10mh
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TERNARY STRUCTURE OF HHAI METHYLTRANSFERASE WITH ADOHCY AND HEMIMETHYLATED DNA CONTAINING 5,6-DIHYDRO-5-AZACYTOSINE AT THE TARGET
OverviewOverview
A key step in the predicted mechanism of enzymatic transfer of methyl, groups from S-adenosyl-l-methionine (AdoMet) to cytosine residues in DNA, is the transient formation of a dihydrocytosine intermediate covalently, linked to cysteine in the active site of a DNA (cytosine, C5)-methyltransferase (DNA C5-MTase). Crystallographic analysis of, complexes formed by HhaI methyltransferase (M.HhaI), AdoMet and a target, oligodeoxyribonucleotide containing 5-fluorocytosine confirmed the, existence of this dihydrocytosine intermediate. Based on the premise that, 5,6-dihydro-5-azacytosine (DZCyt), a cytosine analog with an, sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, could mimic the non-aromatic character of the cytosine ring in this, transition state, we synthesized a series of synthetic substrates for DNA, C5-MTase containing DZCyt. Substitution of DZCyt for target cytosines in, C-G dinucleotides of single-stranded or double-stranded, oligodeoxyribonucleotide substrates led to complete inhibition of, methylation by murine DNA C5-MTase. Substitution of DZCyt for the target, cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had, a similar effect on methylation by M. HhaI. Oligodeoxyribonucleotides, containing DZCyt formed a tight but reversible complex with M.HhaI, and, were consistently more potent as inhibitors of DNA methylation than, oligodeoxyribonucleotides identical in sequence containing, 5-fluorocytosine. Crystallographic analysis of a ternary complex involving, M.HhaI, S-adenosyl-l-homocysteine and a double-stranded 13-mer, oligodeoxyribonucleotide containing DZCyt at the target position showed, that the analog is flipped out of the DNA helix in the same manner as, cytosine, 5-methylcytosine, and 5-fluorocytosine. However, no formation of, a covalent bond was detected between the sulfur atom of the catalytic site, nucleophile, cysteine 81, and the pyrimidine C6 carbon. These results, indicate that DZCyt can occupy the active site of M.HhaI as a transition, state mimic and, because of the high degree of affinity of its interaction, with the enzyme, it can act as a potent inhibitor of methylation.
About this StructureAbout this Structure
10MH is a Single protein structure of sequence from Haemophilus haemolyticus with SAH as ligand. Active as Deleted entry, with EC number 2.1.1.73 Full crystallographic information is available from OCA.
ReferenceReference
Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine., Sheikhnejad G, Brank A, Christman JK, Goddard A, Alvarez E, Ford H Jr, Marquez VE, Marasco CJ, Sufrin JR, O'gara M, Cheng X, J Mol Biol. 1999 Feb 5;285(5):2021-34. PMID:9925782
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