10MH

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


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.55 Å
  • R-Value Work: 0.205 
  • R-Value Observed: 0.205 

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This is version 1.3 of the entry. See complete history


Literature

Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine.

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.

(1999) J Mol Biol 285: 2021-2034

  • DOI: https://doi.org/10.1006/jmbi.1998.2426
  • Primary Citation of Related Structures:  
    10MH

  • 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.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha 68198-4525, USA.


Macromolecules

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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
PROTEIN (CYTOSINE-SPECIFIC METHYLTRANSFERASE HHAI)C [auth A]327Haemophilus haemolyticusMutation(s): 0 
EC: 2.1.1.73
UniProt
Find proteins for P05102 (Haemophilus parahaemolyticus)
Explore P05102 
Go to UniProtKB:  P05102
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP05102
Sequence Annotations
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  • Reference Sequence

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Entity ID: 1
MoleculeChains LengthOrganismImage
DNA (5'-D(P*CP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*AP*C)-3')A [auth B]12N/A
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
DNA (5'-D(P*GP*TP*CP*AP*GP*5NCP*GP*CP*AP*TP*GP*G)-3')B [auth C]12N/A
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
SAH
Query on SAH

Download Ideal Coordinates CCD File 
D [auth A]S-ADENOSYL-L-HOMOCYSTEINE
C14 H20 N6 O5 S
ZJUKTBDSGOFHSH-WFMPWKQPSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.55 Å
  • R-Value Work: 0.205 
  • R-Value Observed: 0.205 
  • Space Group: H 3 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 99.86α = 90
b = 99.86β = 90
c = 325.2γ = 120
Software Package:
Software NamePurpose
X-PLORrefinement
DENZOdata reduction
SCALEPACKdata scaling

Structure Validation

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Entry History 

Revision History  (Full details and data files)

  • Version 1.0: 1999-02-09
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2023-08-09
    Changes: Data collection, Database references, Derived calculations, Refinement description