S-adenosyl-L-methionine-dependent methyl transfer: observable precatalytic intermediates during DNA cytosine methylation
- PMID: 17616174
- DOI: 10.1021/bi7005948
S-adenosyl-L-methionine-dependent methyl transfer: observable precatalytic intermediates during DNA cytosine methylation
Abstract
S-adenosyl-L-methionine- (AdoMet-) dependent methyltransferases are widespread, play critical roles in diverse biological pathways, and are antibiotic and cancer drug targets. Presently missing from our understanding of any AdoMet-dependent methyl-transfer reaction is a high-resolution structure of a precatalytic enzyme/AdoMet/DNA complex. The catalytic mechanism of DNA cytosine methylation was studied by structurally and functionally characterizing several active site mutants of the bacterial enzyme M.HhaI. The 2.64 A resolution protein/DNA/AdoMet structure of the inactive C81A M.HhaI mutant suggests that active site water, an approximately 13 degree tilt of the target base toward the active site nucleophile, and the presence or absence of the cofactor methylsulfonium are coupled via a hydrogen-bonding network involving Tyr167. The active site in the mutant complex is assembled to optimally align the pyrimidine for nucleophilic attack and subsequent methyl transfer, consistent with previous molecular dynamics ab initio and quantum mechanics/molecular mechanics calculations. The mutant/DNA/AdoHcy structure (2.88 A resolution) provides a direct comparison to the postcatalytic complex. A third C81A ternary structure (2.22 A resolution) reveals hydrolysis of AdoMet to adenosine in the active site, further validating the coupling between the methionine portion of AdoMet and ultimately validating the structural observation of a prechemistry/postchemistry water network. Disruption of this hydrogen-bonding network by a Tyr167 to Phe167 mutation does not alter the kinetics of nucleophilic attack or methyl transfer. However, the Y167F mutant shows detectable changes in kcat, caused by the perturbed kinetics of AdoHcy release. These results provide a basis for including an extensive hydrogen-bonding network in controlling the rate-limiting product release steps during cytosine methylation.
Similar articles
-
Active site dynamics of the HhaI methyltransferase: insights from computer simulation.J Mol Biol. 1999 Oct 15;293(1):9-18. doi: 10.1006/jmbi.1999.3120. J Mol Biol. 1999. PMID: 10512711
-
Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase.Nucleic Acids Res. 2005 Jan 13;33(1):307-15. doi: 10.1093/nar/gki175. Print 2005. Nucleic Acids Res. 2005. PMID: 15653631 Free PMC article.
-
Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine.J Mol Biol. 1999 Feb 5;285(5):2021-34. doi: 10.1006/jmbi.1998.2426. J Mol Biol. 1999. PMID: 9925782
-
Structure and function of DNA methyltransferases.Annu Rev Biophys Biomol Struct. 1995;24:293-318. doi: 10.1146/annurev.bb.24.060195.001453. Annu Rev Biophys Biomol Struct. 1995. PMID: 7663118 Review.
-
DNA methyltransferases: mechanistic models derived from kinetic analysis.Crit Rev Biochem Mol Biol. 2012 Mar-Apr;47(2):97-193. doi: 10.3109/10409238.2011.620942. Epub 2012 Jan 20. Crit Rev Biochem Mol Biol. 2012. PMID: 22260147 Review.
Cited by
-
Introduction--Epiphanies in epigenetics.Prog Mol Biol Transl Sci. 2011;101:1-21. doi: 10.1016/B978-0-12-387685-0.00001-9. Prog Mol Biol Transl Sci. 2011. PMID: 21507348 Free PMC article.
-
5-Methylation of cytosine in CG:CG base-pair steps: a physicochemical mechanism for the epigenetic control of DNA nanomechanics.J Phys Chem B. 2013 Dec 27;117(51):16436-42. doi: 10.1021/jp409887t. Epub 2013 Dec 16. J Phys Chem B. 2013. PMID: 24313757 Free PMC article.
-
N-methylation of the amide bond by methyltransferase asm10 in ansamitocin biosynthesis.Chembiochem. 2011 Jul 25;12(11):1759-66. doi: 10.1002/cbic.201100062. Epub 2011 Jun 16. Chembiochem. 2011. PMID: 21681880 Free PMC article.
-
Mammalian DNA methyltransferases: a structural perspective.Structure. 2008 Mar;16(3):341-50. doi: 10.1016/j.str.2008.01.004. Structure. 2008. PMID: 18334209 Free PMC article. Review.
-
DNA cytosine methylation: structural and thermodynamic characterization of the epigenetic marking mechanism.Biochemistry. 2013 Apr 23;52(16):2828-38. doi: 10.1021/bi400163k. Epub 2013 Apr 12. Biochemistry. 2013. PMID: 23528166 Free PMC article.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Molecular Biology Databases