Catalytic Space Engineering as a Strategy to Activate C–H Oxidation on 5‑Methylcytosine in Mammalian Genome

Conditional remodeling of enzyme catalysis is a formidable challenge in protein engineering. Herein, we have undertaken a unique active site engineering tactic to command catalytic outcomes. With ten–eleven translocation (TET) enzyme as a paradigm, we show that variants with an expanded active site significantly enhance multistep C–H oxidation in 5-methylcytosine (5mC), whereas a crowded cavity leads to a single-step catalytic apparatus. We further identify an evolutionarily conserved residue in the TET family with a remarkable catalysis-directing ability. The activating variant demonstrated its prowess to oxidize 5mC in chromosomal DNA for potentiating expression of genes including tumor suppressors.