CHEN Ze-Qin HE Yun-Qing

GUO Lin-Fenga XUE Yingc

a (College of Chemistry and Chemical Engineering, Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, China West Normal University, Nanchong 637002, China)

b (Department of Chemistry and Engineering, Sichuan Key Laboratory of Exploitation and Study of Distinctive Plants, Sichuan University of Arts and Science, Dazhou 635000, China)

c (College of Chemistry, Key Laboratory of Green Chemistry and Technology in Ministry of Education, Sichuan University, Chengdu 610064, China)

1 INTRODUCTION

The living cell is constantly susceptible to the potential damage of reactive oxygen species (ROS),whose origin may be intracellular, such as normal cellular metabolism[1], or extracellular, for instance,ionizing radiation or tar and tobacco smoke[2]. One of the most important and serious ROS-induced damages of the cellular genome is the oxidation of guanine (G)base in DNA, leading to the yield of 7,8-dihydro-8-oxoguanine (8-oxoG)[3-4]. 8-OxoG is arguably the most important mutagenic lesion in DNA because of its tendency to create a harmful faulty pairing with the nearest adenine (A)by Hoogsteen mode, giving rise to an 8-oxoG:A pair.This mismatched base pair causes, in the subsequent replication step, a G:C→T:A transversion mutation[5-6], which results in the loss of genomic integrity and cellular regulation associated with aging and a variety of disease states, including cancer[7-8].

To avoid the loss of information stored in DNA due to the incorrect replication, most aerobic organisms have evolved a complex cellular defense system against 8-oxoG mutagenesis or other single-base lesions in DNA, which includes a large ensemble of repair proteins[9-10]. As is the case for most oxidized bases, the preferred repair mechanism for 8-oxoG residues is the BER pathway[11-12], in which the key components are lesion-specific DNA glycosylases that are able to scan the genome for damaged base sites and catalyze the scission of glycosidic bond[13-14]. The glycosylases and processes involved in the repair of 8-oxoG are by far the most intensively studied of those that repair oxidative DNA damage, attributed to the presumed abundance of this lesion, its mutagenic potential and popularity as biomarker of oxidative DNA damage, and the ease with which it can be detected. In Escherichia coli,two DNA glycosylases prevent the mutagenesis caused by 8-oxoG: the Fpg protein, which excises 8-oxoG from damaged DNA, and the MutY protein,which excises the adenine residues incorporated by DNA polymerases opposite 8-oxoG. In human cells,the bifunctional enzyme hOGG1 is specifically responsible for the 8-oxoG repair and has attracted much attention in the past several decades[15-16].Unlike most bifunctional glycosylases, however,hOGG1 has been shown to mainly operate as a monofunctional glycosylase under physiological concentrations of magnesium or in vivo[17-18].

As can be readily seen, a range of deoxynucleotides and ribonucleotides have been modeled to clarify the mechanistic details of the damaged base excision process[19-29]. For 8-oxo-2?-deoxyguanosine(8-oxo-dG, Fig. 1), theoretical insights into the cleavage of N-glycosidic bond have been reported in the literature, typically including (1)small and large model studies on the cleavage mechanism of N-glycosidic bond using an amine group (lysine in hOGG1)as the nucleophile[22?26], and (2)SN2 hydrolysis mechanism, by which the effects of nucleophile, nucleoside, and phosphate backbone conformation on the energetics of the glycosidic bond cleavage were investigated[28-29]. Nevertheless,many questions remain unaddressed regarding the chemistry of this fundamental biological repair process. Whether the hydrolysis process of 8-oxodG can occur via a stepwise SN1 process? Which mechanism is more favorable, the SN1 or SN2? How the presences of explicit and implicit solvent molecules affect the hydrolysis process? Herein, understanding of the hydrolysis mechanism of 8-oxo-dG is essential for the insight into the catalytic process and has been systematically pursued in the present study.We began our study with the most simplistic hydrolytic model that only includes the nucleoside and one water molecule at the reaction center, which has been widely employed in the literature to investigate DNA hydrolysis mechanisms[19-22]. And then, an expanding water-assisted hydrolysis model with four discrete water molecules was established to determine the structural effects since the inclusion of explicit solvation during optimizations can change the geometries of reaction intermediates due to the charge stabilization[30-32].

Fig. 1. Structure and atomic numbering for 8-oxo-dG

2 COMPUTATIONAL DETAILS

In the present study, two types of computational models were taken into account, including the direct and water-assisted hydrolysis models. Studies have shown that purine hydrolysis model containing three discrete water molecules produces strain in the system and alters the sugar puckering with respect to the nucleoside optimized in fully explicit water[21,33].Herein, considering that the large purine ring system increases the distance of the hydrogen-bond network,four water molecules were chosen in the waterassisted hydrolysis model to connect the sugar moiety with 8-oxo-dG.

All geometry optimizations for potential energy surface (PES)scan were performed at the B3LYP/6-31+G(d)theory of level, thanks to its successful application in the studies of many biological reaction mechanisms[19-21]. For the two computational models,the initial glycosidic bond length C(1?)?N(9)and nucleophile distance C(1?)?O(w)were set to be 1.5 and 3.4 ?, respectively for PES scans. The PES was obtained by incrementally compressing or elongating one of the distances by 0.2 ? and relaxing the remainder of system. On the PESs, the glycosidic bond length C(1?)?N(9)varied from 1.3 to 4.3 ?.The nucleophile distance C(1?)?O(w)spanned 1.2～4.4 ? for the direct hydrolysis model and 1.2～3.8 ? for the water-assisted hydrolysis model. Following PES scan, all the representative structures displayed in PESs were further optimized without any constraints at the B3LYP/6-31+G(d)level of theory.The harmonic frequency analysis was used to confirm the stationary point as a minimum with all positive frequencies or as a transition state with only one imaginary frequency.

The implicit solvation effects of water was estimated through single-point calculations on gasphase geometries using the integral-equation formalism polarizable continuum model (IEF-PCM)with UAHF radii (rmin= 0.5 and ofac= 0.8)[34]. This model of solvent can provide better hydration energies with respect to UAKS radii[35-37], and has been widely used to study the hydrolysis of 2?-deoxyribonucleosides[20-21]. The dielectric constant used in the calculations is ε = 78.4 for water. All of the singlepoint energies in water were corrected by the gasphase thermodynamic quantities. The thermodynamic data reported in this paper are at 298.15 K and 1 atm. All calculations were carried out using Gaussian 09[38].

3 RESULTS AND DISCUSSION

The PESs for the direct and water-assisted hydrolysis models are depicted in Fig. 2. On the PES,the x-axis represents the distance between N(9)of 8-oxo-dG and C(1?)of sugar (i.e., the glycosidic bond length), the y-axis represents the distance between the oxygen of nucleophile and C(1?)of the sugar (i.e., the nucleophile distance), and the relative energy is plotted in color. Preliminary calculations have shown that the top-left corner of the surface with the glycosidic bond length between 1.3 and 2.1 ? and nucleophile distance between 1.2 and 1.8 ? yields highly compressed structures and has much higher energy than the rest of the surface. Thereby,this region was not modeled. Full optimization structures of all the stationary points depicted on the PESs of the direct and water-assisted hydrolysis models are shown in Figs. 3 and 4, respectively. The changes in Gibbs free energies (ΔG)and electronic energies (ΔE)are shown in Table 1.

Table 1. Changes of Gibbs Free Energies (ΔG), Electronic Energies (ΔE)and Dipole Moments (Δμ)for the Hydrolysis of N-glycosidic Cleavage in 8-oxodG (Energy in kcal/mol and Dipole Moment in Debye)

?

Fig. 2. B3LYP/6-31+G(d)potential energy surfaces for the direct (a)and water-assisted (b)hydrolysis models of 8-oxo-dG (atomic distances are in ?; color scale provided in legend, energies are in kcal/mol)

Fig. 3. Full optimization structures of stationary points for the direct hydrolysis of 8-oxo-dG (energies are in kcal/mol;values out of parentheses, ΔG at the B3LYP/6-31+G(d)level in the gas phase; values in parentheses, ΔG in water solution at the corresponding calculation level. Definitions are the same in the following figures)

3. 1 Direct hydrolysis model with one discrete water molecule

As shown in Fig. 2a, the reactant complex lies in the bottom left purple region on the PES. In the reactant complex (RC, Fig. 3), the distances of C(1?)?N(9)and C(1?)?O(w)are 1.46 and 4.30 ?,respectively. The nucleophile water molecule keeps coordinated to the nucleobase through two hydrogen bonds, O(a)?H··N(3)and N(2)?H··O(a). A dissociative SN1 transition state (TSD, Fig. 3)can be located on the PES, which corresponds to the departure of 8-oxoG from the sugar moiety. In TSD,the lengths of C(1?)?N(9)and C(1?)?O(w)are 3.10 and 3.80 ?, respectively. The strong hydrogen bond(O(a)?H··N(3))formed between the water molecule and 8-oxoG anion can efficiently keep the lone pairs on the nucleophile from stabilizing the positive charge on the sugar. Thus, once the glycosidic bond is further lengthened, unrealistic strains on the system are incorporated, whose alleviation requires the spontaneous geometrical rearrangements and lead to drastically different structures and energies(bottom right blue quadrant, Fig. 2a). The blue energy well corresponds to the C(2?)-H abstraction product complex (PCD, Fig. 3). In PCD, the H(2?)atom is completely abstracted by the nucleobase,which is favorable in the gas phase to stabilize a partial negative charge on the base and a partial positive charge on the sugar. The hybridizations of C(1?)and C(2?)atoms both convert from sp3in the reactant complex to sp2, leading to the formation of a C(1?)=C(2?)double bond (1.34 ? in PCD).However, with the increase of glycosidic bond length by more than 3.3 ?, a discontinuous region is encountered, leading to a collapse of the PES. Consequently, the C(2?)?H abstraction structure can’t directly yield a hydrolysis product. The discrete water molecule in this reaction actually acts as solvent rather than nucleophile.

Apart from dissociation process, a continuous concerted (SN2)pathway can also be characterized on the PES (Fig. 2a). The vector of the imaginary vibrational frequency for the SN2 transition state(TSC, Fig. 3)mainly corresponds to the cleavage of N-glycosidic bond with the simultaneous proton transfer from the water nucleophile to the N(9)atom of 8-oxoG. The distances of C(1?)?N(9)and C(1?)?O(w)are 2.26 and 2.99 ?, respectively. In the product complex (PCC, Fig. 3), the proton is completely transferred to 8-oxoG anion, yielding the N(9)protonated 8-oxoG tautomer. The gas-phase ΔG-s of TSCand TSDare 45.52 and 37.94 kcal/mol, respectively, at the B3LYP/6-31+G(d)level of theory.Consequently, even though only the SN2 pathway can be properly identified on the gas-phase optimized surface of the direct hydrolysis model, the path of the slowest ascent still corresponds to SN1 dissociation.

The direct hydrolysis model can give valuable information about the hydrolysis mechanism.However, it is still too simple to afford a full description of the hydrolysis mechanism. Numerous experimental studies on the nonenzymatic deglycosylation of nucleic acids, reported in the literature[39-40],has illustrated that the uncatalyzed hydrolysis of the N-glycosidic bond in 2?-deoxynucleosides or nucleotides occurs via a dissociative (SN1)pathway rather than synchronous (SN2)mechanism. Nevertheless, most computational studies for nucleoside hydrolysis have reported that the synchronous SN2 mechanism is favored due to the prohibitively high activation energies for the dissociation step of SN1 mechanism[19]. Discrepancies between computational and experimental results for the deglycosylation of nucleotide or nucleoside may be partially attributed to the small computational model, which only contains the nucleoside and one nucleophile.Previous reports on some reactions, such as proton transfer, tautomerization, and deamination[30,31,41],have suggested that the presence of explicit solvent molecules contributes to the transfer of proton and reduce the ring constraint of transition states or stabilize the related groups. Therefore, a possible reason for the inability of the complete mechanism depiction by current model may be lack of adequate water molecules to accurately describe a continuous reaction surface. Then, what we will do next is to investigate an expanding hydrolytic model of 8-oxo-dG containing the nucleoside and four explicit water molecules.

3. 2 Water-assisted hydrolysis model with four discrete water molecules

On the PES (Fig. 2b), the reactant complex lies at the bottom left purple quadrant. The SN1 process begins with the departure of 8-oxo-dG from the sugar moiety with a gas-phase dissociative ΔG of 32.45 kcal/mol at the B3LYP/6-31+G(d)level of theory. In the transition state (wTSD, Fig. 4), the distance of C(1?)?N(9)is 2.78 ?, suggesting the partial dissociation of the N-glycosidic bond. Two water molecules stay below the C(1?)atom of the sugar, and thus help stabilize the oxacarbenium cation being formed. The full cleavage of the N-glycosidic bond from the sugar occurs in a well with a depth of 20.67 kcal/mol on the surface. In the intermediate (wIM, Fig. 4), the length of C(1?)?N(9)bond reaches 3.10 ?. With the proceeding of the reaction, the water molecule nucleophile adds to C(1?)of the sugar with the simultaneous proton transfer from water molecule to the nucleobase through a gas-phase ΔG of 12.80 kcal/mol relative to the intermediate. In wTSA(Fig. 4), the distances of C(1?)?N(9)and C(1?)?O(w)are 3.39 and 1.88 ?,respectively. The full dissociative SN1 mechanism has been characterized for the hydrolysis of 8-oxodG for the first time.

Fig. 4. Full optimization structures of stationary points for the water-assisted hydrolysis of 8-oxo-dG

As found for the direct hydrolysis model, a SN2 transition state (wTSC, Fig. 4)is also characterized on the PES of the water-assisted hydrolysis model.In wTSC, the distances of C(1?)?N(9)and C(1?)?O(w)are 2.70 and 2.00 ?, respectively. The ΔG of wTSCis 41.98 kcal/mol in the gas phase at the B3LYP/6-31+G(d)level of theory, which is higher than that reported for the SN1 mechanism by 9 kcal/mol. Thus, the path of the slowest ascent corresponds to the SN1 process in the gas phase.

3. 3 Solvent effect

Single-point energy computations at the SCRF/IEF-PCM model were performed at the gas-phase optimized geometries to estimate the effect of the implicit solvent. Computed single-point energies and dipole moments for all the critical structures in a medium of water are presented in Table 1 for comparison. As shown in Table 1, all the dipole moments of the transition states are higher than their corresponding reactant complexes or intermediates, suggesting that the solvation of implicit solution on transition states is stronger.Thus, the implicit solvent molecules favor to stabilize the transition states and gives rise to the reductions of activation energies.

Taking the solvent effect into account, the activation energies for the rate-determining steps of SN1 and SN2 mechanisms are 27.36 and 34.01 kcal/mol,respectively, which are close to the values reported for the hydrolysis of 2?-deoxyguanosine (25.82 kcal/mol for SN1 and 37.76 kcal/mol for SN2)[21].This mainly results from their structural similarity and therefore the effect of DNA damage on the energetics is relatively small. Our results have reproduced the experimental trend that the dissociative SN1 mechanism is favored over the synchronous SN2 mechanism for the nonenzymatic hydrolytic deglycosylation of nucleic acids. The activation energy of the rate-determining step for the SN1 mechanism agrees well with the experimental value of 31±2 kcal/mol observed previously for the hydrolytic depurination of DNA[42].

4 CONCLUSION

8-oxoG is the most important and serious ROS-induced damage of the cellular genome, which contributes to the loss of genomic integrity and cellular regulation, and thereby must be repaired. Careful analysis of the PESs in this work shows that the expansion hydrolysis model containing four explicit water molecules can characterize both the SN1 and SN2 mechanisms well. It suggests that explicit solvent during optimizations must be incorporated to accurately reflect the entire reaction mechanism. Our findings indicate that studies involving hydrolysis of nucleosides or nucleotides should not be limited to one discrete water molecule in the computational model and emphasizes the importance of explicit solvent molecule in activating the nucleophile and stabilizing the leaving group. Our results agree well with the experimental investigations and provide a baseline for the important deglycosylation biological process, which allows a comparison to future computational studies of enzyme-catalyzed process.

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