Nucleobase Modifiers Identify TET Enzymes as Bifunctional DNA Dioxygenases Capable of Direct N‐Demethylation

Abstract

TET family enzymes are known for oxidation of the 5‐methyl substituent on 5‐methylcytosine (5mC) in DNA. 5mC oxidation generates the stable base 5‐hydroxymethylcytosine (5hmC), starting an indirect, multi‐step process that ends with reversion of 5mC to unmodified cytosine. While probing the nucleobase determinants of 5mC recognition, we discovered that TET enzymes are also proficient as direct N‐demethylases of cytosine bases. We find that N‐demethylase activity can be readily observed on substrates lacking a 5‐methyl group and, remarkably, TET enzymes can be similarly proficient in either oxidation of 5mC or demethylation of N4‐methyl substituents. Our results indicate that TET enzymes can act as both direct and indirect demethylases, highlight the active‐site plasticity of these FeII/α‐ketoglutarate‐dependent dioxygenases, and suggest activity on unexplored substrates that could reveal new TET biology.

Catalytic Mechanism of Human Ten-Eleven Translocation-2 (TET2) Enzyme: Effects of Conformational Changes, Electric Field, and Mutations

“…HAT involves the abstraction of a hydrogen atom from the methyl group of the substrate by the electrophilic Fe­(IV)O intermediate, resulting in Fe­(III)–OH center and a reactive radical formation at the methylene carbon of the substrate. ,, This step has been reported as the rate-limiting one during the substrate oxidation by Fe­(II)/2OG-dependent enzymes. ,− ,, Studies have shown that the relative structure of ferryl to the substrates determine the reaction and the electron transfer mechanism. − We used five snapshots from MD to explore this mechanistic step. The calculations were done at the quintet state of the Fe­(IV)O intermediate as it was previously demonstrated that non-heme Fe­(II)- and 2OG-dependent enzymes favor this spin state. ,− The results revealed that the reaction barrier varies from 16.3 to 19.1 kcal/mol (Table S3) at the B2 + ZPE level of theory, with Boltzmann-weighted averaged , value of 17.1 kcal/mol, which is within the range of previously reported barriers for HAT for 2OG-dependent oxygenases. − ,, The TS HAT Fe–O–H angle varies from 149.4 to 160.1°, suggesting that in all reaction paths, an α-electron is transferred from the substrate to the vacant 3d-orbital of Fe.…”

Bifunctional Role of Fe(II)/2OG-Dependent TET Family 5-Methylcytosine Dioxygenases and ALKBH2,3 in Modified Cytosine Demethylation

“…They also demonstrated the similar proficiency of TET enzymes in either oxidation of 5mC or demethylation of 4mC. This suggests that the TET family of enzymes also possess direct demethylation properties along with oxidative demethylation (Figure 6C) [21]. In many chemical reactions, enzymes require high specificity to distinguish between closely similar substrates.…”

“…This modified cytosine is generated during DNA damage by Surprisingly a recent study has demonstrated that, in addition to direct demethylation, ALKBH2 and 3 are also capable of oxidative demethylation [20]. Furthermore, in another investigation, the TET family of enzymes demonstrated both direct and oxidative demethylation capabilities [21]. This remarkable flexibility of Fe(II)/2OG-dependent nucleic acid-modifying dioxygenases suggests a broader impact in epigenetics.…”

Computational Investigations of Selected Enzymes From Two Iron and α-ketoglutarate-Dependent Families

“…[26,[102][103][104] Recently, it has been shown that TET1-3 enzymes can also carry out direct demethylation with a preference for substrates that lack a 5-methyl group such as 4-N-methyl-5-methylcytosine or 4,4-N,N-dimethyl-5-methylcytosine. [105] The indirect demethylation of each intermediate is similar to the AlkB enzyme mechanism in that these three phases are present: (1) formation of the reactive Fe(IV)=O, (2) hydrogen atom abstraction from the substrate, followed by (3) hydroxyl rebound (for 5hmC and 5caC) or second H abstraction (for 5fC) (Fig. 9A).…”