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. Exchange program: Divergent active‐site residues contribute to the substrate recognition and demethylation specificity of RNA/DNA demethylases AlkB, FTO, and ALKBH5. Swapping these residues results in partially switched demethylation activities (see picture). In the implicated inhibitor design principle, such divergent active‐site sequences could aid the design of selective inhibitors that discriminate these demethylases.