Mechanistic Basis of Bifunctional Oxidation by a Non-Heme Fe(II)/2-Oxoglutarate-Dependent Enzyme CcTET toward N6-Methyladenine and 5-Methylcytosine in Double-Stranded DNA

Document Type

Article

Publication Date

7-17-2026

Abstract

CcTET (Coprinopsis cinerea ten-eleven translocation) is a non-heme Fe(II)/2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the oxidative demethylation of both N6-methyladenine (6mA) and 5-methylcytosine (5mC) in double-stranded DNA, distinguishing it from other TET enzymes that act exclusively on 5mC. Despite this bifunctional activity, the molecular origins of its substrate selectivity and catalytic mechanism remain poorly understood. Here, we employed a multiscale computational approach combining classical molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations to characterize the conformational dynamics and reaction pathways of CcTET. Our results show that CcTET follows the canonical Fe(IV)=O-mediated mechanism, in which hydrogen atom transfer (HAT) is followed by −OH rebound to yield hydroxylated intermediates. We further investigated the post-hydroxylation steps, including the deformylation of 6mA-derived intermediates and the iterative oxidation of 5mC to higher oxidized products. Dynamic cross-correlation analysis reveals that the 5mC-bound system exhibits stronger correlated and anticorrelated motions relative to the 6mA-bound system, consistent with a more preorganized catalytic environment for 5mC. Notably, the D337F mutation, which abolishes 5mC activity while retaining 6mA reactivity, does not significantly alter substrate binding affinity or overall dynamics but disrupts the productive orientation of 5mC relative to the Fe(IV)=O unit. Electric field analysis indicates a substrate-dependent effect, where a more favorable field lowers the HAT barrier for 5mC in the wild-type enzyme, while disruption of this electrostatic environment in the mutant leads to a substantial increase in its barrier; in contrast, 6mA reactivity remains largely unchanged due to compensating effects of field and conformational flexibility. Overall, these results show that substrate positioning and electrostatic preorganization, rather than binding affinity, control catalytic selectivity in CcTET. These insights may help guide the engineering of non-heme Fe(II)/2OG oxygenases for selective transformations of epigenetic DNA modifications for biotechnological and therapeutic applications.

Publication Title

ACS Catalysis

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