10MH

TERNARY STRUCTURE OF HHAI METHYLTRANSFERASE WITH ADOHCY AND HEMIMETHYLATED DNA CONTAINING 5,6-DIHYDRO-5-AZACYTOSINE AT THE TARGET

10MH の概要

• エントリーDOI: 10.2210/pdb10mh/pdb
• 分子名称: DNA (5'-D(P*CP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*AP*C)-3'), DNA (5'-D(P*GP*TP*CP*AP*GP*5NCP*GP*CP*AP*TP*GP*G)-3'), PROTEIN (CYTOSINE-SPECIFIC METHYLTRANSFERASE HHAI), ... (5 entities in total)
• 機能のキーワード: transferase, methyltransferase, restriction system, complex (methyltransferase- dna), transferase-dna complex, transferase/dna
• 由来する生物種: Haemophilus haemolyticus
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 44768.42

構造登録者

Sheikhnejad, G.,Brank, A.,Christman, J.K.,Goddard, A.,Alvarez, E.,Ford Junior, H.,Marquez, V.E.,Marasco, C.J.,Sufrin, J.R.,O'Gara, M.,Cheng, X. (登録日: 1998-08-10, 公開日: 1999-02-09, 最終更新日: 2023-08-09)

主引用文献

Sheikhnejad, G.,Brank, A.,Christman, J.K.,Goddard, A.,Alvarez, E.,Ford Jr., H.,Marquez, V.E.,Marasco, C.J.,Sufrin, J.R.,O'gara, M.,Cheng, X.
Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine.
J.Mol.Biol., 285:2021-2034, 1999

PubMed Abstract: 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.

PubMed: 9925782
DOI: 10.1006/jmbi.1998.2426

実験手法

X-RAY DIFFRACTION (2.55 Å)