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RESEARCH PRODUCT

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

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M.HhaI is a DNA methyltransferase from Haemophilus hemolyticus that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the C5 position of a cytosine. This enzyme is a paradigmatic model for C5 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 analyses 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 Cys81 becomes unprotonated after formation of the ternary complex. This residue then attacks the C6 position of the target cytosine, establishing a fast and reversible equilibrium. Methyl transfer from SAM to position C5 takes place after the previous addition, and the transition state is stabilized by means of hydrogen bond interactions established between the cytosine base and Glu119. Although previous proposals suggest that a hydroxide anion could be the base abstracting the proton from the C5 position, our calculations show that the free energy cost needed to have this anion in the active site is too high. Instead, a crystallographic water molecule can remove the excess proton once the cytosine has been activated by means of a proton transfer from Glu119 to the N3 position. This observation explains the consequences of mutations of this residue on the pre-steady-state and steady-state rate constants. Our proposal then clarifies the role of Glu119 and identifies the nature of the base in charge of proton abstraction, two issues that have been the subject of a long debate in the literature. The authors gratefully acknowledge financial support from FEDER funds and the Ministerio de Economı́a y Competitividad (project CTQ2012-36253-C03-03) and Generalitat Valenciana (ACOMP/2015/239 and GV/2012/053). J.A. and M.R. thank the Ministerio de Economı́a y Competitividad for a FPI fellowship and for a ‘Ramón y Cajal’ contract, respectively. K.Z. acknowledges an FPU fellowship of the Ministerio de Educación. K.S. thanks the Polish Ministry of Science and Higher Education for the “Iuventus Plus” program, project no. 0478/IP3/2015/73 (2015–2016), the Polish National Center for Science (NCN) (grant 2011/02/A/ST4/00246, 2012–2017), and the US National Institutes of Health (ref NIH R01 GM065368). The authors also acknowledge the computational facilities of the Servei d’Informàtica de la Universitat de València in the “Tirant” supercomputer.