The mechanism of M.HhaI DNA C5 cytosine methyltransferase enzyme: a quantum mechanics/molecular mechanics approach.

The mechanism of DNA cytosine-5-methylation catalyzed by the bacterial M.HhaI enzyme has been considered as a stepwise nucleophilic addition of Cys-81-S- to cytosine C6 followed by C5 nucleophilic replacement of the methyl of S-adenosyl-L-methionine to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine. In this study, we show that the reaction is concerted from a series of energy calculations by using the quantum mechanical/molecular mechanical hybrid method. Deprotonation of 5-methyl-6-Cys-81-S-5,6-dihydrocytosine and expulsion of Cys-81-S- provides the product DNA 5-methylcytosine. A required base catalyst for this deprotonation is not available as a member of the active site structure. A water channel between the active site and bulk water allows entrance of solvent to the active site. Hydroxide at 10(-7) mole fraction (pH = 7) is shown to be sufficient for the required catalysis. We also show that Glu-119-CO2H can divert the reaction by protonating cytosine N3 when Cys-81-S- attacks cytosine, to form the 6-Cys-81-S-3-hydrocytosine. The reactants and 6-Cys-81-S-3-hydrocytosine product are in rapid equilibrium, and this explains the observed hydrogen exchange of cytosine with solvent.