Unraveling the Reaction Mechanism of Enzymatic C5-Cytosine Methylation of DNA. A Combined Molecular Dynamics and QM/MM Study of Wild Type and Gln119 Variant

Abstract

M.HhaI is a DNA Methyltransferase from Haemophilus haemolyticus that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the C 5 position of a cytosine. This enzyme is a paradigmatic model for C 5 DNA Methyltransferases due to its major homology to mammalian enzymes and to the availability of high resolution structures of the DNA-enzyme complex. In spite of the number of experimental and theoretical analysis carried out for this system many mechanistic details remain unraveled. We have used full atomistic classical Molecular Dynamics simulations to explore the protein-SAM-DNA ternary complex where the target cytosine base is flipped out into the active site for both the wild type and Glu119Gln mutant. The relaxed structure was used for a combined Quantum Mechanics/Molecular Mechanics exploration of the reaction mechanism using the string method. Exploration of multidimensional Free Energy Surfaces allowed determining a complete picture of the reaction mechanism in agreement with experimental observations. In our proposal

Understanding the R882H mutation effects of DNA methyltransferase DNMT3A: a combination of molecular dynamics simulations and QM/MM calculations

“…With the least-RMSD structure, the energy barriers of the methyl migration step were computed to be 14.9 kcal mol À1 and 15.6 kcal mol À1 for WT and R882H DNMT3A, respectively, similar to the values reported in the literature. 27,31 The catalytic energy barrier increased slightly, by 0.7 kcal mol À1 , in the R882H mutant, which is consistent with the 4-fold reduction in the catalytic ability of monomer DNMT3A from experimental observations. [18][19][20] To validate the robustness of our results, QM/MM calculations with a combination of the M06-2X functional and various basis sets were further carried out (Table S4 †).…”

“…Aranda et al provided a relatively complete reaction energy prole of Haemophilus haemolyticus (M.HhaI) DNA methyltransferase, which shares a conserved catalytic domain with human DNMT3A. 27,28 According to previous studies, the methyl transfer step is turnover-frequency-determining (TOF-determining). 26,27,29 The cytosine activation includes the deprotonation of Cys710 and subsequent addition, and methyl transfer to the ipped cytosine ( Fig.…”

Application of quantum mechanics/molecular mechanics methods in the study of enzymatic reaction mechanisms

“…Up to now, the method has been successful in providing accurate free energies for several enzyme-catalyzed reactions: Hummer and coworkers have combined QM/MM BOMD simulations within an US framework with the string finite-temperature string method to calculate an activation free-energy of approximately 15 kcal mol −1 for the cleavage of RNA by Ribonuclease H, a value in good agreement with the experimental turnover rate of 1-100 min −1 ; 219 the work of Ko ca and coworkers on the O-GlcNAc transferase combined QM/MM CPMD simulations with the linear string method to propose a new mechanism of action for this enzyme with and activation free-energy of 23.5 kcal•mol −1 ; 220 finally, the work of Tuñón and coworkers also combined QM/MM BOMD and the linear string method to describe the mechanism of action of HhaI methyltransferase, proposing that solvent can play an important role in the β-elimination of the hydrogen in the C 5 of the DNA molecule. 221 More recently, Warshel and coworkers developed the paradynamics methodology, [222][223][224] which makes use of two parameters, α k and A k , and Gaussian functions to approximate a less accurate target potential to a more accurate reference one, through a least-squares function that represents the difference in energy between these potentials. Despite that the methodology is not of common use, it has already been used for the study of biomolecular systems such as the haloalkene dehalogenase enzyme and the Ras guanosine-5-triphosphatase (RasGAP) system providing encouraging results.…”

Computational tools for the evaluation of laboratory-engineered biocatalysts

“…QM/MM calculations have been applied to a huge range of applications in enzymatic catalysis during recent years 99 , 130 – 133 and for the study of other systems and properties. 95 , 96 We will focus our discussion on two recent publications 97 , 98 that are based on the application of QM/MM strategies to explain and understand the changes on the enzymatic activities in mutants compared to their respective WT enzymes.…”