Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

Journal name:
Nature
Volume:
468,
Pages:
330–333
Date published:
DOI:
doi:10.1038/nature09497
Received
Accepted
Published online

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.

At a glance

Figures

  1. Oxidative repair of damaged nucleic acid bases by AlkB.
    Figure 1: Oxidative repair of damaged nucleic acid bases by AlkB.

    Oxidative repair of εA, 3-meT and 3-meC by AlkB with intermediates glycol 1, hemiaminal 2 and zwitterion 3 proposed in this study.

  2. Intermediates trapped during in crystallo oxidation of [epsi]A and 3-meT.
    Figure 2: Intermediates trapped during in crystallo oxidation of εA and 3-meT.

    a, Stereo views and electron density maps of the glycol intermediate 1 during εA repair. b, Stereo pairs and density maps of the hemiaminal intermediate 2 during the oxidation demethylation of 3-meT. The blue 2FobsFcal maps are contoured at 1.0σ and the green FobsFcal simulated-annealing omit maps were computed by removing extra atoms of intermediates (compared with the original substrates) and are contoured at 3.0σ. The extra atoms are shown in red. Hydrogen bonds are shown as dashed lines.

  3. A zwitterionic intermediate 3 is proposed for the demethylation of 3-meC.
    Figure 3: A zwitterionic intermediate 3 is proposed for the demethylation of 3-meC.

    a, Oxidation of 3-deazameC in single crystals to yield 3-deazahmC, intermediate 4. The density map and labels are generated and shown as in Fig. 2. b, Quantum mechanical/molecular mechanical calculated structure of 4. c, Optimized structure of model 3 from the oxidized 3-meC crystal. For both calculated structures, carbon atoms are coloured in cyan, nitrogen in blue, oxygen in red, iron in pink and hydrogen in grey. Red natural bond orbital charges are labelled for several base atoms and key distances are marked in black (in ångströms).

Accession codes

References

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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

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 financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

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

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  1. Supplementary Information (10.2M)

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

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