title: Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine.
abstract: 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.
authors.affiliation: Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.|Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Monica Yun
authors.initials: MY
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan, USA.
authors.forename: Hedieh
authors.initials: H
authors.lastname: Torabifard
authors.affiliation: Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.|Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Crawford
authors.affiliation: Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.|Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Jamie E
authors.initials: JE
authors.lastname: DeNizio
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Xing-Jun
authors.initials: XJ
authors.lastname: Cao
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan, USA.
authors.forename: G Andrés
authors.identifier: 0000-0001-6629-3430
authors.initials: GA
authors.lastname: Cisneros
authors.affiliation: Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.|Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Rahul M
authors.initials: RM
authors.lastname: Kohli
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D049308:Dioxygenases; D057809:HEK293 Cells; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation; D010084:Oxidation-Reduction; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D049308:Dioxygenases; C541081:TET2 protein, human