Switching demethylation activities between AlkB family RNA/DNA demethylases through exchange of active-site residues.

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.