A DFT Study of Nucleobase Dealkylation by the DNA Repair Enzyme AlkB

Abstract

Oxidative dealkylation is a unique mechanistic pathway found in the alpha-ketoglutarate-Fe(II)-dependent AlkB family of enzymes to remove the alkylation damage to DNA bases and regenerate nucleobases to their native state. The B3LYP density functional combined with a self-consistent reaction field was used to explore the triplet, quintet, and septet spin-state potential energy surfaces of the multistep catalytic mechanism of AlkB. The mechanism was found to consist of four stages. First, binding of dioxygen to iron in the active-site complex occurs concerted with electron transfer, thereby yielding a ferric-superoxido species. Second, competing initiation for the activation of oxygen to generate the high-valent iron-oxygen intermediates (ferryl-oxo Fe(IV)O and ferric-oxyl Fe(III)O(*) species) was found to occur on the quintet and septet surfaces. Then, conformational reorientation of the activated iron-oxygen ligand was found to be nearly thermoneutral with a barrier of ca. 50 kJ mol(-1). The final stage is the oxidative dealkylation of the damaged nucleobase with the rate-controlling step being the abstraction of a hydrogen atom from the damaging methyl group by the ferryl-oxo ligand. For this step, the calculated barrier of 87.4 kJ mol(-1) is in good agreement with the experimental activation energy of ca. 83 kJ mol(-1) for the enzyme-catalyzed reaction.

Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB

“…Mechanistic characterization focused on experiments at 10°C to minimize interference from protein aggregation, which is enhanced at higher temperature. AlkB catalyzes repair of alkylated nucleobases at a similar rate at either 10 or 37°C (data not shown), consistent with quantum mechanical processes being rate-determining in the reaction cycle (46).…”

Mechanisms of Mutagenic DNA Nucleobase Damages and Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods

“…In 5 TS 1 the cleaving -CH 2 -H bond has lengthened to 1.30 Å while the corresponding FeO … H • distance is quite short at 1.22 Å. (Liu et al 2009) This step is not only the rate limiting step in the actual demethylation of 3me4amPym + , but is also the highest reaction barrier obtained in the overall mechanism of AlkB. (Liu et al 2009) In the related α-KG-Fe(II) non-heme enzyme TauD, which catalyses hydroxylation of propene, abstraction of a methyl hydrogen by the Fe(IV)=O species was also calculated to be the rate limiting step.…”

“…Oxidative repair of alkylated nucleobases: the catalytic mechanism of AlkB A common type of DNA damage is the alkylation of nucleobases at one or more of their oxygen and nitrogen centres. (Liu et al 2009) This can be caused by endo-or exogenous agents such as S-adenosylmethionine or tobacco smoke, respectively. (Hecht 1999;Rydberg & Lindahl 1982) Consequently, cells have developed several methods by which to mediate or repair such damage.…”

“…(Berglund et al 2002;Nakajima & Yamazaki 1987) The O 2 •-moiety formed then attacks the α−KG cosubstrate converting it to succinate with concomitant formation of an oxo-ferryl Fe(IV)=O species, a strong oxidizing agent. (Clifton et al 2006;Kovaleva & Lipscomb 2008;Liu et al 2009) However, the =O group of the oxo-ferryl sits in the apical position and thus is not ideally situated to react with the methylated nucleobase. Hence, it reorients to an equatorial position.…”

“…Calculations were then performed at the IEF-PCM(ε=4.0)-B3LYP/LACV3P+(d,p)//B3LYP/ LACVP(d) level of theory. For complete details on the model and methods used please refer to Liu et al 2009. (Liu et al 2009 In this chapter we focus on key findings of the oxidative demethylation stage of the mechanism, shown schematically in Figure 9.…”