Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 1.14.11.53 extracted from

  • Xu, C.; Liu, K.; Tempel, W.; Demetriades, M.; Aik, W.; Schofield, C.J.; Min, J.
    Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation (2014), J. Biol. Chem., 289, 17299-17311.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
the human ALKBH5 catalytic AlkB domain (residues 74–294) 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 (Commentary)

Crystallization (Comment) Organism
hanging drop vapor diffusion method at 18 °C. The ALKBH5–2-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

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

Inhibitors Comment Organism Structure
citrate modest inhibitor Homo sapiens

Metals/Ions

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

Organism UniProt Comment Textmining
Homo sapiens Q6P6C2
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N6-methyladenine in mRNA + 2-oxoglutarate + O2 the ALKBH5 catalytic domain (residues 74–294) 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 74–294) 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

Synonyms Comment Organism
ALKBH5 demethylase
-
Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
21
-
assay at Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Homo sapiens

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.488
-
pH 7.5, 21°C Homo sapiens citrate