by nucleotides with vari- ribose are important. If they can be suppressed, as in the case
i
4
1812 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 53•DECEMBER 31, 2010

Rcl Active Site
of acyclovir 5Ј-monophosphate, no additional groups can
(39). Paradoxically, c-Myc also activates rcl expression (1),
be introduced at these two positions. The configuration of the whose product hydrolyzes dNMP.
sugar is also crucial as fludarabine does not compete with
dGMP. This differs from the situation of NDT where 2Ј-
fluoro-2Ј-deoxyarabinonucleosides inhibit the transferase
activity. 2Ј-Fluoro-2Ј-deoxyarabinonucleosides have allowed
trapping of the NDT-DFDAP (2,6-diamino-9-(2Ј-deoxy-2Ј-
fluoro-␤-D-arabinofuranosyl)-9H-purine) covalent intermedi-
ate and identification of Glu-98 as the nucleophile (31),
whereas no Rcl-deoxyribose 5-phosphate intermediate was
found.
However, it has to be mentioned that dNTP pools are also
regulated by enzymes of the salvage pathway. Deoxycytidine
(dCK) and deoxyguanosine kinases are constitutively ex-
pressed (40), and dCK activity is positively regulated by phos-
phorylation (41). Both enzyme activities are significantly ele-
vated in cell lines as they start to proliferate (42), and
deoxyguanosine kinase was shown to be relocated from the
mitochondrial matrix to the cytosol at the early step of apo-
ptosis (43). Thus, the anti-apoptotic role of Rcl could be at-
tributed to a counter activity of the deoxycytidine and deox-
yguanosine kinases to maintain cellular homeostasis.
A positively charged pocket composed of Ser-87, Ser-117Ј,
Ser-17, and Arg-19 was proposed to form hydrogen bonds
with the negative charges of the phosphate group. The selec-
tivity of the recognition of the phosphate group is high as it
cannot be replaced by a carboxylate or a sulfate group, and
the strength of the binding is influenced by the net charge.
Several proteomic studies have shown that Rcl is phosphor-
ylated on three different serine residues Ser-12, Ser-28, and
Ser-169 (19–23). According to the rat Rcl structure, Ser-12
and Ser-169 are located in flexible regions that do not interact
with the core structure of the protein. In rat these two flexible
regions can be deleted without affecting the enzymatic activ-
ity (13). According to this, the phosphomimetic S158E has the
same affinity for dGMP as the wild type and a comparable
catalytic efficiency. Whether these flexible regions and the
corresponding phosphorylated serine are involved in protein-
protein interactions (22), protein stability, or cellular localiza-
tion remains to be determined. Ser-28 is conserved in all Rcl
identified so far. Mutation of the corresponding rat serine
Acknowledgments—We thank Val e´ rie Huteau, Amandine Cohen,
Wen Luo for their technical contribution, Gilles Labesse for heplful
discussions and small angle x-ray scattering experiments, Pr. Ioan
9
Lascu for N -deaza-dGMP, and Jason Hargreaves and Yves Janin
for proofreading. Small angle x-ray scattering experiments were re-
corded on the beamline SWING in SOLEIL (Saint-Aubin, France)
with the kind help of Javier Perez.
REFERENCES
1
. Lewis, B. C., Shim, H., Li, Q., Wu, C. S., Lee, L. A., Maity, A., and Dang,
C. V. (1997) Mol. Cell. Biol. 17, 4967–4978
2. Lewis, B. C., Prescott, J. E., Campbell, S. E., Shim, H., Orlowski, R. Z.,
and Dang, C. V. (2000) Cancer Res. 60, 6178–6183
3
. Rhodes, D. R., Barrette, T. R., Rubin, M. A., Ghosh, D., and Chinnaiyan,
A. M. (2002) Cancer Res. 62, 4427–4433
4
. Shin, S., Bosc, D. G., Ingle, J. N., Spelsberg, T. C., and Janknecht, R.
(
2008) J. Cell. Biochem. 105, 866–874
(
Ser-17) to an alanine has no consequence, whereas its change
5. Gorgun, G., Ramsay, A. G., Holderried, T. A., Zahrieh, D., Le Dieu, R.,
Liu, F., Quackenbush, J., Croce, C. M., and Gribben, J. G. (2009) Proc.
Natl. Acad. Sci. U.S.A. 106, 6250–6255
to glutamic acid, to mimic phosphorylation, completely abol-
ishes Rcl activity. Ser-17 is located in the loop between the ␤1
strand and the ␣1 helix and is, thus, accessible to protein ki-
nases. CKII was predicted to phosphorylate rat Ser4–158,
cdc2, or ATM (ataxia telangiectasia mutated) human S169
6
. Peart, M. J., Smyth, G. K., van Laar, R. K., Bowtell, D. D., Richon, V. M.,
Marks, P. A., Holloway, A. J., and Johnstone, R. W. (2005) Proc. Natl.
Acad. Sci. U.S.A. 102, 3697–3702
7. Almon, R. R., DuBois, D. C., and Jusko, W. J. (2007) Endocrinology 148,
(
32). As no protein kinase was proposed for the phosphoryla-
2209–2225
8
. Miki, Y., Suzuki, T., Tazawa, C., Ishizuka, M., Semba, S., Gorai, I., and
Sasano, H. (2005) Cancer Lett. 220, 197–210
tion of the two other serines, further studies are, thus, re-
quired to identify them. It will also be important to determine
whether the expression of Rcl is cell cycle-regulated and if its
phosphorylation state varies during the cycle. The identifica-
tion of a phosphorylated peptide in only M phase-arrested
cells supports this hypothesis (19).
9
. Ghiorghi, Y. K., Zeller, K. I., Dang, C. V., and Kaminski, P. A. (2007)
J. Biol. Chem. 282, 8150–8156
10. Brown, N. S., and Bicknell, R. (1998) Biochem. J. 334, 1–8
11. Kaminski, P. A. (2002) J. Biol. Chem. 277, 14400–14407
1
1
1
1
2. Armstrong, S. R., Cook, W. J., Short, S. A., and Ealick, S. E. (1996) Struc-
ture 4, 97–107
The cell deoxyribonucleotide triphosphate pools are regu-
lated by a network of enzymes involved in their synthesis (de
novo and salvage pathways) and in their degradation (nucle-
otidases). Deregulation of this control leads to imbalance
pools, which has consequences on DNA replication fidelity,
maintenance of the nuclear DNA, and cell death (33, 34).
dNTPs levels are generally elevated in actively dividing cells
compared with normal cells (35), and their accumulation
could be one of the events involved in the mutator phenotype
in cancer (36).
3. Yang, Y., Padilla, A., Zhang, C., Labesse, G., and Kaminski, P. A. (2009) J.
Mol. Biol. 394, 435–447
4. Doddapaneni, K., Mahler, B., Pavlovicz, R., Haushalter, A., Yuan, C., and
Wu, Z. (2009) J. Mol. Biol. 394, 423–434
5. Hirsch, A. K., Fischer, F. R., and Diederich, F. (2007) Angew. Chem. Int.
Ed. Engl. 46, 338–352
16. Tener, G. M. (1961) J. Am. Chem. Soc. 83, 159–168
17. Epp, J. B., and Widlanski, T. S. (1999) J. Org. Chem. 64, 293–295
18. Mizuno, Y., Ikehara, M., Watanabe, K. A., Suzaki, S., and Itoh, T. (1963)
J. Org. Chem. 28, 3329–3331
1
9. Dephoure, N., Zhou, C., Vill e´ n, J., Beausoleil, S. A., Bakalarski, C. E.,
Elledge, S. J., and Gygi, S. P. (2008) Proc. Natl. Acad. Sci. U.S.A. 105,
An increase in the nucleotide pool during phase S is essen-
tial for cell proliferation (37). c-Myc directly activates genes
involved in purine and pyrimidine biosynthesis, and a deregu-
lation of c-Myc leads to an increase in nucleotide pools (38)
1
0762–10767
2
0. Molina, H., Horn, D. M., Tang, N., Mathivanan, S., and Pandey, A.
(
2007) Proc. Natl. Acad. Sci. U.S.A. 104, 2199–2204
21. Gauci, S., Helbig, A. O., Slijper, M., Krijgsveld, J., Heck, A. J., and Mo-
DECEMBER 31, 2010•VOLUME 285•NUMBER 53
JOURNAL OF BIOLOGICAL CHEMISTRY 41813

Rcl Active Site
hammed, S. (2009) Anal. Chem. 81, 4493–4501
33. Kunz, B. A., Kohalmi, S. E., Kunkel, T. A., Mathews, C. K., McIntosh,
E. M., and Reidy, J. A. (1994) Mutat. Res. 318, 1–64
34. Bebenek, K., Roberts, J. D., and Kunkel, T. A. (1992) J. Biol. Chem. 267,
3589–3596
2
2. Mayya, V., Lundgren, D. H., Hwang, S. I., Rezaul, K., Wu, L., Eng, J. K.,
Rodionov, V., and Han, D. K. (2009) Sci. Signal. 2, ra46
3. Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P.,
and Mann, M. (2006) Cell 127, 635–648
2
35. Traut, T. W. (1994) Mol. Cell. Biochem. 140, 1–22
36. Loeb, L. A. (2001) Cancer Res. 61, 3230–3239
37. Mathews, C. K. (2006) FASEB J. 20, 1300–1314
38. Liu, Y. C., Li, F., Handler, J., Huang, C. R., Xiang, Y., Neretti, N., Sedivy,
J. M., Zeller, K. I., and Dang, C. V. (2008) PLoS One 3, e2722
39. Mannava, S., Grachtchouk, V., Wheeler, L. J., Im, M., Zhuang, D.,
Slavina, E. G., Mathews, C. K., Shewach, D. S., and Nikiforov, M. A.
(2008) Cell Cycle 7, 2392–2400
2
2
4. Burcham, P. C. (1999) Mutat. Res. 443, 11–36
5. Hunsucker, S. A., Mitchell, B. S., and Spychala, J. (2005) Pharmacol.
Ther. 107, 1–30
2
2
6. De Bont, R., and van Larebeke, N. (2004) Mutagenesis 19, 169–185
7. Neeley, W. L., and Essigmann, J. M. (2006) Chem. Res. Toxicol. 19,
4
91–505
2
2
8. Barnes, D. E., and Lindahl, T. (2004) Annu. Rev. Genet. 38, 445–476
9. Hah, S. S., Mundt, J. M., Kim, H. M., Sumbad, R. A., Turteltaub, K. W.,
and Henderson, P. T. (2007) Proc. Natl. Acad. Sci. U.S.A. 104,
40. Eriksson, S., Munch-Petersen, B., Johansson, K., and Eklund, H. (2002)
Cell. Mol. Life Sci. 59, 1327–1346
1
1203–11208
41. Smal, C., Vertommen, D., Bertrand, L., Ntamashimikiro, S., Rider, M. H.,
Van Den Neste, E., and Bontemps, F. (2006) J. Biol. Chem. 281,
4887–4893
3
3
0. Holguin, J., and Cardinaud, R. (1975) Eur. J. Biochem. 54, 515–520
1. Porter, D. J., Merrill, B. M., and Short, S. A. (1995) J. Biol. Chem. 270,
1
5551–15556
42. Fyrberg, A., Albertioni, F., and Lotfi, K. (2007) Biochem. Biophys. Res.
Commun. 357, 847–853
3
2. Huang, H. D., Lee, T. Y., Tzeng, S. W., and Horng, J. T. (2005) Nucleic
Acids Res. 33, W226–W229
43. J u¨ llig, M., and Eriksson, S. (2001) J. Biol. Chem. 276, 24000–24004
4
1814 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 53•DECEMBER 31, 2010

Enzymology:
Probing the Active Site of the
Deoxynucleotide N-Hydrolase Rcl Encoded
by the Rat Gene c6orf108
Christelle Dupouy, Chi Zhang, André Padilla,
Sylvie Pochet and Pierre Alexandre Kaminski
J. Biol. Chem. 2010, 285:41806-41814.
doi: 10.1074/jbc.M110.181594 originally published online October 20, 2010
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