DNA cytosine methyltransferases regulate the expression of the genome through 
the precise epigenetic marking of certain cytosines with a methyl group, and 
aberrant methylation is a hallmark of human diseases including cancer. Targeting 
these enzymes for drug design is currently a high priority. We have utilized ab 
initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) 
simulations to investigate extensively the reaction mechanism of the 
representative DNA methyltransferase HhaI (M.HhaI) from prokaryotes, whose 
overall mechanism is shared with the mammalian enzymes. We obtain for the first 
time full free energy profiles for the complete reaction, together with reaction 
dynamics in atomistic detail. Our results show an energetically preferred 
mechanism in which nucleophilic attack of cytosine C5 on the 
S-adenosyl-L-methionine (AdoMet) methyl group is concerted with formation of the 
Michael adduct between a conserved Cys in the active site with cytosine C6. 
Spontaneous and reversible proton transfer between a conserved Glu in the active 
site and cytosine N3 at the transition state was observed in our simulations, 
revealing the chemical participation of this Glu residue in the catalytic 
mechanism. Subsequently, the β-elimination of the C5 proton utilizes as base an 
OH(-) derived from a conserved crystal water that is part of a proton wire water 
channel, and this syn β-elimination reaction is the rate-limiting step. Design 
of novel cytosine methylation inhibitors would be advanced by our structural and 
thermodynamic characterization of the reaction mechanism.