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  • The EMBO Journal (2006) 25, 3389 - 3397
  • doi:10.1038/sj.emboj.7601219

Published online: 6 July 2006

Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage

Ottar Sundheim1,2, Cathrine B Vågbø1, Magnar Bjørås2,3, Mirta ML Sousa1, Vivi Talstad1, Per A Aas1, Finn Drabløs1, Hans E Krokan1, John A Tainer2 and Geir Slupphaug1

  1. Department of Cancer Research and Molecular Medicine, NTNU, Trondheim, Norway
  2. Department of Molecular Biology, The Scripps Research Institute, The Skaggs Institute for Chemical Biology, La Jolla, CA, USA
  3. Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, University of Oslo, Oslo, Norway

Correspondence to:

Geir Slupphaug, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Erling Skjalgssons gt 1, 7006 Trondheim, Norway. Tel.: +47 91825455; Fax: +47 73598801; E-mail: geir.slupphaug@ntnu.no

John A Tainer, Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: +1 858 784 8119; Fax: +1 858 784 2289; E-mail: jat@scripps.edu

Received 9 February 2006; Accepted 9 June 2006

Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 Å resolution and analyse key site-directed mutants. The hABH3 structure reveals the beta-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His1-X-Asp/Glu-Xn-His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.

  • Keywords:

    • alkylation,
    • crystal structure,
    • DNA repair,
    • hABH3,
    • oxidative demethylation