From the Dyson Perrins Laboratory and The Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QY, United Kingdom
Received for publication, October 29, 2002, and in revised form, January 3, 2003
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ABSTRACT |
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AlkB is one of four proteins involved in the
adaptive response to DNA alkylation damage in Escherichia
coli and is highly conserved from bacteria to humans. Recent
analyses have verified the prediction that AlkB is a member of the
Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family
of enzymes. AlkB mediates repair of methylated DNA by direct
demethylation of 1-methyladenine and 3-methylcytosine lesions.
Other members of the Fe(II) and 2OG-dependent oxygenase
family, including those involved in the hypoxic response, are
targets for therapeutic intervention. Assays measuring 2OG turnover were used to investigate the selectivity of AlkB.
1-Methyladenosine, 1-methyl-2'-deoxyadenosine, 3-methylcytidine,
and 3-methyl-2'-deoxycytidine all stimulated 2OG turnover by AlkB but
were not demethylated indicating an uncoupling of 2OG and prime
substrate oxidation and that oligomeric DNA is required for
hydroxylation and subsequent demethylation. In contrast the equivalent
unmethylated nucleosides did not stimulate 2OG turnover indicating that
the presence of a methyl group in the substrate is important in
initiating oxidation of 2OG. Stimulation of 2OG turnover by
1-methyladenosine was highly dependent on the presence of a reducing
agent, ascorbate or dithiothreitol. Following the observation that AlkB
is inhibited by high concentrations of 2OG, analogues of 2OG, including
2-mercaptoglutarate, were found to specifically inhibit AlkB. The
flavonoid quercetin inhibits both AlkB and the 2OG oxygenase
factor-inhibiting hypoxia-inducible factor (FIH) in vitro.
FIH inhibition by quercetin occurs in the presence of excess iron
indicating a specific interaction, while the inhibition of AlkB by
quercetin is, predominantly, due to nonspecific iron chelation.
The integrity of the genome is maintained by a set of DNA repair
enzymes, including those that repair alkylation damage (1). DNA can be
alkylated by a variety of agents that occur both exogenously and
endogenously (2, 3). Alkylating agents are used in some chemotherapy
treatments, and alkylated DNA bases have been detected in the urine of
smokers (4). AlkB catalyzes demethylation of DNA and along with AlkA,
AidB, and Ada is one of four proteins involved in the adaptive response
to alkylation damaged DNA in Escherichia coli (5). Ada,
which has been termed the "suicidal repair protein," contains two
active sites, one of which demethylates O6-methylguanine by irreversible transfer of the
methyl group to a cysteine (5, 6). The second active site removes
methyl groups from S-methyl phosphoesters by
nucleophilic methylation of another cysteine residue (5, 7, 8).
Methylation of the latter cysteine converts Ada into a strong
transcriptional activator of both its own production and the other
three proteins of the adaptive response (9). AlkA is a DNA glycosylase
with a wide substrate selectivity, including excision of cytotoxic 3-methyladenine residues (10). AlkA "flips" the damaged base out of
the DNA double helix by inserting Leu-125 into the position occupied by
the damaged base (11). The glycosidic bond is then cleaved, and the
abasic site repaired by the standard base excision repair pathway. AidB
is poorly characterized, but it may be involved in the destruction of
certain alkylating agents (5).
AlkB is highly conserved from bacteria to mammals (12). AlkB
knockout mutants are defective at repairing DNA methylation damage
induced by SN2 type methylating agents including dimethyl sulfate, methyl iodide, and methyl methane sulfonate
(MMS)1 (13-15). Recently,
two groups have independently confirmed the predictions based upon
sequence analyses (16) that AlkB is a member of the non-heme
FeII family of oxygenases, requiring a 2-oxoglutarate (2OG)
molecule as a cosubstrate (17, 18). It was demonstrated that AlkB
repairs the cytotoxic 1-methyladenine and 3-methylcytosine lesions in both single and double-stranded DNA by an oxidative process (Fig. 1). The reaction most probably proceeds
by hydroxylation of the methyl group, leading to its extrusion as
formaldehyde. Such a direct repair mechanism is likely to be efficient
as unlike the nucleotide excision repair and base excision repair
pathways, it is not reliant on a complementary strand for maintenance
of genetic information.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Oxidative demethylation of DNA by
AlkB.
The 2OG-dependent family of oxygenase enzymes are involved in a range of metabolic processes in humans including the biosynthesis of collagen and carnitine (19). They also play a key role in the hypoxic response, where it has been proposed that enzymes catalyzing hydroxylations of the hypoxia-inducible factor (HIF) act as oxygen sensors (20, 21). Inhibition of the HIF hydroxylases offers potential to develop pro-angiogenic agents; however, inhibitors of the asparagine and prolyl hydroxylases will need to be selective for the target 2OG oxygenases.
Here we report work that supports the identification of AlkB as a
2OG-dependent oxygenase and demonstrate its inhibition by 2OG analogues. Further, we report that in the presence of the nucleoside substrates 1-methyladenosine, 1-methyl-2'-deoxyadenosine, 3-methylcytidine, and 3-methyl-2'-deoxycytidine, there is a stimulation of 2OG turnover by AlkB. However, the 2OG reaction does not appear to
be coupled to hydroxylation of the prime substrate.
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MATERIALS AND METHODS |
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Materials-- All chemicals were purchased from Sigma; oligonucleotides (Sigma-Genosys); restriction enzymes (New England Biolabs); Pfu Turbo, ligation kit, and competent cells (Stratagene); pET-24a(+) vector (Novagen); Gentra systems kit for purification of E. coli DNA, 1-[14C]-2-oxoglutarate (PerkinElmer Life Sciences); hyamine hydroxide (ICN Radiochemicals); and Opti-phase "SAFE" scintillant (Fisher).
Cloning-- AlkB was cloned from genomic E. coli DNA by PCR using the primers: forward 5'-GGTGGTCATATGTTGGATCTGTTTGCC-3' and reverse 5'-GGTGGTGGATCCTTATTCTTTTTTACCTGC-3'. The PCR product was digested with NdeI and BamHI and cloned into pET-24a(+).
Cell Growth--
The pET-24a(+)-alkB plasmid was transformed
into E. coli BL21(DE3) Gold competent cells, and growth
conditions were optimized. Initially cultures were grown at 37 °C
until the A600 reached 0.6, where
isopropyl--D-thiogalactoside was added to a final concentration of 0.2 mM and the temperature was dropped to
28 °C. Cells were harvested after 4 h by centrifugation. AlkB
was produced as approximately 10% of the total soluble protein (by SDS-PAGE analysis).
Cell Lysis and Protein Purification--
Cells (15 g) were
resuspended on ice in 50 ml of 100 mM MES, pH 5.8, 1 mM dithiothreitol, 40 µg ml1 lysozyme, 4 µg ml
1 DNaseI, then sonicated (Ultrasonics Inc., W-380)
with 4 × 30-s pulses and centrifuged (Beckman JA25-50) for 20 min at 19,000 rpm. The supernatant was filtered through a 0.2-µm
membrane and loaded on to a (50 ml) S-Sepharose column
(Amersham Biosciences), run at 10 ml min
1 in 100 mM MES, pH 5.8. AlkB was eluted with a gradient of 0-1 M NaCl over 400 ml. AlkB-containing fractions were pooled
and diluted 3-fold prior to loading on a (6 ml) Resource-S column (Amersham Biosciences) run at 8 ml min
1; again AlkB was
eluted with a gradient of 0-1 M NaCl over 180 ml.
AlkB-containing fractions were concentrated and loaded onto a (720 ml,
Superdex-75) gel filtration column at 3 ml min
1 in 150 mM TRIS, pH 7.5. This yielded ~30 mg of AlkB with >95% purity (by SDS-PAGE analysis).
Methylation of Oligonucleotides with MMS--
Methylated DNA
oligonucleotides were prepared using a modified version of literature
procedures (22, 23). Polydeoxyadenine (poly(dA)) and polydeoxycytosine
(poly(dC)) were both 15 mer. The 27 mer and 33 mer were of random
sequence and contained all four DNA bases. Prior to reaction with MMS
oligomers were dissolved at 0.05 mM in 10 mM
TRIS, pH 7.5. MMS (240 mM in EtOH) was added to a final
concentration of 24 mM. Reactions were carried out at room
temperature for 24 h. The DNA was precipitated by addition of 3 volumes of ice-cold EtOH (0.3 M acetate, pH 5.2) and
placing at 80 °C for 30 min. The sample was then centrifuged, the
supernatant was removed, and the precipitated DNA was washed six
times with EtOH. DNA concentration was measured by UV spectroscopy.
Methylation of 2'-Deoxyadenosine--
1-Methyl-2'-deoxyadenosine
was prepared using the procedure of Singer et al. (24).
1-Methyl-2'-deoxyadenosine was the major product, produced in ~20%
yield (~75% of starting material remained). Yield was estimated by
integration of the HPLC trace at 260 nm. 1-Methyl-2'-deoxyadenosine was
purified by HPLC using a Phenomenex LUNA C-18 column (250 × 10 mm, 5 µm). All solvents were filtered through 0.2-µm filters and
sparged with He (g) at 100 ml min1 for 20 min before use.
The column was run at 3.2 ml min
1 in 50 mM
ammonium acetate, pH 6.4, and 5% acetonitrile for 25 min, and then a
gradient was run to 30% acetonitrile over 5 min and held there for 10 min. Isolated 1-methyl-2'-deoxyadenosine m/z (ESI+)
266 Da; UV:
max(pH 7) 259 nm,
max(pH 1)
259 nm,
max(pH 13) 260 nm,
inflection(pH
13) 279 nm. As expected these UV profiles were the same as those
obtained for the commercial 1-methyladenosine.
Methylation of 2'-Deoxycytidine--
3-Methyl-2'-deoxycytidine
was prepared using the same procedure as for methylation of
2'-deoxyadenosine. Isolated 3-methyl-2'-deoxycytidine m/z(ESI+) 242 Da; UV: max(pH 7) 278 nm,
max(pH 1) 278 nm,
max(pH 13) 267 nm.
As expected these UV profiles were the same as those obtained for the
commercial 3-methylcytidine.
1-[14C]2-Oxoglutarate Assays--
This assay is
based on the method used to measure 14CO2
release by -ketoisocaproate oxygenase (25). Standard assay
conditions comprise a total volume of 100 µl, 50 mM TRIS, pH 7.5, 4 mM ascorbate, 160 µM 2OG (2.5% 1-[14C]), 80 µM
(NH4)2SO4.FeSO4.6H2O,
0.48 mg ml
1 catalase, 12.5 µM AlkB.
Generally two stocks were made, one total volume of 25 µl contained
AlkB and FeII, the second total volume of 75 µl contained
all other reagents. Assays were started by the addition of the
FeII, AlkB stock to the other stock. A tube containing 200 µl of hyamine hydroxide was added, and the vial sealed. The assays
were incubated at 37 °C for 5 min and quenched with 20% (v/v)
trifluoroacetic acid (300 µl). They were then left on ice for 30 min
to collect 14CO2 gas before the hyamine
hydroxide was removed and treated with scintillant for counting
(Beckman, LS6500). Assay points were performed in triplicate unless
otherwise stated, values quoted are an average with standard deviation
given as the error. DNA oligomers were incubated at a final
concentration of 10 µM. Nucleoside and base substrates
were dissolved at 20 mM in Me2SO, these were added to a final concentration of 400 µM, and AlkB
activity was not affected by the presence of 2% (v/v)
Me2SO. For the inhibitor, replacement of ascorbate and
variation of 2OG concentration assays 800 µM
1-methyladenosine was present. Inhibitors were assayed at five
concentrations around the approximate IC50 value; the maximum concentration of inhibitor tested was 4 mM. Initial
rate was plotted against inhibitor concentration in an Excel worksheet, a curve was fitted to the data, and the equation for the curve was
solved for half the rate in the absence of inhibitor to give an
approximate IC50 value. For assays in which
FeII concentration was varied with inhibitor concentration
held at IC50, FeII was added to a final
concentration of 40, 80, 160, 250, and 500 µM. For the
assays using an alternative reducing agent a 100-mM stock
of reducing agent was made and added to a final concentration of 4 mM. For variation of ascorbate concentration assays,
ascorbate was added to a final concentration of 0, 0.25, 0.5, 1, and 4 mM. For variation of 2OG concentration assays, 2OG was
added to a final concentration 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500, and 700 µM; these assays were single
points and carried out at both 80 and 250 µM
FeII. Graphs showing variation of ascorbate and 2OG
concentration were plotted in SigmaPlot.
HPLC/LCMS Assays--
HPLC was carried out using a
Synergi C-18 Hydro column (250 × 4.6 mm 4 µm) from Phenomenex,
and the elutant was analyzed using a Photodiode Array Detector. All
solvents were filtered through 0.2-µm filters and sparged with He (g)
at 100 ml min1 for 20 min before use. Prior to injection
1% (v/v) acetic acid was added to samples, and they were centrifuged
at 13,000 rpm for 10 min. The column was run at 0.8 ml
min
1 in 50 mM ammonium acetate, pH 6.4, and
2% acetonitrile for 25 min, and then a gradient was run to 30%
acetonitrile over 5 min and held there for 10 min. AlkB assays for HPLC
were carried out using the same concentrations of reagents as above
except that no radiolabeled 2OG was present, and they were quenched
with 1 volume of MeOH after 40 min and placed on ice to precipitate the protein. HPLC runs for 1-methlyadenosine, 1-methyl-2'-deoxyadenosine, 3-methlycytidine, and 3-methyl-2'-deoxycytidine assays were also analyzed with some demethylated nucleoside added to confirm that product would be detected if demethylation had occurred. The same conditions were used for the LCMS assays with products being
analyzed with a Micromass ZMD mass spectrometer electrospray
ionization (+).
Factor-inhibiting HIF Assays--
Factor-inhibiting HIF (FIH)
was purified and assayed as described previously with the C-terminal
fragment of HIF1-(775-826) as a glutathione
S-transferase fusion protein was used as a substrate (21).
Synthesis of Inhibitors--
N-Oxalylglycine,
N-oxalyl-4S-alanine,
N-oxalyl-4R-alanine (26) and 2-hydroxyglutarate
(27) were prepared according to literature procedures.
2-Thiono(N-oxalylglycine) was prepared as follows:
the dimethyl ester of 2-thiono(N-oxalylglycine) was prepared
as reported (28) and subsequently deprotected to give the desired
product (26) (1.7 mmol, 85%) as a yellow solid, mp 105-106 °C;
max (NaCl, MeOH)/cm
1 1730, 1697 (C=O);
H(200 MHz; Me2SO-d6)
4.27 (2 H, d, 3JHH 6.0, CH2), 10.9 (1 H, br t, NH);
C(50 MHz;
Me2SO-d6) 47.9 (CH2),
163.4, 169.4, 189.5 (C=O, C=S); m/z(APCI
) 162 (M-H+, 30%), 118 (M-H+-CO2,
100%); HRMS 161.9861 calculated for M-H+
(C4H4NO4S), 161.9861 found.
(±)-2-Mercaptoglutarate was prepared according to the literature
procedure (29). 2-Acetylsulphanyl-pentanedioic acid diethylester was
prepared as reported (29, 30) then deprotected by mixing 10 mmol
(2.62g) of 2-acetylsulphanyl-pentanedioic acid diethylester with 40 ml
of 2 M aqueous sodium hydroxide solution to give 1.37 g (8.3 mmol, 83%) of (±)-2-mercaptoglutarate as a colorless solid (spectroscopic data not previously reported): mp 95-96 °C;
max (NaCl, MeOH)/cm
1 1708 (C=O);
H (200 MHz; Me2SO-d6)
1.70-2.13 (2 H, m,
SCHCH2CH2), 2.35 (2 H, t,
3JHH 7.5, SCHCH2CH2), 3.37 (1 H, t,
3JHH 7.5, SCH), 12.59 (2 H, br s,
COOH), SH signal probably obscured by Me2SO;
C(50 MHz; Me2SO-d6)
31.0, 32.0 (CH2CH2, SCH signal obscured by
Me2SO resonance), 174.5, 174.8 (C=O);
m/z (APCI
) 163 (M-H+,
100%).
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RESULTS AND DISCUSSION |
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Expression and Purification-- The alkB gene was cloned from E. coli and inserted into the pET-24a(+) vector. A three column protocol was developed for purification of AlkB based on cation exchange and gel filtration chromatography, giving the desired protein at >95% purity by SDS-PAGE analysis.
Substrate Assays and Structural Recognition-- AlkB activity was analyzed by incubating AlkB with 1-[14C]2OG and measuring the release of 14CO2 upon formation of succinate (25). The results were consistent with the conclusions of Falnes et al. and Trewick et al. that AlkB is a 2OG-dependent FeII dioxygenase (17, 18). Several oligonucleotides including poly(dA) and poly(dC) were reacted with the SN2 methylating agent MMS. When assayed with AlkB, the unmethylated oligonucleotides did not increase 2OG turnover significantly above the uncoupled rate (Table I). However, the methylated oligonucleotides gave rise to a significant increase in decarboxylation of 2OG by AlkB. The methylated oligomers used to ascertain the identity of the substrates of AlkB (18) are not precisely defined in a chemical sense as they are made by nonspecific methylation of an oligomeric substrate. Consequently, monomeric, potential substrates were assayed to probe the structural features of DNA that are required for AlkB catalysis.
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Various methylated bases, nucleotides, and nucleosides were assayed as
potential substrates for AlkB, including those with the 1-methyladenine
and 3-methylcytosine bases that are substrates for AlkB when
incorporated into oligomeric DNA (Table
II) (17, 18). Incubation with
1-methyladenosine or 1-methyl-2'-deoxyadenosine was found to induce up
to a 7-fold increase in 2OG turnover. 3-Methylcytidine and
3-methyl-2'-deoxycytidine only brought about a 2- and 3-fold increase,
respectively. This suggests that 1-methyladenine rather than
3-methylcytosine lesions may be preferred substrates for AlkB. An
HPLC-based assay was developed to investigate the production of
nucleosides from their methylated counterparts by AlkB. Neither analysis by photodiode array HPLC or by ESI-LCMS led to the detection of any demethylated nucleoside for the incubation of either
1-methyladenosine, 1-methyl-2'-deoxyadenosine, 3-methylcytidine, or
3-methyl-2'-deoxycytidine with AlkB. Thus, unlike the situation with
oligomeric DNA the turnover of 2OG and hydroxylation of the prime
substrate appear to be predominantly uncoupled in the presence of
nucleoside substrate analogues. This is of interest with regard to
substrate recognition by AlkB. Crystallographic, spectroscopic, and
solution studies of other 2OG oxygenases have indicated that binding of
the prime substrate to the enzyme complex is required to initiate
dioxygen binding and subsequent reaction of 2OG (31-33). It appears
that 1-methyladenosine and 3-methylcytidine nucleosides are recognized by the AlkB active site and can stimulate reaction of 2OG with dioxygen, but the reactive oxidizing species, believed to be
[FeIV=O FeIII-O·], does
not react with the methyl group. It may be that when the requisite
connections are not made to the DNA oligomer the reactive oxidized
species is not positioned correctly to effect hydroxylation. To effect
further 2OG catalysis the original FeII center must be
regenerated by an alternative pathway possibly involving ascorbate (see
below).
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To further investigate the structural motifs recognized by AlkB, assays with 1-methyladenine and various non-methylated bases and nucleosides were carried out (Table II). The rate of 2OG turnover remained consistent at the level observed in the absence of substrate when AlkB was incubated with adenosine, 2'-deoxyadenosine, or adenine, i.e. in the absence of the 1-methyl group. For both 1-methyladenine, 1-methyl-2'-deoxyadenosine, and 1-methyladenosine there was an increase in the rate of 2OG turnover, indicating that the 1-methyl group is an absolute requirement for stimulation of the reaction between 2OG and O2. 2OG conversion was lower for 1-methyladenine than for 1-methyladenosine and 1-methyl-2'-deoxyadenosine, indicating that the presence of a sugar moiety improves substrate recognition by AlkB, possibly by direct contacts between the enzyme and the sugar giving rise to tighter enzyme/substrate analogue binding. Similarly, cytidine, 2'-deoxycytidine and cytosine all failed to bring about an increase in the rate of 2OG conversion, indicating that AlkB activity is stimulated by binding the methyl group of 3-methylcytidine nucleosides.
Stimulation of 2OG oxidation without coupling to hydroxylation of the prime substrate has previously been observed for procollagen prolyl-4-hydroxylase with substrate analogues (34). Further, mutagenesis studies on deacetoxycephalosporin C synthase have resulted in an uncoupling of 2OG turnover and prime substrate hydroxylation (35). For procollagen prolyl-4-hydroxylase the uncoupled oxidation of 2OG has been linked to the requirement for ascorbate as well as FeII and 2OG. It has been proposed that an ascorbate molecule regenerates the FeII center in the event of uncoupled turnover of 2OG to the ferryl intermediate and succinate (36).
Role of Ascorbate--
Uncoupled 2OG turnover by AlkB was found to
be highly dependent on ascorbate both in the presence and absence of
1-methyladenosine, but more so in the former case (Fig.
2). However, the amount of ascorbate
required for optimal uncoupled 2OG turnover was far in excess of
stoichiometry to 2OG. The role of ascorbate was investigated further by
attempting to replace it in the assay with alternative reducing agents
(Table III). Activity was maintained with
D-isoascorbate and dithiothreitol,
the latter giving approximately 90% of the activity with
L-ascorbate. All other agents tested
resulted in a significant loss of activity, though when compared with
the rate of reaction in the absence of reducing agent,
-mercaptoethanol gave a moderate increase in activity while addition
of dithionite and 4-nitrocatechol brought about a reduction in
rate.
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For prolyl-4-hydroxylase, in the presence of poly(L-proline) (a substrate that stimulates uncoupled 2OG turnover but is not hydroxylated) a stoichiometric reaction of 2OG with disappearance of ascorbate has been reported (36). Recent work on anthocyanidin synthase, a 2OG-dependent oxygenase involved in the biosynthesis of flavonoids in plants, has suggested that an ascorbate molecule may bind at the active site in the presence of the prime substrate and might be involved in catalysis (37). Elucidation of the exact role of ascorbate in AlkB catalysis will require further work. However, the observation that it can be replaced by dithiothreitol suggests that its role may not be entirely specific.
Inhibition of AlkB--
During the course of this work it was
found that 2OG concentrations greater than 200 µM caused
a reduction in the initial rate of 2OG turnover (Fig.
3). This inhibition was unaffected by an
increase in FeII concentration, and the same pattern was
observed at both 80 µM and 250 µM
FeII. Therefore, AlkB inhibition at high 2OG concentrations
is unlikely to be a result of FeII chelation by 2OG. The
2OG-dependent dioxygenases deacetoxycephalosporin C
synthase and thymine-7-hydroxylase have also been observed to be
inhibited by high concentrations of 2OG (33, 38). Enzyme inhibition by
high substrate concentrations is often thought to be a result of two
molecules of substrate binding to the enzyme to produce an inactive
complex (39). It is possible, though unlikely, that inhibition of AlkB
by 2OG has physiological significance. The inhibition of AlkB by high
concentrations of 2OG prompted us to investigate the inhibition of AlkB
by a variety of structural analogues of 2OG.
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The ability to selectively inhibit enzymes that repair alkylation
damage to DNA could potentially allow a reduction in the amount of
cytotoxic alkylating agents used in cancer chemotherapy. Various
compounds were therefore assayed as inhibitors of AlkB (Table
IV). N-Oxalylglycine differs
from 2OG by the replacement of the 3-CH2 with an NH and has
previously been reported to be a competitive inhibitor of both the
procollagen (26) and the HIF hydroxylases (20, 21).
N-Oxalylglycine was found to be a moderate inhibitor of AlkB
with an IC50 of 0.70 mM. The 2 thiono derivative of N-oxalylglycine gave a similar
IC50 of 0.81 mM. Both R- and
S-enantiomers of N-oxalylalanine were then tested as inhibitors. Neither stereochemistry was found to be a good inhibitor
with IC50 values of 2.4 and 3.3 mM for the
S- and R-enantiomers, respectively, both
significantly higher than that for N-oxalylglycine. This
observation implies that space within the 2OG binding pocket of AlkB
may be limited compared with some other 2OG oxygenases. N-Oxalyl-4S-alanine was also found to be a better
inhibitor than the 4R of procollagen prolyl-4-hydroxylases
(26). Inhibitors in which the 2-keto of 2OG was replaced with a thiol
or an alcohol were then tested. As these compounds lack the carbonyl
group that reacts with dioxygen they are relatively resistant to
nucleophilic attack by an activated dioxygen molecule. The C-2 thiol,
(±)-2-mercaptoglutarate had the lowest IC50 (0.12 mM) of the 2OG analogues tested; in contrast the C-2
alcohol showed no inhibition up to a concentration of 4 mM. The significant difference between inhibition by the alcohol and the thiol is interesting and is possibly a result of the
high affinity of the thiol for FeII. Changing the
concentration of FeII had no effect on inhibition by
(±)-2-mercaptoglutarate, indicating that simple solution-based
FeII chelation was not responsible for inhibition. It is
also noteworthy that, during catalysis by isopenicillin N
synthase, an oxidase with a close structural relationship to the 2OG
oxygenases, ligation of its thiol substrate
(L)--(
-aminoadipyl)-(L)-cysteinyl-(D)-valine to the FeII center is a key step in catalysis (40). Thiols
are present in a number of pharmaceuticals, therefore it may be of
interest to pursue their use in attempts to prepare generic templates
for inhibition of 2OG-dependent oxygenases.
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Recently, it has been proposed that the naturally occurring flavonoid
quercetin, (a compound used in dietary supplements) can regulate the
hypoxic response by inhibition of the HIF hydroxylases involved in the
degradation of the HIF-1 protein (41). HIF-1
is hydroxylated by
members of the FeII, 2OG-dependent dioxygenase
family at an asparagine residue by FIH and two proline residues by the
prolyl hydroxylase enzymes. Quercetin is a good iron chelator, and its
regulatory role could in part be due to its effect on iron
concentrations and consequently the activity of the FeII
dioxygenases. Under the standard AlkB assay conditions, quercetin was
found to have an IC50 of 0.08 mM for inhibition
of 2OG turnover by AlkB. However, AlkB activity could be returned to
normal levels by addition of excess FeII (160 µM) (data not shown). This indicates that there
is unlikely to be any specific and tight binding
interaction between quercetin and AlkB. However it may still be
possible to modulate the activity of AlkB using nonspecific iron
chelators. For comparison the effect of quercetin on the asparagine
hydroxylase FIH was also explored. Quercetin was found to inhibit FIH
with an IC50 of 0.6 mM. Increasing the
FeII concentration failed to return FIH activity to the
normal rate, indicating a specific interaction between FIH and
quercetin. The specificity of quercetin for FIH inhibition over that of
AlkB could aid the design of inhibitors that specifically inhibit
members of the 2OG-dependent family of dioxygenases
involved in the hypoxic response or in repair of alkylated DNA. The
inhibition of FIH by quercetin may also reflect a role for flavonoids
in human metabolism, but to conclude such at present is premature.
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ACKNOWLEDGEMENTS |
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We thank the Engineering and Physical Sciences Research Council, Biotechnology and Biological Research Council, Wellcome Trust, European Union, and Deutscher Akademischer Austauschdienst for financial support of the work and Dr. N. J. Oldham for mass spectrometry analyses.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-1865-275625;
Fax: 44-1865-275625; E-mail:
christopher.schofield@chem.ox.ac.uk.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211058200
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ABBREVIATIONS |
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The abbreviations used are: MMS, methyl methane sulfonate; 2OG, 2-oxoglutarate; FIH, factor-inhibiting HIF; HIF, hypoxia-inducible factor; MES, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; LCMS, liquid chromatography mass spectrometry; poly(dA), polydeoxyadenine; poly(dC), polydeoxycytosine.
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