Cloned (Comment) | Organism |
---|---|
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 16°C as an N-terminal His-tagged protein | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
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 | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
E153A | mutant enzyme displays activity similar to wild type | Homo sapiens |
R130A | no detectable activity toward m6A RNA | Homo sapiens |
Y139A | no detectable activity toward m6A RNA | Homo sapiens |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
citrate | modest inhibitor | Homo sapiens |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | the enzyme requires binding of the cosubstrate 2-oxoglutarate and Fe2+ for catalysis | Homo sapiens | |
Mn2+ | 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 | Homo sapiens |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | Q6P6C2 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
N6-methyladenine in mRNA + 2-oxoglutarate + O2 | the ALKBH5 catalytic domain (residues 74294) is active and can demethylate ssDNA and ssRNA with similar activity | Homo sapiens | adenine in mRNA + formaldehyde + succinate + CO2 | - |
? | |
N6-methyladenine in single-stranded DNA + 2-oxoglutarate + O2 | 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 | Homo sapiens | adenine in single-stranded DNA + formaldehyde + succinate + CO2 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
ALKBH5 demethylase | - |
Homo sapiens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
21 | - |
assay at | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Homo sapiens |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
0.488 | - |
pH 7.5, 21°C | Homo sapiens | citrate |