Abstract
AlkB is the title enzyme of a family of DNA dealkylases that catalyze the direct oxidative dealkylation of nucleobases. The conventional mechanism for the dealkylation of N1-methyl adenine (1-meA) catalyzed by AlkB after the formation of FeIV–oxo is comprised by a reorientation of the oxo moiety, hydrogen abstraction, OH rebound from the Fe atom to the methyl adduct, and the dissociation of the resulting methoxide to obtain the repaired adenine base and formaldehyde. An alternative pathway with hydroxide as a ligand bound to the iron atom is proposed and investigated by QM/MM simulations. The results show OH– has a small impact on the barriers for the hydrogen abstraction and OH rebound steps. The effects of the enzyme and the OH– ligand on the hydrogen abstraction by the FeIV–oxo moiety are discussed in detail. The new OH rebound step is coupled with a proton transfer to the OH– ligand and results in a novel zwitterion intermediate. This zwitterion structure can also be characterized as Fe–O–C complex and facilitates the formation of formaldehyde. In contrast, for the pathway with H2O bound to iron, the hydroxyl product of the OH rebound step first needs to unbind from the metal center before transferring a proton to Glu136 or other residue/substrate. The consistency between our theoretical results and experimental findings is discussed. This study provides new insights into the oxidative repair mechanism of DNA repair by nonheme FeII and α-ketoglutarate (α-KG) dependent dioxygenases and a possible explanation for the substrate preference of AlkB.
Highlights
E. coli AlkB is a member of the FeII and α-KG dependent dioxygenase superfamily of enzymes
The Molecular Dynamics (MD) simulations were performed with the pmemd program in AMBER11.11 The snapshot with the lowest QM/MM energy among ten selected snapshots was chosen for further optimization on all reactants, intermediates, and products
The differences between these two pathways on the OH rebound step and the unbinding of the DNA base from the metal and the formation of formaldehyde step are discussed in sections 3.2 and 3.3, respectively
Summary
E. coli AlkB is a member of the FeII and α-KG dependent dioxygenase superfamily of enzymes. AlkB can repair alkylated bases such as 1-meA and N3-methyl cytosine (3-meC) via an oxidative dealkylation.[1] The proposed mechanism, based on the mechanism of the related enzyme TauD,[2] involves a series of steps that can be separated in two parts. The first part is composed of the formation of an FeIV O (ferryl) intermediate along with the release of CO2 and formation of succinate. After the formation of the iron(IV)−oxo, the oxo moiety undergoes a reorientation from an axial to an equatorial position. The subsequent steps comprise the second part, which involve the oxidation of the methyl moiety on the base as shown in Scheme 1 (see Supporting Information (SI) Scheme S1 for the full mechanism including part 1)
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