Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 400, Issue 3, Pages 632-644Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.05.021
Keywords
DNA cytosine methyltransferase; methylation reaction mechanism; DNA as cofactor; DNA phosphate group; quantum mechanical calculations
Categories
Funding
- Spanish MICINN [CTQ 2007-67528, CSD 2007-00006]
- UPV/EHU [IT-324-07]
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Methylation of cytosine residues in the DNA is one of the most important epigenetic marks central to the control of differential expression of genes. We perform quantum mechanical calculations to investigate the catalytic mechanism of the bacterial HhaI DNA methyltransferase. We find that the enzyme nucleophile, Cys81, can attack C6 of cytosine only after it is deprotonated by the DNA phosphate group, a reaction facilitated by a bridging water molecule. This finding, which indicates that the DNA acts as both the substrate and the cofactor, can explain the total loss of activity observed in an analogous enzyme, thymidylate synthase, when the phosphate group of the substrate was removed. Furthermore, our results displaying the inability of the phosphate group to deprotonate the side chain of serine is in agreement with the total, or the large extent of, inactivity observed for the C81S mutant. In contrast to results from previous calculations, we find that the active site conserved residues, Glu119, Arg163, and Arg165, are crucial for catalysis. In addition, the enzyme DNA adduct formation and the methyl transfer from the cofactor S-adenosyl-L-methionine are not concerted but proceed via stepwise mechanism. In many of the different steps of this methylation reaction, the transfer of a proton is found to be necessary. To render these processes possible, we find that several water molecules, found in the crystal structure, play an important role, acting as a bridge between the donating and accepting proton groups. (C) 2010 Elsevier Ltd. All rights reserved.
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