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. 2014 Nov 11;10(11):5136-5148.
doi: 10.1021/ct500572t. Epub 2014 Sep 25.

Alternative Pathway for the Reaction Catalyzed by DNA Dealkylase AlkB from Ab Initio QM/MM Calculations

Dong Fang  1 , G Andrés Cisneros  1
Affiliations

Alternative Pathway for the Reaction Catalyzed by DNA Dealkylase AlkB from Ab Initio QM/MM Calculations

Dong Fang et al. J Chem Theory Comput. .

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.

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Figures

Scheme 1
Scheme 1. Proposed Mechanism for the Steps Starting from H Abstraction in the Dealkylation Catalyzed by AlkB Based on the TauD Mechanism
Scheme 2
Scheme 2. Proposed Detailed H2O Pathway Starting from the Hydrogen Abstraction
Scheme 3
Scheme 3. OH– Pathway: The Newly Proposed Mechanism with OH– Coordinated to the Iron (ROH)
Figure 1
Figure 1
Active site of AlkB with 1-meA in the crystal structure (PDB ID: 2FDG).
Figure 2
Figure 2
Relative energies (in kcal/mol, with ISFeIII–OF as the reference state) of reactant, TS and I1 and Mülliken spin populations of key atoms (Fe, the first O denotes the oxo, the second O denotes the O of OH– bound to the iron, and C denotes the carbon of methyl group of 1-meA) for the hydrogen abstraction step for OH– pathway in quintet (ISFeIII–OF and HSFeIII–OAF) and triplet states (LSFeIII–OF and ISFeIII–OAF).
Figure 3
Figure 3
α-LUMO and β-LUMO (canonical orbitals, isovalue = 0.05 au) of the quintet reactants, MECP and TS structures along the H2O pathway. Carbon atoms are colored in gray, hydrogen in white, nitrogen in blue, oxygen in red, iron in purple, and boundary carbon atoms for pseudo-bond in cyan.
Figure 4
Figure 4
α-LUMO and β-LUMO (canonical orbitals, isovalue for the surface is 0.05 au) of the quintet reactants, TOH (Table 1) and TS structures along the OH– pathway. Carbon atoms are colored in gray, hydrogen in white, nitrogen in blue, oxygen in red, iron in purple, and boundary carbon atoms for pseudo-bond in cyan.
Figure 5
Figure 5
Relative energies (kcal/mol) of the structures along the minimum energy path (MEP) for the OH rebound step in the quintet state for H2O pathway (a) and OH– pathway (b). The numbers in the parentheses are reaction barriers, which are the energy differences between intermediates and their corresponding TSs. The energy of the corresponding ISFeIII–OF reactant (Figure 2) is taken as zero for each pathway. Carbon atoms are colored in gray, hydrogen in white, nitrogen in blue, oxygen in red, iron in purple, and boundary carbon atoms for pseudo-bond in cyan.
Figure 6
Figure 6
NCI surface (isovalue 0.5 au and a color scale −0.1 < sign(λ2)ρ < 0.1 au) of the zwitterion structure (I2OH) for the OH– pathway.
Figure 7
Figure 7
(a) Chemical structures of 1-meA and 1-deazameA. (b, c, d) 1-DeazameA-related intermediates. Carbon atoms are colored in gray, hydrogen in white, nitrogen in blue, oxygen in red, iron in purple, and boundary carbon atoms for pseudo-bond in cyan.
Figure 8
Figure 8
Relative energies (in kcal/mol) for the structures along the minimum energy path (MEP) for the detachment of the DNA base from Fe and the formation of formaldehyde in the quintet state for the H2O pathway (a) and OH– pathway (b). The numbers in the parentheses are reaction barriers, which are the energy differences between intermediates and their corresponding TSs. The ISFeIII–OF reactant (Figure 2) is taken as the reference for each pathway. Carbon atoms are colored in gray, hydrogen in white, nitrogen in blue, oxygen in red, iron in purple, and boundary carbon atoms for pseudo-bond in cyan.
Figure 9
Figure 9
Relative energies (kcal/mol) for the structures along the minimum energy path (MEP) for the proton transferred to Glu136 in the quintet state for the H2O pathway. (Arg210, Glu136, and a bridging water were added to the QM subsystem for these structures). Carbon atoms are colored in gray, hydrogen in white, nitrogen in blue, oxygen in red, iron in purple, and boundary carbon atoms for pseudo-bond in cyan.
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