Crystal structure of AlkB D135I/E136H mutant protein with cofactors bound to dsDNA containing m6A/A
Classification:
OXIDOREDUCTASE/DNA
Technique:
X-Ray Diffraction
Resolution:
1.52
R value free:
0.212
R value observed:
1.81
R value work:
1.80
Abstract of the PDB Structure's related Publication:
The AlkB family demethylases AlkB, FTO, and ALKBH5 recognize differentially methylated RNA/DNA substrates, which results in their distinct biological roles. Here we identify key active-site residues that contribute to their substrate specificity. Swapping such active-site residues between the demethylases leads to partially switched demethylation activities. Combined evidence from X-ray structures and enzyme kinetics suggests a role of the active-site residues in substrate recognition. Such a divergent active-site sequence may aid the design of selective inhibitors that can discriminate these homologue RNA/DNA demethylases.
Belongs to AlkB family of nonheme Fe(II)/a-ketoglutarate (a-KG)-dependent dioxygenases (same protein family as FTO demethylase). In vitro demethylates m6A in RNA and DNA however m6A in mRNA is a physiologically relevant substrate of ALKBH5 inside cells. Strongly prefers single-stranded substrates, exhibits an almost unnoticeable demethylation activity toward m6A in a 26-mer double-stranded RNA. ALKBH5 and its demethylation activity play important roles in mRNA export as well as roles in the association of the nuclear speckle proteins and RNA metabolism. The Alkbh5 deficiency leads to aberrant spermatogenesis and apoptosis in mouse testes. Transcriptome analysis revealed differentially expressed genes associated with spermatogenesis and the p53 functional interaction network, in line with the impaired fertility observed for the Alkbh5-/- mouse.
ALKBH5 Is a Mammalian RNA Demethylase that Impacts RNA Metabolism and Mouse Fertility.
Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, Vagbo CB, Shi Y, Wang WL, Song SH, Lu Z, Bosmans RP, Dai Q, Hao YJ, Yang X, Zhao WM, Tong WM, Wang XJ, Bogdan F, Furu K, Fu Y, Jia G, Zhao X, Liu J, Krokan HE, Klungland A, Yang YG, He C...