Letter | Published:

Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

Nature volume 468, pages 330333 (11 November 2010) | Download Citation

Abstract

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology1,2, including the oxidative demethylation of methylated nucleic acids and histones3,4. Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids5,6, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes7,8,9,10,11. Here we present structural characterization of oxidation intermediates for these demethylases. Using a chemical cross-linking strategy12,13, complexes of AlkB–double stranded DNA (dsDNA) containing 1,N6-etheno adenine (εA), N3-methyl thymine (3-meT) and N3-methyl cytosine (3-meC) are stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo. Glycol (from εA) and hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.

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Accessions

Data deposits

Atomic coordinates are deposited in the Protein Data Bank under accessionnumbers 3O1M,3O1O,3O1P, 3O1R,3O1S,3O1T,3O1Uand3O1V.

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Acknowledgements

This study was supported by the National Institutes of Health (GM071440 to C.H.; GM084028 to Q.C.), beamlines 23ID-B (General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT)), 19BM-D (Structual Biology Center (SBC-CAT)), 14BM-C (BioCARS) and 21ID-D (Life Sciences Collaborative Access Team (LS-CAT)) at the Advanced Photon Source at Argonne National Laboratory, the National Institutes of Health and the United States Department of Energy. Computational resources from the National Center for Supercomputing Applications at the University of Illinois and the Center of High Throughput Computing at UW–Madison are appreciated. We also thank X. Yang, Z. Ren and E. Duguid for crystallographic discussions.

Author information

Affiliations

  1. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA

    • Chengqi Yi
    • , Guifang Jia
    • , Wen Zhang
    • , Guanqun Zheng
    • , Xing Jian
    • , Cai-Guang Yang
    •  & Chuan He
  2. Department of Chemistry and Theoretical Chemistry Institute, University of Wisconsin, 1101 University Avenue, Madison, Wisconsin 53706, USA

    • Guanhua Hou
    •  & Qiang Cui
  3. Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA

    • Qing Dai
  4. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China

    • Cai-Guang Yang

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Contributions

C.Y., G.J. and C.H. designed the experiments. Experiments were performed by C.Y., G.J., Q.D., W.Z., G.Z., X.J. and C.-G.Y.; computational analyses were performed by G.H. and Q.C. C.Y. and C.H. wrote the paper and G.H., Q.D. and Q.C. contributed to editing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Chuan He.

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    Supplementary Information

    This file contains Supplementary Tables 1-2, Supplementary Figures 1-22 with legends, Supplementary Notes and additional references.

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https://doi.org/10.1038/nature09497

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