Enzymatic C5-cytosine methylation of DNA: mechanistic implications of new crystal structures for HhaL methyltransferase-DNA-AdoHcy complexes

O'Gara, M., Klimasauskas, S., Roberts, R. J., Cheng, X. (September 1996) Enzymatic C5-cytosine methylation of DNA: mechanistic implications of new crystal structures for HhaL methyltransferase-DNA-AdoHcy complexes. Journal of Molecular Biology, 261 (5). pp. 634-45. ISSN 0022-2836 (Print)

URL: http://www.ncbi.nlm.nih.gov/pubmed/8800212
DOI: 10.1006/jmbi.1996.0489


The refined crystal structures of HhaI methyltransferase complexed with cognate unmethylated or methylated DNA together with S-adenosyl-L-homocysteine, along with the previously-solved binary and covalent ternary structures, offer a detailed picture of the active site at individual stages throughout the reaction cycle. This picture supports and extends a proposed mechanism for C5-cytosine methylation that may be general for the whole family of C5-cytosine methyltransferases. The structures of the two new complexes have been refined to crystallographic R-factors of 0.189 and 0.178, respectively, at 2.7 A resolution. We observe that both unmethylated 2'-deoxycytidine and 5-methyl-2'-deoxycytidine flip out of the DNA helix and fit into the active site of the enzyme. The catalytic sulfur atom of Cys81 interacts strongly with C6. The C5 methyl group of the flipped 5-methyl-2'-deoxycytidine is bent approximately 50 degrees out of the plane of the cytosine ring and towards the sulfur atom of S-adenosyl-L-homocysteine. This unusual position is probably due to partial sp3 character at C5 and C6 and to steric effects of the conserved amino acid residues Pro80 and Cys81. Two water molecules are held near the hydrophobic edge (C5 and C6) of the flipped cytosine by two conserved amino acid residues (Gln82 and Asn304) and the phosphoryl oxygen atom of the phosphate group 3' to the flipped nucleotide, and one of them may serve as the general base for eliminating the proton from C5. Protonation of the cytosine N3 during the methylation reaction may involve Glu119, which itself might be protonated via a water-mediated interaction between the terminal carboxyl group of Glu119 and the amino group of the methionine moiety of S-adenosyl-L-methionine. The cofactor thus plays two key roles in the reaction.

Item Type: Paper
Uncontrolled Keywords: Base Sequence Comparative Study Crystallography, X-Ray Cytosine/chemistry/ metabolism DNA/ chemistry/ metabolism DNA Methylation Methionine/analogs & derivatives/chemistry/metabolism Models, Molecular Molecular Sequence Data Nucleic Acid Conformation Protein Conformation Protons Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. S-Adenosylhomocysteine/ metabolism Site-Specific DNA Methyltransferase (Cytosine-Specific)/ chemistry/metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA methylation
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > methyltransferase
Investigative techniques and equipment > x ray crystallography
CSHL Authors:
Communities: CSHL labs
Depositing User: Kathleen Darby
Date: 6 September 1996
Date Deposited: 13 May 2014 14:57
Last Modified: 13 May 2014 14:57
Related URLs:
URI: https://repository.cshl.edu/id/eprint/30096

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