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.