Structure ofNaegleriaTet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA

Abstract

The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solved NgTet1–5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably because the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate.

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

“…T1372A produces 65% fC/caC, while T1372E and T1372V are able to produce only 8% and 3% fC, respectively. When starting with fC substrate under the same conditions, WT enzymes convert about half of fC to caC, corroborating that the final step of oxidation is the least efficient 13,23 . T1372A generates 19% caC, ~1/3 of the WT level, while E/V mutants make <3% caC, near or below the detection limits of our assays.…”

“…Using a structure-based algorithm 28 recently borne out by crystal structures 23 , we found that Thr1372 and Tyr1902 align with Ala212 and Phe295 in NgTetl, respectively (Supplementary Fig. 1c), making NgTetl analogous to our T1372A/Y1902F double mutant.…”

“…1c), making NgTetl analogous to our T1372A/Y1902F double mutant. In NgTetl, it was proposed that these residues form a hydrophobic pocket that accommodates hmC as it rotates from a product to a substrate conformation for further oxidation 23 . The active site mutations A212V/N, which could sterically hinder hmC binding within this pocket, were found to partially stall hmC oxidation.…”

“…We note, however, that other related mechanisms could also play a role and are not mutually exclusive with this model. Such mechanisms include restriction of substrate/product rotation 15,23 , changes in protein dynamics (Supplementary Fig. 9), and/or altered accessibility of the active site to DNA and α-KG—all of which could occur in concert with aberrant hydrogen bonding to hmC.…”

Distributive Processing by the Iron(II)/α-Ketoglutarate-Dependent Catalytic Domains of the TET Enzymes Is Consistent with Epigenetic Roles for Oxidized 5-Methylcytosine Bases

“…The enzyme interacts almost exclusively with the strand bearing the oxidation target, and it makes hydrogen bonds exclusively with the CpG dinucleotide, with the 5m C/ 5hm C/ 5f C base flipped into the active site to project toward the Fe(II) and αKG. [34][35][36][37] The disposition of the substrate and co-substrate sub-sites may thus dictate that partially oxidized species and DNA dissociate to allow for net replacement of succinate by αKG.…”