**6. 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. One approach is to use enzymes known as DNA glycolsylases to simply excise the damaged nucleobase via an acid-base cleavage of its N-glycosidic bond.(Mishina et al. 2006; Sedgwick et al. 2007) Alternatively, repair enzymes may use a non-redox mechanism to remove only the alkyl group. For example, O6–methylguanine– DNA methyltransferase transfers the methyl of O6–methylguanine onto an active site cysteinyl residue.(Lindahl et al. 1988) However, a third approach is alkyl group removal using a redox mechanism catalysed by the AlkB family of enzymes.

Mechanisms of Mutagenic DNA Nucleobase Damages and

model 3me4amPym+ in the triplet and quintet spin states.

corresponding FeO…H• distance is quite short at 1.22 Å.(Liu et al. 2009)

kcal mol–1; in good agreement with the present calculated barrier height for **5TS1**.

In **5IC2** the Fe(IV)=O has been reduced to Fe(III)—OH while the substrate now contains a methylene-carbon centered radical (see **Figure 9**). The next step is a 'rebound', transfer, of the hydroxyl group from the Fe–OH moiety to the substrate's radical centre. This reaction is almost barrierless proceeding via **5TS2** with a cost of only 0.6 kcal mol–1. Significantly, the resulting intermediate **5IC3** lies markedly lower in energy than **5IC1** by –44.8 kcal mol–1. Thus, effectively, the result of the first two steps in demethylation are exothermic insertion of an oxygen into a C—H bond of the substrates methyl group with reduction of the Fe(IV) centre to Fe(II). The much lower relative energy of **5IC3** may also reflect the structural rearrangement that occurs at the Fe centre which has gone from penta– to tetra-coordinate.

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 407

surface; the next lowest spin state, the triplet, at all times being higher in energy. Thus, herein, only the results for the quintet spin state are discussed. However, the energies for the

Fig. 9. Potential energy surface for the dealkylation of the methylated adenine nucleobase

In the apical position, the Fe(IV)=O oxygen is approximately 4.12 Å from the nearest target methyl hydrogen of the 3me4amPym+ substrate. However, in **5IC1**, where it now lies in the equatorial position trans to His187, this distance has been reduced dramatically to 2.68 Å. Demethylation is initiated by abstraction of a hydrogen (H•) from the target methyl group of the substrate by the Fe(IV)=O oxygen. This step proceeds via the transition structure **5TS1** at a cost of 20.9 kcal mol–1 to give intermediate **5IC2** lying just 1.0 kcal mol–1 higher in energy than **5IC1**. In **5TS1** the cleaving -CH2—H bond has lengthened to 1.30 Å while the

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.(de Visser 2006) The barrier in AlkB, however, is significantly higher. It has been observed that in C—H activating heme enzymes the barrier for such an abstraction is linearly related to the BDE of the substrates C—H bond being cleaved.(de Visser et al. 2004; Kaizer et al. 2004) Thus, the significantly higher barrier observed in AlkB compared to TauD is likely due to the 14.3 kcal mol–1 greater –CH2—H bond energy in its substrate, 3me4amPym+. It should be noted that experimentally kcat for AlkB has been measured to be 4.5 min–1,(Koivisto et al. 2003; Yu et al. 2006) corresponding to a rate–limiting barrier of 19.8

triplet surface have been included in **Figure 9** for purposes of comparison.

Although included in the α–ketoglutarate–Fe(II)–dependant dioxygenase superfamily, the AlkB family is the only one known to oxidatively dealkylate nucleobases.(Falnes et al. 2002; Trewick et al. 2002) Specifically, under physiological conditions they oxidatively repair the methylated nucleobases 1–meA, 1–meG, 3–meC and 3–meT (**Figure 8**).(Yang et al. 2008) It is noted, however, that they have been demonstrated to also be able to remove longer alkyl chains.(Delaney et al. 2005; Duncan et al. 2002; Koivisto et al. 2003; Mishina et al. 2005)

Fig. 8. Examples of methylated substrates that are dealkylated by the AlkB family of enzymes. The methyl groups to be removed have been colored in red.

Several X-ray crystal structures have been obtained of AlkB complexed with, for instance, cosubstrate or coproducts.(Yang et al. 2008; Yu et al. 2006) From these structures it was concluded that the active site of AlkB contains an Fe(II) ion ligated via two histidyl (His131, His187) and an aspartyl (Asp133) residue. Furthermore, the α–ketoglutarate (α−KG) cosubstrate bidentately ligates to the Fe(II) centre via carboxyl and ketone oxygens,(Falnes et al. 2002; Mishina et al. 2005; Trewick et al. 2002) while the methylated nucleobase sits adjacent to the Fe(II) centre. Based on these structures it was proposed that the mechanism of AlkB begins with activation of O2: on binding to the Fe(II) centre the O2 moiety is reduced to O2 •– while the Fe centre is oxidized to Fe(III).(Berglund et al. 2002; Nakajima & Yamazaki 1987) The O2•– 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.(Yu et al. 2006) An X-ray crystal structure (PDB: 2FD8) has been obtained under anaerobic conditions of AlkB with the single-strand trinucleotide dT-(1-me-dA)-dT bound within its active site.(Yu et al. 2006) Based on this experimental structure we obtained an appropriate chemical model for our computational studies. In particular, key active site residues, the Fe(II) centre, and α−KG modeled by pyruvate were included. A crystal structure water observed to be ligated to the Fe(II) centre in the apical position was replaced by O2 while the substrate 1 methyladenine was modeled as 3–methyl–4–amino pyrimidinyl cation (3me4amPym+). 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**. However, it should be noted that the initial O2 activation process preferentially occurs in the quintet spin state with a rate-limiting barrier of just 9.1 kcal mol–1. The lowest energy pathway for the subsequent reorientation of the Fe(IV)=O species was also found to occur for the quintet spin state. Furthermore, it was found to proceed via a concerted mechanism with a barrier of 11.3 kcal mol–1. For the oxidative demethylation itself, the lowest energy pathway also occurred on the quintet

Although included in the α–ketoglutarate–Fe(II)–dependant dioxygenase superfamily, the AlkB family is the only one known to oxidatively dealkylate nucleobases.(Falnes et al. 2002; Trewick et al. 2002) Specifically, under physiological conditions they oxidatively repair the methylated nucleobases 1–meA, 1–meG, 3–meC and 3–meT (**Figure 8**).(Yang et al. 2008) It is noted, however, that they have been demonstrated to also be able to remove longer alkyl chains.(Delaney et al. 2005; Duncan et al. 2002; Koivisto et al. 2003; Mishina et al. 2005)

Fig. 8. Examples of methylated substrates that are dealkylated by the AlkB family of

Several X-ray crystal structures have been obtained of AlkB complexed with, for instance, cosubstrate or coproducts.(Yang et al. 2008; Yu et al. 2006) From these structures it was concluded that the active site of AlkB contains an Fe(II) ion ligated via two histidyl (His131, His187) and an aspartyl (Asp133) residue. Furthermore, the α–ketoglutarate (α−KG) cosubstrate bidentately ligates to the Fe(II) centre via carboxyl and ketone oxygens,(Falnes et al. 2002; Mishina et al. 2005; Trewick et al. 2002) while the methylated nucleobase sits adjacent to the Fe(II) centre. Based on these structures it was proposed that the mechanism of AlkB begins with activation of O2: on binding to the Fe(II) centre the O2 moiety is reduced

•– while the Fe centre is oxidized to Fe(III).(Berglund et al. 2002; Nakajima & Yamazaki

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.(Yu et al. 2006) An X-ray crystal structure (PDB: 2FD8) has been obtained under anaerobic conditions of AlkB with the single-strand trinucleotide dT-(1-me-dA)-dT bound within its active site.(Yu et al. 2006) Based on this experimental structure we obtained an appropriate chemical model for our computational studies. In particular, key active site residues, the Fe(II) centre, and α−KG modeled by pyruvate were included. A crystal structure water observed to be ligated to the Fe(II) centre in the apical position was replaced by O2 while the substrate 1 methyladenine was modeled as 3–methyl–4–amino pyrimidinyl cation (3me4amPym+).

•– moiety formed then attacks the α−KG cosubstrate converting it to succinate

ε

LACVP(d) level of theory. For complete details on the model and methods used please refer

In this chapter we focus on key findings of the oxidative demethylation stage of the mechanism, shown schematically in **Figure 9**. However, it should be noted that the initial O2 activation process preferentially occurs in the quintet spin state with a rate-limiting barrier of just 9.1 kcal mol–1. The lowest energy pathway for the subsequent reorientation of the Fe(IV)=O species was also found to occur for the quintet spin state. Furthermore, it was found to proceed via a concerted mechanism with a barrier of 11.3 kcal mol–1. For the oxidative demethylation itself, the lowest energy pathway also occurred on the quintet

=4.0)-B3LYP/LACV3P+(d,p)//B3LYP/

enzymes. The methyl groups to be removed have been colored in red.

Calculations were then performed at the IEF-PCM(

to Liu et al. 2009.(Liu et al. 2009)

to O2

1987) The O2

surface; the next lowest spin state, the triplet, at all times being higher in energy. Thus, herein, only the results for the quintet spin state are discussed. However, the energies for the triplet surface have been included in **Figure 9** for purposes of comparison.

Fig. 9. Potential energy surface for the dealkylation of the methylated adenine nucleobase model 3me4amPym+ in the triplet and quintet spin states.

In the apical position, the Fe(IV)=O oxygen is approximately 4.12 Å from the nearest target methyl hydrogen of the 3me4amPym+ substrate. However, in **5IC1**, where it now lies in the equatorial position trans to His187, this distance has been reduced dramatically to 2.68 Å. Demethylation is initiated by abstraction of a hydrogen (H•) from the target methyl group of the substrate by the Fe(IV)=O oxygen. This step proceeds via the transition structure **5TS1** at a cost of 20.9 kcal mol–1 to give intermediate **5IC2** lying just 1.0 kcal mol–1 higher in energy than **5IC1**. In **5TS1** the cleaving -CH2—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.(de Visser 2006) The barrier in AlkB, however, is significantly higher. It has been observed that in C—H activating heme enzymes the barrier for such an abstraction is linearly related to the BDE of the substrates C—H bond being cleaved.(de Visser et al. 2004; Kaizer et al. 2004) Thus, the significantly higher barrier observed in AlkB compared to TauD is likely due to the 14.3 kcal mol–1 greater –CH2—H bond energy in its substrate, 3me4amPym+. It should be noted that experimentally kcat for AlkB has been measured to be 4.5 min–1,(Koivisto et al. 2003; Yu et al. 2006) corresponding to a rate–limiting barrier of 19.8 kcal mol–1; in good agreement with the present calculated barrier height for **5TS1**.

In **5IC2** the Fe(IV)=O has been reduced to Fe(III)—OH while the substrate now contains a methylene-carbon centered radical (see **Figure 9**). The next step is a 'rebound', transfer, of the hydroxyl group from the Fe–OH moiety to the substrate's radical centre. This reaction is almost barrierless proceeding via **5TS2** with a cost of only 0.6 kcal mol–1. Significantly, the resulting intermediate **5IC3** lies markedly lower in energy than **5IC1** by –44.8 kcal mol–1. Thus, effectively, the result of the first two steps in demethylation are exothermic insertion of an oxygen into a C—H bond of the substrates methyl group with reduction of the Fe(IV) centre to Fe(II). The much lower relative energy of **5IC3** may also reflect the structural rearrangement that occurs at the Fe centre which has gone from penta– to tetra-coordinate.

Mechanisms of Mutagenic DNA Nucleobase Damages and

*J. Chem. Phys.*, 98(7), 5648-5652.

scission." *Chem. Rev.*, 98(3), 1109-1151.

inflammation processes." *Nat. Chem. Biol.*, 2(7), 348-349.

Acids, H. Morrison, ed., Wiley, New York, 1-272.

yl radical." *J. Am. Chem. Soc.*, 122(39), 9525-9533.

c substitutions." *J. Biol. Chem.*, 267(1), 166-172.

*Chem. Phys.*, 98, 1372.

417(6887), 463-468.

41(8), 1075-1083.

23(3-4), 309-326.

*Chem.-Eur. J.*, 6(3), 475-484.

110(29), 9200-9211.

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 409

Becke, A. D. (1993a). "A Mixing of Hartree-Fock and Local Density-Functional Theories." *J.* 

Becke, A. D. (1993b). "Density-Functional Thermochemistry .3. The Role of Exact Exchange."

Berglund, G. I., Carlsson, G. H., Smith, A. T., Szoke, H., Henriksen, A., & Hajdu, J. (2002).

Burrows, C. J., & Muller, J. G. (1998). "Oxidative nucleobase modifications leading to strand

Cadet, J., Douki, T., & Ravanat, J. L. (2006). "One-electron oxidation of DNA and

Cadet, J., Douki, T., & Ravanat, J. L. (2008). "Oxidatively generated damage to the guanine

Cadet, J., & Vigny, P. (1990). Bioorganic Photochemistry. Photochemistry and the Nucleic

Cammi, R., & Tomasi, J. (1995). "Remarks on the use of the apparent surface-charges (ASC)

Cances, E., Mennucci, B., & Tomasi, J. (1997). "A new integral equation formalism for the

Candeias, L. P., & Steenken, S. (2000). "Reaction of HO center dot with guanine derivatives

Cauet, E., Dehareng, D., & Lievin, J. (2006). "Ab initio study of the ionization of the DNA

Chatgilialoglu, C., Ferreri, C., Bazzanini, R., Guerra, M., Choi, S. Y., Emanuel, C. J., Horner,

Cheng, K. C., Cahill, D. S., Kasai, H., Nishimura, S., & Loeb, L. A. (1992). "8-

Clifton, I. J., McDonough, M. A., Ehrismann, D., Kershaw, N. J., Granatino, N., & Schofield,

Cullis, P. M., Malone, M. E., & MersonDavies, L. A. (1996). "Guanine radical cations are

immediate strand breaks in DNA." *J. Am. Chem. Soc.*, 118(12), 2775-2781.

stranded beta-helix fold proteins." *J. Inorg. Biochem.*, 100(4), 644-669.

and anisotropic dielectrics." *J. Chem. Phys.*, 107(8), 3032-3041.

"The catalytic pathway of horseradish peroxidase at high resolution." *Nature*,

moiety of DNA: Mechanistic aspects and formation in cells." *Accounts Chem. Res.*,

methods in solvation problems - Iterative versus matrix-inversion procedures and the renormalization of the apparent charges." *J. Comput. Chem.*, 16(12), 1449-1458. Cances, E., & Mennucci, B. (1998). "New applications of integral equations methods for

solvation continuum models: ionic solutions and liquid crystals." *J. Math. Chem.*,

polarizable continuum model: Theoretical background and applications to isotropic

in aqueous solution: Formation of two different redox-active OH-Adduct radicals and their unimolecular transformation reactions. Properties of G(-H)(center dot)."

bases: Ionization potentials and excited states of the cations." *J. Phys. Chem. A*,

J. H., & Newcomb, M. (2000). "Models of DNA C1 ' radicals. Structural, spectral, and chemical properties of the thyminylmethyl radical and the 2 '-deoxyuridin-1 '-

hydroxyguanine, an abundant form of oxidative dna damage, causes g -> t and a ->

C. J. (2006). "Structural studies on 2-oxoglutarate oxygenases and related double-

precursors of 7,8-dihydro-8-oxo-2'-deoxyguanosine but are not precursors of

The final step in the demethylation of 3me4amPym+ is decomposition of the hydroxylated intermediate to give 4–amino pyrimidine, the model for the demethylated nucleobase, and formaldehyde (**Figure 9**). In this step an active site Glu136 residue acts as a general base and deprotonates the Ade–CH2OH hydroxyl with concomitant cleavage of the AdeN—CH2OH bond.(Liu et al. 2009) This reaction proceeds via **5TS3** at the modest cost of 10.3 kcal mol–1. It is noted that in **5TS3** the AdeN…CH2OH and Ade-CH2O—H bonds have lengthened to 1.58 and 1.22 Å, respectively, highlighting their concomitant cleavage. The product-bound complex **5PC** lies 45.1 kcal mol–1 lower in relative energy than the initial reactant complex **5IC1**. Thus, demethylation of 1-methyladenine is calculated to be a quite exothermic process.
