Information on EC 1.14.11.53 - mRNA N6-methyladenine demethylase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.11.53
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RECOMMENDED NAME
GeneOntology No.
mRNA N6-methyladenine demethylase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
N6-methyladenine in mRNA + 2-oxoglutarate + O2 = adenine in mRNA + formaldehyde + succinate + CO2
show the reaction diagram
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SYSTEMATIC NAME
IUBMB Comments
mRNA-N6-methyladenosine,2-oxoglutarate:oxygen oxidoreductase (formaldehyde-forming)
Contains iron(II). Catalyses oxidative demethylation of mRNA N6-methyladenine. The FTO enzyme from human can also demethylate N3-methylthymine from single stranded DNA and N3-methyluridine from single stranded RNA [1,2] with low activity [3].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
5'-CUCGAUACG(m6A)UCCGGUCAAA-3' + 2-oxoglutarate + O2
5'-CUCGAUACGAUCCGGUCAAA-3' + formaldehyde + succinate + CO2
show the reaction diagram
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?
5'-UACACUCGAUCUGG(m6A)CUAAAGCUGCUC-3'-biotin + 2-oxoglutarate + O2
5'-UACACUCGAUCUGGCUAAAGCUGCUC-3'-biotin + formaldehyde + succinate + CO2
show the reaction diagram
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-
?
CCCC(m6A)CCCCCCCCC + 2-oxoglutarate + O2
+ formaldehyde + succinate + CO2
show the reaction diagram
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30% demethylation
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-
?
GA(m6A)CA + 2-oxoglutarate + O2
GAACA + formaldehyde + succinate + CO2
show the reaction diagram
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39% demethylation
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-
?
GCGG(m6A)CUCCAGAUG + 2-oxoglutarate + O2
GCGGACUCCAGAUG + formaldehyde + succinate + CO2
show the reaction diagram
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31% demethylation
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-
?
GG(m6A)CU + 2-oxoglutarate + O2
GGACU + formaldehyde + succinate + CO2
show the reaction diagram
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37% demethylation
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-
?
N3-methylcytosine in single-stranded DNA + 2-oxoglutarate + O2
cytosine in single-stranded DNA + formaldehyde + succinate + CO2
show the reaction diagram
N3-methylthymine in single-stranded DNA + 2-oxoglutarate + O2
thymine in single-stranded DNA + formaldehyde + succinate + CO2
show the reaction diagram
N3-methyluracil in single-stranded mRNA + 2-oxoglutarate + O2
uracil in single-stranded mRNA + formaldehyde + succinate + CO2
show the reaction diagram
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
show the reaction diagram
N6-methyladenine in NANOG mRNA + 2-oxoglutarate + O2
adenine in NANOG mRNA + formaldehyde + succinate + CO2
show the reaction diagram
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?
N6-methyladenine in single-stranded DNA + 2-oxoglutarate + O2
adenine in single-stranded DNA + formaldehyde + succinate + CO2
show the reaction diagram
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the ALKBH5 catalytic domain (residues 74294) is active and can demethylate ssDNA and ssRNA with similar activity. m6A ssDNA may not be a physiologically relevant ALKBH5 substrate
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?
N6-methyladenine in single-stranded DNA oligonucleotide + 2-oxoglutarate + O2
adenine in single-stranded DNA oligonucleotide + formaldehyde + succinate + CO2
show the reaction diagram
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
N6-methyladenine in mRNA + 2-oxoglutarate + O2
adenine in mRNA + formaldehyde + succinate + CO2
show the reaction diagram
N6-methyladenine in NANOG mRNA + 2-oxoglutarate + O2
adenine in NANOG mRNA + formaldehyde + succinate + CO2
show the reaction diagram
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
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the enzyme requires binding of the cosubstrate 2-oxoglutarate and Fe2+ for catalysis. In the crystallization trial, Fe2+ is replaced with Mn2+ to obtain a catalytically inactive form of the enzyme
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(1-chloro-4-hydroxyisoquinoline-3-carbonyl)glycine
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2-oxoglutarate
citrate
dithiothreitol
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IOX1
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competitive inhibition
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N-oxalylglycine
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Pyridine 2,4-dicarboxylate
moderate inhibitor
succinate
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0025
2-oxoglutarate
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at pH 8.0 and 37C
0.000192
5'-UACACUCGAUCUGG(m6A)CUAAAGCUGCUC-3'-biotin
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at pH 8.0 and 37C
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2.583
CCCC(m6A)CCCCCCCCC
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at pH 7.4 and 37C
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2.251
GA(m6A)CA
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at pH 7.4 and 37C
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1.755
GCGG(m6A)CUCCAGAUG
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at pH 7.4 and 37C
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2.334
GG(m6A)CU
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at pH 7.4 and 37C
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0.00095 - 0.00192
N3-methylthymine in single-stranded DNA
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0.00208 - 0.00851
N3-methyluracil in single-stranded mRNA
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0.00166
N6-methyladenine in mRNA
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pH and temperature not specified in the publication
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.3
2-oxoglutarate
Homo sapiens
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at pH 8.0 and 37C
0.0023
CCCC(m6A)CCCCCCCCC
Homo sapiens
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at pH 7.4 and 37C
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0.0027
GA(m6A)CA
Homo sapiens
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at pH 7.4 and 37C
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0.0029
GCGG(m6A)CUCCAGAUG
Homo sapiens
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at pH 7.4 and 37C
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0.0023
GG(m6A)CU
Homo sapiens
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at pH 7.4 and 37C
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0.000117 - 0.00148
N3-methylthymine in single-stranded DNA
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0.0019 - 0.0033
N3-methyluracil in single-stranded mRNA
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0.0029
N6-methyladenine in mRNA
Homo sapiens
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pH and temperature not specified in the publication
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
120
2-oxoglutarate
Homo sapiens
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at pH 8.0 and 37C
34
0.88
CCCC(m6A)CCCCCCCCC
Homo sapiens
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at pH 7.4 and 37C
213982
1.2
GA(m6A)CA
Homo sapiens
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at pH 7.4 and 37C
213980
1.6
GCGG(m6A)CUCCAGAUG
Homo sapiens
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at pH 7.4 and 37C
213981
1
GG(m6A)CU
Homo sapiens
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at pH 7.4 and 37C
213979
0.77 - 1.23
N3-methylthymine in single-stranded DNA
210622
0.223 - 1.58
N3-methyluracil in single-stranded mRNA
210623
1.75
N6-methyladenine in mRNA
Homo sapiens
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pH and temperature not specified in the publication
210772
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.488 - 0.628
citrate
0.026
N-oxalylglycine
Homo sapiens
Q6P6C2
pH 7.2, 21C
0.347
Pyridine 2,4-dicarboxylate
Homo sapiens
Q6P6C2
pH 7.2, 21C
0.03
succinate
Homo sapiens
Q6P6C2
pH 7.2, 21C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 7.5
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7
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assay at
7.2
assay at
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 33750, calculated from amino acid sequence; x * 35000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with manganese(II) and 2-oxoglutarate, sitting drop vapor diffusion method, using 0.2 M sodium iodide, pH 7.0, 20% (w/v) polyethylene glycol 3350
ALKBH566292 is crystallized in sitting drops at 20C by the vapour diffusion method in the presence of Mn2+ and (1-chloro-4-hydroxyisoquinoline-3-carbonyl)glycine. Crystallization drops contain 0.2 ml of a protein solution containing a final concentration of 10 mg/ml hexahistidine-tagged ALKBH566292, 0.5 mM MnCl2 and 2 mM IOX3 mixed with 0.1 ml of well solution containing 125 mM potassium nitrate and 15% (w/v) polyethylene glycol 3350. Crystals (size 100 x 50 x 50 mM) appeared after 3 months. Crystals are harvested using nylon loops and cryoprotected using well solution diluted with 25% (v/v) glycerol and flashcooled in liquid nitrogen. Crystal structure of human ALKBH5 (residues 66-292) to 2.0 A resolution. ALKBH566292 has a double-stranded beta-helix core fold. The active site metal is octahedrally coordinated by an HXD...H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other ferrous iron-dependent nucleic acid oxygenase
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crystallizations are performed at 24 and 4C using both the hanging drop and sitting drop vapor diffusion methods. Five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. These findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members
hanging drop vapor diffusion method at 18 C. The ALKBH52-oxoglutarate-Mn2+ is crystallized in a buffer containing 0.2 M ammonium dihydrogen phosphate, 20% PEG 3350. The ALKBH5 is crystallized with citrate in a buffer containing ammonium citrate, 20% PEG 3350. Before flashfreezing crystals in liquid nitrogen, crystals are soaked in a cryoprotectant consisting mother liquor plus 12% glycerol
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hanging drop vapor diffusion method, using 0.2 M ammonium citrate dibasic pH 5.1, 20% (w/v) PEG 3350
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hanging-drop vapour-diffusion method, crystal structure of FTO in complex with the mononucleotide 3-methylthymine
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the purified recombinant mRNA N6-methyladenine demethylase appears to be more stable than the purified recombinant murine mRNA N6-methyladenine demethylase in vitro
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DE52 column chromatography, Ni-NTA column chromatography, and Superdex 200 gel filtration
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Ni affinity column chromatography, HiTrap Q column chromatography, and Superdex 75 gel filtration
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Ni-NTA column chromatography, MonoS column chromatography, and Superdex 200 gel filtration
Ni-NTA His-Bind resin column chromatography, Source 15S column chromatography, and Superdex 200 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a plasmid with the vector backbone pNIC28-Bsa4 encoding a hexahistidine-tagged ALKBH566292 construct is transformed into Escherichia coli BL21 (DE3) cells
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3) Rosetta cells
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expression in Escherichia coli BL21 Star (DE3)
expression in the BL21 (DE3) Escherichia coli strain
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mutants of human Alkbh5 are amplified by PCR and subcloned into a modified pET-28a (Novagen) vector encoding a tobacco etch virus protease recognition site. The final clones are verified by DNA sequencing. All of the recombinant plasmids are transformed into Escherichia coli strain BL21(DE3)
the human ALKBH5 catalytic AlkB domain (residues 74294) is subcloned into pET28a-MHL vector and expressed. The cloned vector is transformed into Escherichia coli BL21-V2R-pRARE2. Recombinant protein is produced at 16C as an N-terminal His-tagged protein
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Wild-type and various mutant enzymes cloned into pET-15b are expressed in Escherichia coli strain BL21
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
ALKBH5 is upregulated under hypoxia
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ALKBH5 mRNA levels are significantly increased under hypoxic conditions by greater than 1.6fold in SUM-159 and MDA-MB-435 cells and greater than 2fold in MDA-MB-231 and MCF-7 cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
269A/Q271A
double mutation shows no impact on the repair efficiency of Alkbh5
E153A
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mutant enzyme displays activity similar to wild type
F232A/F234A
mutant exhibits 41% activity toward m6A-containing ssDNA
F232D/Q233D/F234E
the mutant enzyme displays a severe loss of activity, demonstrating only 13.5% of wild-type activity
H204A
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inactive mutant enzyme
H231A/D233A
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mutant completely loses m6A demethylation activity
H266A
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activity is significantly compromised
I209D
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the mutant shows decreased activity compared to the wild type enzyme
I209E
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the mutant shows decreased activity compared to the wild type enzyme
K231A/K235A
double mutation shows no impact on the repair capacity of Alkbh5
K231E/K235E
double mutation shows no impact on the repair capacity of Alkbh5
Q146A/K147A/R148A
mutant retains 44% of the wild-type activity
R130A
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no detectable activity toward m6A RNA
R269E/Q271E
mutant with greatly reduced catalytic activity to 50%
R316Q
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mutation abolishes 80% of the wild-type activity towards m6A demethylation in vitro
R316Q/R322Q
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mutant completely loses m6A demethylation activity
Y139A
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no detectable activity toward m6A RNA