Computations Reveal a Rich Mechanistic Variation of Demethylation

Research Article pubs.acs.org/acscatalysis

Computations Reveal a Rich Mechanistic Variation of Demethylation of N‑Methylated DNA/RNA Nucleotides by FTO Binju Wang,† Zexing Cao,‡ Dina A. Sharon,† and Sason Shaik*,† †

Institute of Chemistry and The Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel ‡ State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 360015, People’s Republic of China S Supporting Information *

ABSTRACT: The fat-mass and obesity-associated (FTO) protein employs an iron(IV) oxo species to demethylate Nmethylated nucleic acids. Herein, we use atomistic-theoretical calculations to study the demethylation of the N-methylated DNA/RNA bases 6-methylated adenine (m6A), 3-methylated thymine (m3T), and 3-methylated uracil (m3U). The mechanisms involve in-enzyme hydroxylation of the methyl group, followed by hydrolysis of the oxidized intermediates in aqueous solution to demethylate the bases. The in-enzyme reactions have been studied using quantum mechanical/ molecular mechanical (QM/MM) calculations, while the hydrolytic reactions occurring outside the enzyme have been explored with hybrid cluster-continuum (HCC) calculations. When the results obtained with these different methods are combined, the calculated barrier for the overall transformation is consistent with the experimental free energy barrier for the major route of m6A demethylation: in this pathway, adenine’s N1 site acts as an internal base catalyst in the rate-determining hydrolysis of the hydroxylated hemiaminal intermediate hm6A to a demethylated A and formaldehyde. This N1-catalyzed mechanism makes m6A the most reactive substrate in comparison to other bases we tested. In the minor, slower, route, two oxidation steps by FTO generate an amide intermediate (f6A) that undergoes in-water hydrolysis, producing A and formic acid, as found experimentally. In contrast, since m3T and m3U lack internal basic catalytic sites, their hemiaminals decompose with high barriers. The mechanism instead involves two sequential oxidations, leading to demethylated bases and formic acid. Thus, our results, obtained using a holistic approach combining modeling the enzyme and the surrounding aqueous solution, suggest revisions of the experimental mechanisms for m3T and m3U demethylation. KEYWORDS: DNA repair, demethylation, FTO enzyme, QM/MM calculations, cluster-continuum model, reaction mechanism, nonheme enzymes fat-mass and obesity-associated (FTO) enzyme,10−14 which is encoded by the Fto gene and is a homologue of the DNA demethylation enzyme AlkB. Both enzymes are members of the superfamily of iron α-ketoglutarate-dependent dioxygenases, which can activate O2 and generate the high-valent iron(IV) oxo intermediate that performs a variety of oxidative processes.15−24 AlkB is well-known for its demethylation activity primarily in DNA demethylation. Similarly, FTO was recently discovered as a demethylating enzyme in DNA base demethylation10,25 but more effectively so in RNA base demethylation.26−28 A common methylation of single-stranded DNA (ssDNA)10 involves the formation of 3-methylthymine (m3T; see Scheme 1a). Similarly, one of the most abundant methylations in

1. INTRODUCTION One of the prevalent modifications of nucleic acid bases (nucleobases) in DNA and RNA is methylation mediated either by methyl transferases or by various methylating molecules.1−9 These modifications of nucleobases may in turn result in cytotoxic and/or mutagenic consequences,1−8 and hence the repair of these modifications is essential. However, the process of creating modifications and then removing them also serves an important functional purpose. For instance, the reversible methylation/demethylation of DNA and RNA bases provides a means of regulating gene expression in transcriptional epigenetics and in post-translational processes (influencing factors such as RNA stability and lifetime).1−8 As such, the demethylation process also constitutes a key tool of the genome, and unraveling the corresponding reaction mechanism is of utmost interest. The demethylation of methylated DNA/RNA is carried out by a variety of enzymes. Our target enzyme in this work is the © XXXX American Chemical Society

Received: August 23, 2015 Revised: October 8, 2015

7077

DOI: 10.1021/acscatal.5b01867 ACS Catal. 2015, 5, 7077−7090

Research Article

ACS Catalysis

demethylation of m3T is achieved through a single-round oxidation and single pathway.10 On the other hand, the demethylation of m6A proceeds via two competing pathways (Scheme 1b), even though the demethylation pathway via f6A is only a minor pathway.28 Herein we investigate the reactivity of FTO toward m3T and m6A, with the aim of understanding the root causes of the mechanistic variation and the origins of the higher demethylation reactivity of m6A.3b In addition, since the decomposition mechanism of the hemiaminal intermediates is a key step in N-methylated DNA/RNA demethylation,3,10,25−28 we wish to gain atomistic insight into this step for both systems, considering possibilities of both reactions occurring in water solution and inside of FTO. To elucidate the nature of the demethylation mechanisms of FTO in Scheme 1, we used here quantum mechanical/ molecular mechanical (QM/MM) and hybrid cluster-continuum (HCC) calculations. QM/MM calculations can yield atomistic information on structures and mechanisms within the native environment of the protein.36−46 Similarly, the HCC calculations have been demonstrated to be quite useful for studying water-mediated reactions, such as hydration and hydrolysis reactions.47,48 As such, we will use the combination of the QM/MM calculations and the HCC model calculations to investigate the full demethylation mechanisms of Nmethylated nucleobases of DNA/RNA. The manuscript is organized as follows: first, we explore the demethylation of m6A by FTO, which was experimentally found to proceed via two competing pathways (Scheme 1b).28 As shall be seen, our calculations reproduce the reaction kinetics very well for the FTO-mediated demethylation of m6A. Moreover, from the calculations we deduce that the reorganization needed for the preparation of hm6A/iron(IV) oxo in the second-round oxidation (Scheme 1b) is the slow and rate-determining step for f6A formation. This discussion is followed by a description of the m3T demethylation process. Our calculations demonstrate that m3T cannot possibly be demethylated by the mechanism proposed in the original experimental investigation.10 The computationally derived mechanism involves two rounds of oxidation of m3T to form the f3T intermediate, which is subsequently hydrolyzed into the final demethylated base in water solution. The final section of the paper compares the demethylation mechanisms and kinetics for a variety of DNA and RNA nucleotides, namely m6A, m3T, m3U, and m3C, and this section explains why m6A is a major substrate of FTO.

Scheme 1. Proposed Mechanisms for FTO-Mediated Demethylation of (a) m3T and (b) m6A

mRNA (mRNA)27−29 is the formation of N6-methyladenosine (m6A; see Scheme 1b), which plays a vital role in regulating numerous biological processes in RNA.30−33 In contrast to the AlkB family, FTO contains an extra loop in the substrate entry site, which may inhibit the binding of the DNA duplex to the active site of FTO,25 leading thereby to poor demethylation of double-stranded DNA (dsDNA). Initial experimental investigations implied that the FTO protein has a preference for the demethylation of m3T in ssDNA.10 Subsequent elegant studies by He and co-workers indicated that FTO has a low demethylation efficiency toward m3T in dsDNA,26 whereas m3T is efficiently demethylated in both ssRNA and ssDNA.26 Furthermore, their later biological studies supported the claim that m6A in nuclear RNA is a major physiological substrate of FTO.27 Previous literature has experimentally explored possible mechanisms of demethylation by FTO. For the demethylation of m3T in ssDNA, Schofield et al.10 proposed the general mechanism shown in Scheme 1a, in which formaldehyde and thymine (T) products are formed via the decomposition of the hemiaminal intermediate hm3T. Such a catalytic mechanism is analogous to the oxidative demethylation of DNA bases by AlkB.21,34,35 However, recent experiments by He et al.28 showed that FTO demethylates its major substrate m6A via the novel mechanism shown in Scheme 1b. FTO not only converts m6A to the N6-hydroxymethyladenosine (hm6A) intermediate, but it also converts hm6A further to N6formyladenosine (f6A) via a second-round oxidation depicted in Scheme 1b. Such a mechanism is quite similar to the TETmediated oxidation of 5-methylcytosine.2,3 Subsequently, both hm6A and f6A can be hydrolytically decomposed in water, with a half-life of 3 h under physiological conditions. Kinetic experiments28 indicate that f6A formation is a relatively slow step, which involves release of the initially formed hm6A and its rebinding to the newly formed high-valent iron(IV) oxo species of the enzyme. As such, the demethylation mechanisms of FTO are intriguing: according to experimental observations, the

2. COMPUTATIONAL STRATEGIES AND METHODS 2.1. Strategies. Scheme 2a shows the QM subsystem used in the QM/MM calculations, whereas Scheme 2b,c shows the respective steps for QM/MM or HCC calculations on the demethylation of m3T and m6A. The QM subsystem consists of the active species of the enzyme and the substrate (note that two different substrates were studied). For the substrate m6A, the QM region contains 72 atoms during the first round of oxidation and 73 atoms during the second round of oxidation. For the substrate m3T, the QM region contains 71 atoms during the first round of oxidation and 72 atoms during the second round of oxidation. In all cases, the total charge of the QM region is 0. The active enzymatic species contains the FeIVO moiety as well as its ligands: His231, His307, Asp233, succinate, and a water molecule. The substrates in Scheme 2a contain the m3T and 7078


Research Article

ACS Catalysis

and all the other histidine residues were doubly protonated. All glutamic acid and aspartic acid residues were deprotonated. The resulting system had a net charge of −5, which was neutralized by protonating titratable residues on the surface of the protein. After adding all hydrogens, the positions of the hydrogen atoms were optimized with 200 steps of steepest descent and 200 steps of the adapted basis Newton−Raphson method using the CHARMM27 force fields implemented in the CHARMM program.51 The resulting protein was solvated with a 16 Å layer of TIP3P water. Then, to attain equilibrium of the inner solvent layer, we followed these four steps: (1) optimization of the inner solvent layer for 1000 steps of steepest descent and 1000 steps of the adapted basis Newton−Raphson method, (2) slow heating of the protein from 0 to 300 K for 15 ps with a 1 fs time step, (3) equilibration of the solvent layer for 15 ps at 300 K with a 1 fs time step, and (4) resolvation of the protein to fill up the interspace of the solvent layer. These four steps were repeated four times. After these procedures, a productive molecular dynamics (MD) run was performed for 2.5 ns. During all these MD simulations, the coordinates of the entire Fe(IV) oxo unit and the metal-ligating residues as well as the outer 8 Å of the solvent layer were kept frozen. For the m6A substrate, the base part of m3T was replaced by that of m6A, and the deoxyribose sugar of m3T was replaced by the ribose sugar of m6A. All other features were the same as in the case of m3T. 2.3. QM/MM Methodology. All QM/MM calculations were performed using ChemShell,52 combining Turbomole53 for the QM region and DL_POLY54 for the MM region. The CHARMM27 force field was employed throughout this study for the MM region.51 The electronic embedding scheme55 was used to account for the polarizing effect of the enzyme’s environment on the QM region. Hydrogen link atoms with the charge-shift model52 were applied to treat the QM/MM boundary. During QM/MM geometry optimizations, the QM region was studied with the hybrid UB3LYP56 density functional with two levels of theory. For geometry optimization, the double-ζ basis set LACVP for iron and the 6-31G(d) basis set for all other atoms, collectively labeled as B1, were used. The energies were further corrected with the larger basis set LACV3P++** for all atoms, labeled as B2. The computed electronic energy barrier and computed free energy barrier are generally close for H-abstraction. For example, Habstraction from m6A has an electronic barrier of 17.7 kcal/ mol, while the frequency analysis shows that the zero-point energy (ZPE) correction will reduce the barrier by 2.6 kcal/ mol, whereas the molecular entropy correction (−TΔS) will increase the barrier by 1.6 kcal/mol (the third part is vibrational enthalpy: −0.6 kcal/mol). The net effect is a close match between the computed electronic energy barrier of 17.7 kcal/ mol and the computed free energy barrier of 16.1 kcal/mol. Such a close match was proven by Thiel and co-workers, who carried out careful sampling on a variety of H-abstraction processes in P450.43b,c Thus, given the prohibitively high computational cost of QM/MM MD-based free energy calculations, we treated the electronic energy barriers as estimates of the free energy barriers in the enzyme. B3LYP has already been proven to be a successful21,23,24,41−44 functional for studying iron-based metalloenzymes. A recent extensive benchmark study of H-abstraction by an iron(IV) oxo species found UB3LYP was the best functional for such systems.57 All of the calculations focused on the

Scheme 2. (a) QM Subsystems Used for QM/MM Calculations (High-Valent Iron(IV) Oxo Species and the Two Nucleotides m3T and m6A with Their Methylated Bases (in Red)), (b) QM/MM and HCC Studies of the Demethylation Mechanisms form3T, and (c) QM/MM and HCC Studies of the Demethylation Mechanisms for m6A

m6A bases and sugars, which are partially truncated from the respective nucleotides (m3C and m3U are treated analogously). 2.2. Setup of the QM/MM System. The initial structure of the enzyme−substrate complex was prepared on the basis of the recently determined X-ray structure of the m3T-containing FTO enzyme (PDB code 3LFM, with a resolution of 2.50 Å).25 However, this PDB file contains no crystal waters, while another structure (PDB code 4IDZ, with a resolution of 2.46 Å)49 contains crystal waters. Therefore, the missing inner crystal waters were added to 3LFM by superimposing the two crystal structures of 3LFM and 4IDZ. We assigned the protonation states of titratable residues (His, Glu, Asp) on the basis of pKa values from the PROPKA software50 in combination with careful visual inspection of local hydrogenbonded networks. The histidine residue His271 was protonated at the δ position, while His30 was protonated at the ε position, 7079


Research Article

ACS Catalysis

bond cleavage step in the present study (see Table S1 in the Supporting Information). Our tests indicated that BMK/6311++G(d,p)62 can yield results quite similar to those of CCSD(T)/6-311++G(d,p) calculations for both carbonyl group hydration and C−N bond cleavage (see Table S1). As such, the relative energies of the B3LYP/6-31G(d)-optimized structures were further refined by single point calculations at the BMK/6-311++G(d,p) level, labeled as B2.

quintet spin state, since it is both the ground state and the only reactive state for the iron(IV) oxo species of interest in this work.21−24 All of the transition states (TSs) were located by relaxed potential energy surface (PES) scans followed by full TS optimizations using the P-RFO optimizer implemented in the HDLC code.58 2.4. Hybrid Cluster-Continuum (HCC) Methodology. Most studies of reactions in solution use the dielectric continuum model to evaluate the solvation energy. However, implicit solvation models cannot account for specific solute− solvent interactions, such as the strong hydrogen-bonding interactions between the solvent and the solute during reactions. Such interactions are especially important in proton transfer-related reactions.47,48 To overcome this problem, some of us recently adopted the hybrid cluster-continuum (HCC) model,47,48 where the central solute is solvated explicitly by some solvent molecules and the resulting cluster is treated by a dielectric continuum model. The explicit solvent molecules can cover the most important solute−solvent interactions in the reactions, while the continuum part accounts for long-range electrostatic interactions of the solute with the bulk solvent. This HCC model was verified to be very useful for studying chemical reactions in aqueous solutions, such as hydration and hydrolysis reactions.48 A recent study on the hydration of CO2 in aqueous solution indicated that the HCC model can yield thermodynamic properties and mechanistic results quite similar to those obtained from more advanced ab initio molecular dynamics simulations.48d In order to estimate Gibbs free energies, we directly used the solution-phase vibrational frequencies from the cluster-continuum model calculations. First, the frequency calculations in the gas phase may overestimate the entropic contribution arising from translational and rotational motions, because the suppression of these motions in solution is not captured by gasphase calculations. More importantly, many TSs and intermediates cannot be located by gas phase-only calculations, as many charged species cannot be stabilized in the gas phase without the bulk solvent effect.48 Finally, a recent study59 clearly showed that using solution-phase vibrational frequencies to calculate free energies is a practical approach, especially when liquid- and gas-phase structures differ significantly or when stationary points present in liquid solution do not exist in the gas phase. All of the HCC calculations were performed with the Gaussian 09 software package.60 The geometries of all of the TSs, reactants, and intermediates involved in the reaction were fully optimized using a hydrated cluster in conjunction with the SMD continuum solvation model61 at the B3LYP/6-31G(d) level of theory, labeled as B1. Harmonic frequency calculations were performed using the equilibrium geometries to confirm the existence of first-order saddle points and local minima on the potential energy surfaces and to estimate the zero-point energies, as well as the thermal and entropic corrections. The connections between the stable structures and the transition states were ascertained by analyzing the corresponding imaginary frequency modes, as well as by limited intrinsic reaction coordinate (IRC) calculations. For single point energy corrections, we tested various popular density functional theoretical methods on two key reaction steps: hydration of the carbonyl group and C−N bond cleavage. In accordance with our previous study,48b B3LYP provided quite accurate results for the hydration of the carbonyl group, but it underestimated the calculated barrier for the C−N

3. RESULTS AND DISCUSSION 3.1. The Active Species of FTO and the Mode by Which It Binds the Substrate. The well-characterized crystal structure of m3T-bound FTO25 was used as a starting point for generating the reactant clusters (RCs) of the corresponding iron(IV) oxo species with the methylated DNA/RNA nucleotides. Figure 1 shows, as a typical example, the MD

Figure 1. QM/MM optimized reactant cluster taken from a representative snapshot in the equilibrated MD trajectory of the iron(IV) oxo species with bound m3T.

equilibrated active site structure of the iron(IV) oxo species of FTO in a complex with m3T, which is quite similar to the initial crystal structure (PDB 3LFM).25 Similar to the iron center in the AlkB enzyme,24 in FTO, the Fe center is hexacoordinated, with two His residues, one oxo group, one Asp residue, one succinate, and one water molecule. Arg322 is H-bonded to the oxo group and the Asp ligand of the iron(IV) oxo species. The substrate m3T is stabilized by hydrogen-bonding interactions with Arg96 and Glu234. Such hydrogen-bonding interactions should be important in controlling the substrate’s binding mode, placing the substrate relative to the iron(IV) oxo moiety such that the N-methyl group can be selectively oxidized. Tyr108 tends to keep m3T in place through a steric barricade. The methyl group of m3T points directly to the oxo of the iron(IV) oxo moiety, making it quite available for H-abstraction by the iron oxo moiety. Other RCs have very similar features. Furthermore, FTO encloses all of the substrate except the sugar. This sugar is in contact with the solvating water molecules outside the enzyme. As shown in Figure 1, the base 7080


Research Article

ACS Catalysis

Figure 2. (a) QM/MM (UB3LYP/B2) relative energies (kcal/mol) for the first-round oxidation of m6A by FTO. Key intermediates along the reaction energy profile are schematically drawn. The red energy profile depicts putative follow-up transformations of IC1b. Due to the prohibitively high reaction barriers, the red pathway will not be taken, and reactions will stop at IC1b. (b) QM/MM optimized structures for the reactant complexes of iron(IV) oxo/m6A (RC1) and the hemiaminal intermediate hm6A (IC1b). The key bond lengths are given in Å.

therefore proceeded to consider the decomposition of the hm6A intermediate in aqueous solution outside of the enzyme. The results are described in the following section. 3.2.2. HCC Study of the Decomposition of hm6A in Water Solution. We first considered the single bridging water chainmediated decomposition of hm6A with zero to two explicit water molecules. These results are presented in detail in the Supporting Information. The calculated pathway without the assistance of any water has a remarkably high free energy barrier of 59.0 kcal/mol (Figure S3 in the Supporting Information). The assistance of one or two water molecules decreases the free energy barrier to 29.5 kcal/mol (Figure S4 in the Supporting Information) and 25.6 kcal/mol (Figure S5 in the Supporting Information), respectively. As noted before,48 a single water chain comprised of one to four waters is not capable of stabilizing highly charged intermediates such as hydronium (H3O+) and hydroxide (OH−) ions in proton transfer processes. For instance, previous theoretical calculations on the hydration free energy of the proton suggest that in aqueous solution the proton should be represented as an Eigen cluster (H9O4+), in which three water molecules are required to saturate the first hydration shell of H3O+.48a,63,64 In such a cluster, the calculated hydration free energy of the proton is around 263 kcal/mol, in agreement with the available experimental values of 262.465 and 264.0 kcal/mol.66 The loss of two water molecules from the first hydration shell of H3O+, as in the H5O2+ Zundel cluster, will underestimate the hydration Gibbs free energy of the proton by about 10 kcal/ mol.63 Such a large deviation can lead to biased descriptions of mechanistic and thermodynamic properties for proton transferrelated processes. Therefore, we incorporated more water

component of the substrate binds quite compactly with the surrounding residues, thus leaving no room for the outside water molecules (residing near the sugar) to enter into the active site, as confirmed by our MD simulations. 3.2. QM/MM and HCC Studies of the Demethylation of m6A. Since m6A is a major substrate of FTO, we begin the exploration of the demethylation mechanism with this substrate. 3.2.1. QM/MM Study of the First-Round Oxidation of m6A by FTO’s Active Species. Figure 2 shows the QM/MM calculated relative energy profile for the first-round oxidation of m6A by FTO. With the reactant cluster (RC1) as the starting point, H-abstraction from the methyl group of m6A proceeds with a barrier of 17.7 kcal/mol via TS1a and generates a carbon radical intermediate (IC1a). Mulliken population analysis of IC1a shows that C6 has a spin density of 0.81, indicating that the radical is significantly localized on C6. The following rebound step is quite facile, yielding the hemiaminal intermediate hm6A (IC1b) with a barrier of 7.7 kcal/mol. Starting from IC1b, the C6−N6 bond breakage is coupled to a proton transfer from O2 to N1, leading to formaldehyde and the N1-protonated intermediate IC1c. A subsequent proton transfer from N1 to N6 leads to the final demethylated base product (PC1). The calculated overall barrier is much too high (70.6 kcal/mol from IC1b to TS1d) to be feasible. There are not any residues in the active site that could feasibly serve as acid or base catalysts to lower the barrier. Furthermore, there is not a sufficient amount of water molecules to facilitate the hydrolysis reaction in the active site. Thus, it is clear that the hm6A intermediate will not undergo the last step of the demethylation within the active site of the FTO enzyme. We 7081


Research Article

ACS Catalysis

Figure 3. (a) BMK/B2//B3LYP/B1 relative QM energies (in kcal/mol) for the decomposition of the hm6A intermediate in aqueous solution, shown along with schematic drawings of key intermediates along the reaction pathway. The relative energies are given as electronic energy first and then free energy in parentheses. The red arrows highlight the direction of proton transfer. (b) Optimized structures of key species. The key bond lengths are given in Å.

molecules and constructed a reactant complex of hm6A with water molecules well-connected by three-dimensional hydrogen bond networks. Figure 3 presents the calculated relative energy profile for the decomposition of hm6A as well as the optimized structures of key species involved in the these reactions. With the reactant complex of hm6A (RC2) as the starting point, N1 abstracts a proton from an adjacent water molecule, generating a hydroxide ion in IC2a, which is stabilized by Hbonding interactions with neighboring water molecules, with three H-bond lengths of 1.44, 1.52, and 1.69 Å. The resulting hydroxide ion can further abstract a proton as indicated by the red arrows in IC2a, yielding the N1-protonated zwitterion IC2b. The subsequent C6−N6 bond cleavage via TS2c leads to IC2c. The following H-bond reorientations and a solventassisted proton shift from N1 to N6 will form the demethylated adenosine (PC2). We also considered C−N bond cleavage in IC2a, but this pathway was found to be unfeasible, and the C− N bond elongation scans invariably led to the formation of IC2b. Therefore, the decomposition of hm6A has a specific self-

catalyzed mechanism, in which N1 acts as an internal base catalyst to deprotonate the hydroxyl group of hm6A and form the N1-protonated intermediate IC2a. The decomposition of the hm6A intermediate in aqueous solution has a ratedetermining free energy barrier of 23.1 kcal/mol (via TS2c), which is in good agreement with the experimental value of ∼24 kcal/mol.28 We also considered the N8-catalyzed mechanism as a potential pathway for the decomposition of the hemiaminal intermediate hm6A. However, this mechanism was found to have a very high electronic energy barrier (estimated from scanning as >34.8 kcal/mol, as shown in Figure S7 in the Supporting Information), in comparison with the much lower electronic energy barrier of 25.3 kcal/mol for the N1-catalyzed mechanism. Moreover, we could not locate the N8-protonated intermediate during the C−N bond cleavage potential energy scan (Figure S8 in the Supporting Information). The reason is that the N8-protonated intermediate is quite unstable: it is 13.6 kcal/mol higher in energy than the N1-protonated intermediate 7082


Research Article

ACS Catalysis

Figure 4. (a) QM/MM (UB3LYP/B2) relative energies (kcal/mol) for the oxidation of the hm6A intermediate by the iron(IV) oxo active species, with schematic drawings of key intermediates along the reaction path. The red energy prof ile for the nucleophilic attack on f6A is the road not taken. (b) QM/MM optimized structures for the reactant complexes of iron(IV) oxo/hm6A (RC3) and the intermediate f6A (IC3b). The key bond lengths are given in Å.

nucleophilic attack on the carbonyl group of f6A. However, a ferric coordinated water molecule is not a powerful nucleophile, and in addition our calculations show that the hydration is not facile for the single water-mediated pathway.48b,d Indeed, as shown by the red energy profile, the nucleophilic attack proceeds with a very high barrier of 46.7 kcal/mol (TS3c). Clearly, the f6A (IC3b) intermediate represents a dead end, which will not undergo any further transformations inside the active site of the FTO enzyme. As suggested in experimental work,28 f6A will exit the active site and undergo a transformation in the aqueous phase, which will be described in the next subsection. 3.2.4. HCC Study of f6A Hydrolysis in Water Solution. Since 6 f A cannot undergo the final demethylation step in the enzyme, we turn again to the investigation of this step in the aqueous solution outside the enzyme. The current HCC model follows our previous studies48 of the hydration of carbonyl groups in aqueous solution. Figure 5 displays the calculated relative energy profile for the hydrolysis of f6A in aqueous solution, while Figure 6 presents the optimized structures of key species involved in the these reactions. With f6A (RC4) in Figure 5 as the starting point, the initial nucleophilic attack of an adjacent water molecule on the carbonyl group at C6 via TS4a leads to the water adduct intermediate IC4a. Subsequently, two competing proton transfer pathways can occur, as indicated on IC4a, using red and blue arrows. The pathway marked by the red arrows involves proton transfer to N1 via TS4c, leading to the N1protonated intermediate IC4c. The other pathway, marked by the blue arrow, involves proton transfer to the neighboring

(Figure S9 in the Supporting Information). Therefore, the decomposition of hm6A will proceed exclusively via the N1catalyzed mechanism. 3.2.3. QM/MM Study of the Oxidation of hm6A to f6A by the Iron(IV) Oxo Species of FTO. According to a recent experimental study,28 the FTO-mediated demethylation of m6A involves a novel second-round oxidation pathway, in which the corresponding hemiaminal intermediate hm6A is converted to the N6-formyladenosine intermediate, f6A (Scheme 1b). Thus, we proceeded to consider the FTO-mediated oxidation of hm6A by the second iron(IV) oxo species. Figure 4 shows the QM/MM calculated relative energy profile as well as the optimized key species involved in the reactions. As shown in the QM/MM optimized structure of the reactant complex, iron(IV) oxo/hm6A (RC3), the hydroxyl group of hm6A forms a good H bond with the oxo group. As shown in Figure 4b, the hm6A substrate is stabilized in RC3 by H-bonding interactions with two neighboring Arg residues. With RC3 as the starting point, the hydrogen abstraction of H1 by the iron(IV) oxo species via TS3a is facile and results in the O-based radical intermediate IC3a. The Fe−OH moiety in IC3a can further abstract a second hydrogen (H3) from the −CH2O• group via TS3b, with a small barrier, yielding the amide intermediate f6A and a ferrous water complex (IC3b). Thus, the FTO-mediated oxidation process of hm6A to f6A is by itself very facile. What about the follow-up decomposition reaction of f6A within the enzyme? As shown in the QM/MM optimized structure of IC3b, a water molecule is located just below the carbonyl group of f6A, and it is in principle well-positioned for 7083


Research Article

ACS Catalysis

Figure 5. BMK/B2//B3LYP/B1 relative QM energies (in kcal/mol) for the hydrolysis of the f6A intermediate in an aqueous solution, shown along with schematic drawings of key intermediates along the reaction pathway: (a) competing pathways leading from RC4 to IC4b or IC4c; (b) competing pathways leading from IC4b to IC4f and IC4h. The red sections of the profiles represent the higher-energy pathways, while the black portions are the lower-energy pathways which are followed. The relative energies are given as electronic energy first and then free energy in parentheses. The arrows highlight the direction of proton transfer.

water, yielding the intermediate IC4b with an H3O+ core. As shown in Figure 5a, formation of IC4b is kinetically more favorable than IC4c formation; thus, we proceeded to consider further reactions of IC4b. With IC4b (Figure 5b) as the starting point, the subsequent proton transfer from the H3O+ core to the neighboring water affords another Eigen intermediate (IC4d), in which the H3O+ core is strongly H-bonded to three surrounding water molecules, with three H-bond lengths of 1.51, 1.55, and 1.58 Å, respectively (Figure 6). With IC4d as the starting point, we investigated the two competing reactions shown in Figure 5b. One, in the red profile, involves a direct C6−N6 bond cleavage

via TS4f, leading to the intermediate IC4f. This pathway requires a very high overall free energy barrier of 34.7 kcal/mol. The second pathway, in the black section of the profile, involves protonation of O2 by the H3O+ ion via TS4e, leading to the gem-diol intermediate IC4e. The subsequent proton abstraction by N1 assisted by a water molecule (via TS4g) leads to the N1protonated intermediate IC4g, which dissociates by C6−N6 bond cleavage via TS4h, leading to formic acid and the N1protonated isomer of adenosine (IC4h). This is a facile process, and the N1-protonated isomer of adenosine can easily isomerize to adenosine via a proton shift from N1 to N6. The isomerization pathway assisted by two water molecules has 7084


Research Article

ACS Catalysis

Figure 6. Optimized structures of key species involved in the decomposition of the f6A intermediate in water solution. The key bond lengths are given in Å.

have a barrier of 17.7 kcal/mol. FTO-mediated decomposition of the hm6A intermediate to form the demethylated base requires a prohibitively high overall reaction barrier of 70.6 kcal/mol (starting from IC1b). In the following steps, hm6A can either undergo decomposition in aqueous solution to yield A and formaldehyde or a second-round oxidation by the FTO enzyme leading to f6A, which is subsequently hydrolyzed to yield A and formic acid. The calculated free energy barriers for the decomposition of hm6A and hydrolysis of f6A in aqueous solution are 23.1 and 22.7 kcal/mol, respectively, in good agreement with the experimental value of ∼24 kcal/mol.28 In order to accomplish the second-round oxidation of hm6A, the system has to reorganize to prepare the new reactant complex of hm6A/iron(IV) oxo.67 The term “reorganize” involves dissociation of hm6A from FTO and its subsequent rebinding, as well as the formation of a new iron(IV) oxo active species starting from the iron in the resting state. As can be seen from Scheme 3, once hm6A/iron(IV) oxo is formed, f6A formation in the second-round oxidation has actually a small barrier of 13.7 kcal/mol. As such, the inefficient formation of f6A does not originate in H-abstraction by the iron(IV) oxo species, and it can only be due to the reorganization steps for the reactant complex (hm6A/iron(IV) oxo) formation. Therefore, the major demethylation pathway of m6A proceeds via the decomposition of hm6A in water solution, while the secondround oxidation of hm6A followed by the subsequent hydrolysis of the resulting f6A is a minor and inefficient demethylation pathway due to the slow reorganization step. The fact that the hydrolysis of f6A is still observable, even if as a minor route, means that the effective free energy barrier of the reorganization step is only slightly higher than the ratedetermining barrier, 23.1 kcal/mol, for the major pathway. For instance, a barrier of 24.8 kcal/mol would mean that 10% of the reactions follow the minor path, while 90% follow the major path at physiological temperature. Clearly, the present QM/ MM and HCC calculations are quite reliable for the FTOmediated oxidations of m6A and the subsequent watermediated decomposition of hm6A and hydrolysis of f6A. At the same time, the results for the minor route allow us to bracket the corresponding effective barrier as approximately 25 kcal/mol, due mainly to the reorganizational steps of the enzyme en route to the formation of the second iron(IV) oxo active species.

a small Gibbs free energy barrier of 7.9 kcal/mol (Figure S12 in the Supporting Information). As shown in the black energy profiles in Figure 5, the hydrolysis of f6A has a rate-determining free energy barrier of 22.7 kcal/mol. This is also consistent with the experimental value of ∼24 kcal/mol.28 3.2.5. FTO-Mediated Demethylation of m6A: Comparison between Theory and Experiment. The reaction mechanisms and kinetics of the FTO-mediated demethylation of m6A were thoroughly studied by experimental means.28 The proposed demethylation reaction proceeds either through the decomposition of the hemiaminal hm6A or through hydrolysis of the formyl-amide intermediate f6A. Both intermediates have halflives of ∼3 h in aqueous solution under physiological conditions. These half-lives correspond to a free energy barrier of ∼24 kcal/mol (based on Eyring’s transition state theory). Moreover, experiment indicates that the formation of hm6A in the first-round oxidation is much faster than f6A formation in the second-round oxidation.28 Scheme 3 summarizes the calculated barriers for the demethylation of m6A in the respective transformation pathways. Starting from m6A, FTO-mediated hm6A formation via the H-abstraction/rebound mechanism was calculated to Scheme 3. QM/MM and HCC Calculated Demethylation Mechanism of m6Aa

a

The barriers (the values in parentheses are free energy barriers from HCC calculations) near the respective arrows are in kcal/mol and belong to the rate-determining step in the respective forward transformations. 7085


Research Article

ACS Catalysis 3.3. Computed Demethylation Mechanism of m3T by FTO. The above conclusion that the second oxidation round is minor merely due to slow reorganizational steps allows us to proceed to the m3T demethylation process and then generalize to other bases. Scheme 4 summarizes the calculated barriers for the ratedetermining steps in the respective forward transformations

to form thymine (T) and formic acid, with a low free energy barrier of 16.4 kcal/mol (Scheme 4). Consequently, our mechanism differs from the experimental proposal (see Scheme 1a).10 As such, we have to conclude that the “formaldehyde” product proposed by experiment is likely formic acid, as revealed by our calculations. 3.4. What Factors Control the Efficiency of FTOMediated Demethylation of Methylated Bases? Figure 7

Scheme 4. QM/MM and HCC Calculated Demethylation Mechanism of m3Ta

Figure 7. N-Methylated bases considered in this work and their calculated N−CH3 bond lengths, given in Å. The calculated free energy barriers (in kcal/mol, with the HCC model) for the decomposition of the corresponding hemiaminal intermediates in aqueous solutions are shown below the structures. a

The barriers (values in parentheses are HCC free energy barriers) near the respective arrows are in kcal/mol and belong to the ratedetermining step in the respective forward transformations. Due to an overly high barrier, the pathway marked in red is not taken.

shows the four bases studied by us along with their C−N bond lengths. These bond lengths suggest that either the C−N bond energies in the methylated bases are similar or m6A may have the strongest C−N bond by a slight margin. Nevertheless, according to experimental results,3,10,26,27 the FTO enzyme demethylates the various methylated bases in the following order of efficiency: m6A > m3T = m3U > m3C. This highlights that factors other than C−N bond strength may determine demethylation reactivity. Figure 7 also summarizes the activation free energies (listed underneath the bases) for the decomposition of the corresponding hemiaminals in aqueous solutions. Looking at these barriers (see Figure 7), one would expect the efficiency of the FTO-mediated demethylation reactions to follow the order m3C > m6A ≫ m3T = m3U, which is completely inconsistent with the experimental trend. Why is m 3 C, which seems to have the best prospects for demethylation, in fact the least reactive in the series? In addition, what are the factors that make m6A a major substrate of FTO with the highest demethylation efficiency? 3.4.1. Why Does m3C Exhibit the Poorest Demethylation Efficiency in FTO? The hemiaminal hm3C has an N+−CH2OH linkage, which should be able to easily undergo heterolytic cleavage. Indeed, our calculations indicate that the decomposition of the hemiaminal intermediate hm3C (Figure S30 in the Supporting Information) follows a mechanism very similar to that of hm3T decomposition (H3O+-mediated C−N bond cleavage), but with a much smaller free energy barrier of 20.4 kcal/mol (in comparison to 30.3 kcal/mol for hm3T). The result supports the experimental deduction68 that the positively charged cytosine base is a much better leaving group than the neutral thymidine. Indeed, m3C is much more efficiently demethylated than m3T by the AlkB enzyme.69 However, in FTO, the demethylation reactivity of m3C is quite poor, apparently because m3C is not able to be effectively bound within the active site of FTO, and it escapes quickly, as shown by previous MD studies.28 This is probably due to the electrostatic repulsion between the positive charge of m3C and the active site environment.28 Therefore, the low demethylation reactivity of m3C in FTO is not due to an intrinsic lack of

during the demethylation of m3T, while all the details behind these results are relegated to Figures S13−S28 in the Supporting Information. It is instructive to compare these data to the results presented in Scheme 3 for m6A. Thus, with the methylated base m3T as the starting point, the first-round oxidation by FTO-iron(IV) oxo, in Scheme 4, produces the hemiaminal intermediate hm3T, with a small barrier of 13.9 kcal/mol. The subsequent FTO-mediated decomposition of hm3T requires a prohibitively high overall reaction barrier of 55.1 kcal/mol (Figure S13). Thereafter, there are two available pathways. One is the decomposition of hm3T in aqueous solution, to yield T and formaldehyde, and the other is a second-round oxidation followed by water-assisted decomposition of f3T to yield T and formic acid. Comparison to Scheme 3 shows that the water-assisted decomposition of hm6A has a moderate free energy barrier of 23.1 kcal/mol, whereas the same process for hm3T in Scheme 4 requires a high free energy barrier of 30.3 kcal/mol. With such a high barrier under physiological conditions, the reaction would be exceedingly slow (k ≈ 2.7 × 10−9 s−1). It is instructive to compare this computed barrier to the ratedetermining free energy barrier in Figure 3 and Scheme 3 for the decomposition of the corresponding hm6A intermediate, which has a value of 23.1 kcal/mol (k ≈ 3.2 × 10−4 s−1). Thus, while postponing discussion of why the barrier for the waterassisted hm3T decomposition is so high (see full profile in Figure S22 in the Supporting Information), it is clear that hm3T and hm6A undergo f undamentally dif ferent decomposition mechanisms in water, with rate constants that dif fer by 5 orders of magnitude. Considering our above estimated effective barrier of ∼25 kcal/mol for the reorganizational step of a secondrebound oxidation by FTO (which we can reasonably assume to be the same for any FTO substrate), we may conclude that m3T demethylation will prefer to proceed by two rounds of oxidation by FTO, leading to f3T, which in turn is hydrolyzed 7086


Research Article

ACS Catalysis

Figure 8. Comparison of the calculated mechanisms for the decomposition of (a) hm6A and (b) hm3T. The results are taken from Figure 3 for hm6A and Figure S22 in the Supporting Information for hm3T, respectively. The calculated relative free energies are given in kcal/mol.

reactivity, but rather purely due to its fleeting presence in the active site of FTO. 3.4.2. Why Is m6A a Major Substrate for FTO? As shown in Figure 3, hm6A undergoes a water-assisted decomposition to adenine and formaldehyde, with a rather moderate barrier (23.1 kcal/mol). In contrast, the same decomposition process for hm3T has a large barrier (30.3 kcal/mol in Figure S22 in the Supporting Information). Similarly, our calculations for hm3U show that the water-assisted demethylation barrier (30.2 kcal/ mol in Figure S29 in the Supporting Information) is virtually identical to that of hm3T. The question then is why is the barrier for water-assisted decomposition of the hm6A hemiaminal so much smaller than those barriers computed for the decomposition of hm3T and hm3U? Figure 8 compares the key steps in the decomposition of hm6A vs hm3T. For hm6A, our calculations reveal that the demethylation reaction follows a self-catalyzed mechanism in which N1 acts as a base catalyst that facilitates the decomposition of the hemiaminal intermediate hm6A in comparison with the decomposition of hm3T. Thus, how does this self-catalysis reduce the barrier for the decomposition of hm6A in comparison with hm3T? Figure 8 breaks down the process into two steps and shows their energy demands. Thus, the water-catalyzed deprotonation of hm3T requires a free energy of 18.5 kcal/mol, whereas the same process for hm6A requires a free energy of only 12.2 kcal/mol, more than 6 kcal/ mol lower due to the internal catalysis by N1 deprotonation. In contrast, the subsequent C−N bond cleavage reactions require almost the same free energy barriers: 11.8 kcal/mol for hm3T vs 10.9 kcal/mol for hm6A. Therefore, according to our results, the N1-catalyzed mechanism makes m6A the most reactive substrate (among the substrates studied in this work) in the decomposition of the hemiaminal intermediate, and hence m6A is a major substrate of FTO. The opportunity or lack thereof for internal catalysis during the N−CH2OH deprotonation of the hemiaminals determines the chosen mechanistic path from the hemiaminal onward. Both methylated bases, m6A (Scheme 3) and m3T (Scheme 4), undergo a facile first step of hydroxylation by FTO to form the corresponding hemiaminals hm6A and hm3T. Subsequently, hm6A, which has an internal catalytic site for deprotonation of N−CH2OH, undergoes mostly a water-assisted decomposition

to adenine and formaldehyde, with a rather moderate barrier (23.1 kcal/mol). In contrast, hm3T, which lacks an internal catalyst, avoids the large decomposition barrier (30.3 kcal/mol in Figure S22 in the Supporting Information) and instead undergoes a second-round FTO-mediated oxidation (with an estimated effective barrier of ∼25 kcal/mol). The so-produced f3T then undergoes a facile water-assisted hydrolysis.

4. CONCLUSION This work reports comprehensive QM/MM and hybrid clustercontinuum (HCC) calculations on the mechanistic nature of FTO- and water-mediated overall demethylation of four different N-methylated DNA/RNA bases: m6A, m3T, m3U, and m 3 C. The QM/MM calculations show that the hydroxylation of the N-methylated bases by the iron(IV) oxo active species of FTO is a fast reaction. The slow step during enzymatic oxidation is the reorganization event necessary for the preparation of the iron(IV) oxo/hemiaminal reactant complexes for the second-round oxidation. From experimental data and our calculations, we estimated the effective free energy barrier of the reorganizational step of the enzyme to be ∼25 kcal/mol. This barrier determines the mechanistic fate past the first oxidation round, and Scheme 5 summarizes these choices, using the label B for the base and mnB, hmnB, and fnB for the nth position methylated base and the corresponding hemiaminal and formyl-amide intermediates, respectively. Thus, in a nutshell, the choice is made after the formation of the hemiaminal hmnB. If the direct demethylation by the watermediated decomposition of the hemiaminal intermediate, to the left in Scheme 5, has a barrier significantly smaller than 25 kcal/ mol, the demethylation will produce the free base, B, along with formaldehyde, without passing through the second-round FTO-mediated oxidation. If, however, the water-mediated decomposition of hmnB has a barrier exceeding 25 kcal/mol, the corresponding hemiaminal hmnB will undergo, to the right in Scheme 5, a second-round FTO-mediated oxidation, to form the corresponding formyl-amide fnB, which is hydrolyzed in the aqueous phase to generate formic acid and the demethylated base. Using this dividing line, we find that the major mechanism for m6A demethylation is the direct water-mediated decomposition of hm6A (Scheme 3), while the minor mechanism 7087


Research Article

ACS Catalysis

complete set of demethylation reactions of the N-methylated DNA/RNA nucleotides.

Scheme 5. Schematic Representation of the Mechanistic Choices for Methylated Base Demethylation by the FTO Enzymea

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.5b01867. Total QM/MM energies, QM energies, thermal corrections, and Cartesian coordinates of all computed species (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail for S.S.: [email protected]. mnB symbolizes the base methylated at the nth site, hmnB is the corresponding hemiaminal, wherein the N-methyl group is oxidized to N−CH2OH, and fnB is the corresponding formyl-amide N−CHO. The numbers near arrows are free energy barriers (in kcal/mol). a

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by an Israel Science Foundation grant (ISF-1183/13) to S.S. D.A.S. thanks the United States-Israel Educational Foundation and the Fulbright U.S. Student Program for her fellowship.

proceeds by two rounds of oxidation followed by amide hydrolysis. Our calculated barrier for the decomposition of hm6A in water solution is 23.1 kcal/mol, while the barrier for the hydrolysis of the amide intermediate f6A is 22.7 kcal/mol. Both values are in good agreement with the experimental value of ∼24 kcal/mol.28 On the other hand, for m3T (Scheme 4), the decomposition of the hemiaminal intermediate hm3T has a high free energy barrier of 30.3 kcal/mol, much larger than the dividing line (∼25 kcal/mol). As such, m3T demethylation is diverted to undergo two sequential FTO-mediated oxidation cycles followed by hydrolysis of the amide intermediate f3T to yield T and formic acid. What determines the different behaviors of m6A and m3T is the ability of the N1 site of adenine to act as a base catalyst and enable the direct decomposition of hm6A with a moderate barrier of 23.1 kcal/mol. The lack of this internal base catalyst in thymine raises the barrier for the direct hydrolysis of hm3T to 30.3 kcal/mol and diverts the mechanism toward two sequential oxidation cycles, as in the right of Scheme 5. The behavior of m3U is perfectly analogous to m3T, in that both are lacking an internal base catalyst and must proceed to the right branch in Scheme 5. The opportunity for self-catalyzed hydrolysis of the hemiaminal makes the m6A substrate the most reactive, and hence it is a major substrate for the FTO enzyme. The methylated cytosine, m3C, is poorly demethylated because FTO cannot efficiently bind this positively charged substrate. With appropriate mutations in the active site, FTO may demethylate m3C as efficiently as it demethylates m6A. Finally, our results suggest a revision of the experimental mechanism for m3T and m3U demethylation, which were proposed to proceed via the single-round mechanism. However, the calculations show the corresponding hemiaminal decompositions encounter high barriers due to lack of an internal basic catalytic site, and the mechanism is diverted to follow a route involving two sequential FTO-mediated oxidations. The AlkB protein68 is capable of promoting the decomposition of the hemiaminal intermediate of m3T (hm3T) because of the Glu136 residue in its active site, which serves as a base catalyst that can facilitate the C−N bond cleavage reaction.21 However, the FTO enzyme lacks any such base catalyst in the active site (and it does not have a feasible acid catalyst either). That is why FTO requires the cooperation of water catalysis outside of the enzyme to accomplish the

■

REFERENCES

(1) Cedar, H.; Bergman, Y. Nat. Rev. Genet. 2009, 10, 295−304. (2) (a) Shen, L.; Song, C. X.; He, C.; Zhang, Y. Annu. Rev. Biochem. 2014, 83, 585−614. (b) He, Y. F.; Li, B. Z.; Li, Z.; Liu, P.; Wang, Y.; Tang, Q. Y.; Ding, J. P.; Jia, Y. Y.; Chen, Z. C.; Li, L.; Sun, Y.; Li, X. X.; Dai, Q.; Song, C. X.; Zhang, K. L.; He, C.; Xu, G. L. Science 2011, 333, 1303−1307. (c) Ito, S.; Shen, L.; Dai, Q.; Wu, S. C.; Collins, L. B.; Swenberg, J. A.; He, C.; Zhang, Y. Science 2011, 333, 1300−1303. (d) Pfaffeneder, T.; Hackner, B.; Truss, M.; Münzel, M.; Müller, M.; Deiml, C. A.; Hagemeier, C.; Carell, T. Angew. Chem., Int. Ed. 2011, 50, 7008−7012. (3) (a) Zheng, G.; Fu, Y.; He, C. Chem. Rev. 2014, 114, 4602−4620. (b) Lu, L.; Zhu, C.; Xia, B.; Yi, C. Chem. - Asian J. 2014, 9, 2018− 2029. (4) Wu, H.; Zhang, Y. Cell 2014, 156, 45−68. (5) Bergman, Y.; Cedar, H. Nat. Struct. Mol. Biol. 2013, 20, 274−281. (6) Shrivastav, N.; Li, D.; Essigmann, J. M. Carcinogenesis 2010, 31, 59−70. (7) Law, J. A.; Jacobsen, S. E. Nat. Rev. Genet. 2010, 11, 204−220. (8) Spruijt, C. G.; Gnerlich, F.; Smits, A. H.; Pfaffeneder, T.; Jansen, P. W. T. C.; Bauer, C.; Münzel, M.; Wagner, M.; Müller, M.; Khan, F.; Eberl, H. C.; Mensinga, A.; Brinkman, A. B.; Lephikov, K.; Müller, U.; Walter, J.; Boelens, R.; van Ingen, H.; Leonhardt, H.; Carell, T.; Vermeulen, M. Cell 2013, 152, 1146−1159. (9) Aranda, J.; Zinovjev, K.; Roca, M.; Tuñoń , I. J. Am. Chem. Soc. 2014, 136, 16227−16239. (10) Gerken, T.; Girard, C. A.; Tung, Y. C. L.; Webby, C. J.; Saudek, V.; Hewitson, K. S.; Yeo, G. S. H.; McDonough, M. A.; Cunliffe, S.; McNeill, L. A.; Galvanovskis, J.; Rorsman, P.; Robins, P.; Prieur, X.; Coll, A. P.; Ma, M.; Jovanovic, Z.; Farooqi, I. S.; Sedgwick, B.; Barroso, I.; Lindahl, T.; Ponting, C. P.; Ashcroft, F. M.; O’Rahilly, S.; Schofield, C. J. Science 2007, 318, 1469−1472. (11) Frayling, T. M.; Timpson, N. J.; Weedon, M. N.; Zeggini, E.; Freathy, R. M.; Lindgren, C. M.; Perry, J. R. B.; Elliott, K. S.; Lango, H.; Rayner, N. W.; Shields, B.; Harries, L. W.; Barrett, J. C.; Ellard, S.; Groves, C. J.; Knight, B.; Patch, A. M.; Ness, A. R.; Ebrahim, S.; Lawlor, D. A.; Ring, S. M.; Ben-Shlomo, Y.; Jarvelin, M. R.; Sovio, U.; Bennett, A. J.; Melzer, D.; Ferrucci, L.; Loos, R. J. F.; Barroso, I.; Wareham, N. J.; Karpe, F.; Owen, K. R.; Cardon, L. R.; Walker, M.; Hitman, G. A.; Palmer, C. N. A.; Doney, A. S. F.; Morris, A. D.; Smith, G. D.; Hattersley, A. T.; McCarthy, M. I.; Control, W. T. C. Science 2007, 316, 889−894. 7088


Research Article

ACS Catalysis

(43) (a) Sen, K.; Hackett, J. C. J. Am. Chem. Soc. 2010, 132, 10293− 10305. (b) Senn, H. M.; Thiel, S.; Thiel, W. J. Chem. Theory Comput. 2005, 1, 494−505. (c) Senn, H. M.; Kästner, J.; Breidung, J.; Thiel, W. Can. J. Chem. 2009, 87, 1322−1337. (44) Lonsdale, R.; Houghton, K. T.; Åżurek, J.; Bathelt, C. M.; Foloppe, N.; de Groot, M. J.; Harvey, J. N.; Mulholland, A. J. J. Am. Chem. Soc. 2013, 135, 8001−8015. (45) (a) Karasulu, B.; Thiel, W. ACS Catal. 2015, 5, 1227−1239. (b) Zhu, W. Y.; Liu, Y. J. ACS Catal. 2015, 5, 3953−3965. (c) Zhou, J. W.; Wu, R. B.; Wang, B. J.; Cao, Z. X.; Yan, H. G.; Mo, Y. R. ACS Catal. 2015, 5, 2805−2813. (d) Abad, E.; Rommel, J. B.; Kästner, J. J. Biol. Chem. 2014, 289, 13726−13738. (46) (a) Loewen, P. C.; Carpena, X.; Vidossich, P.; Fita, I.; Rovira, C. J. Am. Chem. Soc. 2014, 136, 7249−7252. (b) Ardèvol, A.; Rovira, C. J. Am. Chem. Soc. 2015, 137, 7528−7547. (47) (a) Pliego, J. R.; Riveros, J. M. J. Phys. Chem. A 2001, 105, 7241−7247. (b) Sunoj, R. B.; Anand, M. Phys. Chem. Chem. Phys. 2012, 14, 12715−12736. (48) (a) Wang, B.; Cao, Z. J. Phys. Chem. A 2010, 114, 12918− 12927. (b) Wang, B.; Cao, Z. Angew. Chem., Int. Ed. 2011, 50, 3266− 3270. (c) Wang, B.; Cao, Z. Chem. - Eur. J. 2011, 17, 11919−1192. (d) Wang, B.; Cao, Z. J. Comput. Chem. 2013, 34, 372−378. (e) Wang, Y.; Deng, W.; Wang, B.; Zhang, Q.; Wan, X.; Tang, Z.; Wang, Y.; Zhu, C.; Cao, Z.; Wang, G.; Wan, H. Nat. Commun. 2013, 4, 2141. (49) Aik, W.; Demetriades, M.; Hamdan, M. K.; Bagg, E. A.; Yeoh, K. K.; Lejeune, C.; Zhang, Z.; McDonough, M. A.; Schofield, C. J. J. Med. Chem. 2013, 56, 3680−3688. (50) Olsson, M. H.; Søndergard, C. R.; Rostkowski, M.; Jensen, J. H. J. Chem. Theory Comput. 2011, 7, 525−537. (51) Brooks, B. R.; Brooks, C. L.; MacKerell, A. D., Jr.; Nilsson, L.; Petrella, R. J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S.; Caflisch, A.; Caves, L.; Cui, Q.; Dinner, A. R.; Feig, M.; Fischer, S.; Gao, J.; Hodoscek, M.; Im, W.; Kuczera, K.; Lazaridis, T.; Ma, J.; Ovchinnikov, V.; Paci, E.; Pastor, R. W.; Post, C. B.; Pu, J. Z.; Schaefer, M.; Tidor, B.; Venable, R. M.; Woodcock, H. L.; Wu, X.; Yang, W.; York, D. M.; Karplus, M. J. Comput. Chem. 2009, 30, 1545−1614. (52) (a) Sherwood, P.; de Vries, A. H.; Guest, M. F.; Schreckenbach, G.; Catlow, C. R. A.; French, S. A.; Sokol, A. A.; Bromley, S. T.; Thiel, W.; Turner, A. J.; Billeter, S.; Terstegen, F.; Thiel, S.; Kendrick, J.; Rogers, S. C.; Casci, J.; Watson, M.; King, F.; Karlsen, E.; Sjovoll, M.; Fahmi, A.; Schäfer, A.; Lennartz, C. J. Mol. Struct.: THEOCHEM 2003, 632, 1−28. (b) Metz, S.; Kästner, J.; Sokol, A.; Keal, T.; Sherwood, P. WIREs Comput. Mol. Sci. 2014, 4, 101−110. (53) Ahlrichs, R.; Bär, M.; Häser, M.; Horn, H.; Kölmel, C. Chem. Phys. Lett. 1989, 162, 165−169. (54) Smith, W.; Forester, T. R. J. Mol. Graphics 1996, 14, 136−141. (55) Bakowies, D.; Thiel, W. J. Phys. Chem. 1996, 100, 10580−10594. (56) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652. (57) Altun, A.; Breidung, J.; Neese, F.; Thiel, W. J. Chem. Theory Comput. 2014, 10, 3807−3820. (58) Billeter, S. R.; Turner, A. J.; Thiel, W. Phys. Chem. Chem. Phys. 2000, 2, 2177−2186. (59) Ribeiro, R. F.; Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. J. Phys. Chem. B 2011, 115, 14556−14562. (60) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .;

(12) Dina, C.; Meyre, D.; Gallina, S.; Durand, E.; Korner, A.; Jacobson, P.; Carlsson, L. M. S.; Kiess, W.; Vatin, V.; Lecoeur, C.; Delplanque, J.; Vaillant, E.; Pattou, F.; Ruiz, J.; Weill, J.; Levy-Marchal, C.; Horber, F.; Potoczna, N.; Hercberg, S.; Le Stunff, C.; Bougneres, P.; Kovacs, P.; Marre, M.; Balkau, B.; Cauchi, S.; Chevre, J. C.; Froguel, P. Nat. Genet. 2007, 39, 724−726. (13) Fischer, J.; Koch, L.; Emmerling, C.; Vierkotten, J.; Peters, T.; Brüning, J. C.; Rüther, U. Nature 2009, 458, 894−898. (14) Church, C.; Moir, L.; McMurray, F.; Girard, C.; Banks, G. T.; Teboul, L.; Wells, S.; Brüning, J. C.; Nolan, P. M.; Ashcroft, F. M.; Cox, R. D. Nat. Genet. 2010, 42, 1086−1092. (15) Que, L., Jr. Acc. Chem. Res. 2007, 40, 493−500. (16) Nam, W. Acc. Chem. Res. 2007, 40, 522−531. (17) Costas, M. Coord. Chem. Rev. 2011, 255, 2912−2932. (18) Borovik, A. S. Chem. Soc. Rev. 2011, 40, 1870−1874. (19) Krebs, C.; Fujimori, D. G.; Walsh, C. T.; Bollinger, J. M., Jr. Acc. Chem. Res. 2007, 40, 484−492. (20) Wong, S. D.; Srnec, M.; Matthews, M. L.; Liu, L. V.; Kwak, Y.; Park, K.; Bell, C. B., III; Alp, E. E.; Zhao, J. Y.; Yoda, Y.; Kitao, S.; Seto, M.; Krebs, C.; Bollinger, J. M., Jr.; Solomon, E. I. Nature 2013, 499, 320−323. (21) Liu, H.; Llano, J.; Gauld, J. W. J. Phys. Chem. B 2009, 113, 4887−4898. (22) Fang, D.; Lord, R. L.; Cisneros, G. A. J. Phys. Chem. B 2013, 117, 6410−6420. (23) Quesne, M. G.; Latifi, R.; Gonzalez-Ovalle, L. E.; Kumar, D.; de Visser, S. P. Chem. - Eur. J. 2014, 20, 435−446. (24) Wang, B.; Usharani, D.; Li, C.; Shaik, S. J. Am. Chem. Soc. 2014, 136, 13895−13901. (25) Han, Z. F.; Niu, T. H.; Chang, J. B.; Lei, X. G.; Zhao, M. Y.; Wang, Q.; Cheng, W.; Wang, J. J.; Feng, Y.; Chai, J. J. Nature 2010, 464, 1205−1209. (26) Jia, G.; Yang, C.; Yang, S.; Jian, X.; Yi, C.; Zhou, Z.; He, C. FEBS Lett. 2008, 582, 3313−3319. (27) Jia, G.; Fu, Y.; Zhao, X.; Dai, Q.; Zheng, G.; Yang, Y.; Yi, C.; Lindahl, T.; Pan, T.; Yang, Y. G.; He, C. Nat. Chem. Biol. 2011, 7, 885−887. (28) Fu, Y.; Jia, G.; Pang, X.; Wang, R.; Wang, X.; Li, C.; Smemo, S.; Dai, Q.; Bailey, K. A.; Nobrega, M. A.; Han, K. L.; Cui, Q.; He, C. Nat. Commun. 2013, 4, 1798. (29) Roost, C.; Lynch, S. R.; Batista, P. J.; Qu, K.; Chang, H. Y.; Kool, E. T. J. Am. Chem. Soc. 2015, 137, 2107−2115. (30) Fu, Y.; Dominissini, D.; Rechavi, G.; He, C. Nat. Rev. Genet. 2014, 15, 293−306. (31) Meyer, K. D.; Jaffrey, S. R. Nat. Rev. Mol. Cell Biol. 2014, 15, 313−326. (32) Wang, X.; Lu, Z.; Gomez, A.; Hon, G. C.; Yue, Y.; Han, D.; Fu, Y.; Parisien, M.; Dai, Q.; Jia, G.; Ren, B.; Pan, T.; He, C. Nature 2013, 505, 117−120. (33) Liu, N.; Dai, Q.; Zheng, G. Q.; He, C.; Parisien, M.; Pan, T. Nature 2015, 518, 560−564. (34) Falnes, P. O.; Johansen, R. F.; Seeberg, E. Nature 2002, 419, 178−182. (35) Trewick, S. C.; Henshaw, T. F.; Hausinger, R. P.; Lindahl, T.; Sedgwick, B. Nature 2002, 419, 174−178. (36) QM/MM was originally conceived in: Warshel, A.; Levitt, M. J. Mol. Biol. 1976, 103, 227−249. (37) Warshel, A. Angew. Chem., Int. Ed. 2014, 53, 10020−10031. (38) Senn, H. M.; Thiel, W. Angew. Chem., Int. Ed. 2009, 48, 1198− 1229. (39) Lin, H.; Truhlar, D. G. Theor. Chem. Acc. 2007, 117, 185−199. (40) van der Kamp, M. W.; Mulholland, A. J. Biochemistry 2013, 52, 2708−2728. (41) Kumar, D.; Thiel, W.; de Visser, S. P. J. Am. Chem. Soc. 2011, 133, 3869−3882. (42) (a) Schyman, P.; Lai, W. Z.; Chen, H.; Wang, Y.; Shaik, S. J. Am. Chem. Soc. 2011, 133, 7977−7984. (b) Wang, B.; Li, C.; Dubey, K. D.; Shaik, S. J. Am. Chem. Soc. 2015, 137, 7379−7390. 7089


Research Article

ACS Catalysis Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision B.01; Gaussian, Inc., Wallingford, CT, 2009. (61) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. J. Phys. Chem. B 2009, 113, 6378−6396. (62) Boese, A. D.; Martin, J. M. L. J. Chem. Phys. 2004, 121, 3405− 3416. (63) Tawa, G. J.; Topol, I. A.; Burt, S. K.; Caldwell, R. A.; Rashin, A. A. J. Chem. Phys. 1998, 109, 4852−4863. (64) Zhan, C. G.; Dixon, D. A. J. Phys. Chem. A 2001, 105, 11534− 11540. (65) Klots, C. E. J. Phys. Chem. 1981, 85, 3585−3588. (66) Tissandier, M. D.; Cowen, K. A.; Feng, W. Y.; Gundlach, E.; Cohen, M. H.; Earhart, A. D.; Coe, J. V. J. Phys. Chem. A 1998, 102, 7787−7794. (67) The reorganization includes all the necessary steps for the preparation of the new reactant complex, including the releasing and rebinding of the hm6A intermediate as suggested by experiment,28 as well as a new cycle that uses a new O2 molecule to generate the iron(IV) oxo active species. This complex reorganization is not the main focus of our present study. (68) Yi, C.; Jia, G.; Hou, G.; Dai, Q.; Zhang, W.; Zheng, G.; Jian, X.; Yang, C. G.; Cui, Q.; He, C. Nature 2010, 468, 330−333. (69) Delaney, J. C.; Essigmann, J. M. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14051−14056.

7090


Computations Reveal a Rich Mechanistic Variation of Demethylation

Recommend Documents