Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine

Saturation mutagenesis, molecular modeling and biochemical analysis revealed that active site interactions involving Thr1372 of TET2 are responsible for controlling its proficiency for stepwise oxidation of 5-methylcytosine residues within DNA. Ten-eleven translocation (TET) enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) to yield 5-hydroxymethylcytosine (hmC) and the rarer bases 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Stepwise oxidation obscures how each individual base forms and functions in epigenetic regulation, and prompts the question of whether TET enzymes primarily serve to generate hmC or are adapted to produce fC and caC as well. By mutating a single, conserved active site residue in human TET2, Thr1372, we uncovered enzyme variants that permit oxidation to hmC but largely eliminate fC and caC. Biochemical analyses, combined with molecular dynamics simulations, elucidated an active site scaffold that is required for wild-type (WT) stepwise oxidation and that, when perturbed, explains the mutants' hmC-stalling phenotype. Our results suggest that the TET2 active site is shaped to enable higher-order oxidation and provide the first TET variants that could be used to probe the biological functions of hmC separately from fC and caC.