title: The mechanism of M.HhaI DNA C5 cytosine methyltransferase enzyme: a quantum mechanics/molecular mechanics approach.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Bruice
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003596:Cytosine / Q000096:biosynthesis / Q000737:chemistry; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry*; D015257:DNA-Cytosine Methylases / Q000737:chemistry*; D004789:Enzyme Activation; D011789:Quantum Theory; D055672:Static Electricity
chemical_list: D003596:Cytosine; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases