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. 2010 Nov 11;468(7321):330-3.
doi: 10.1038/nature09497.

Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

Chengqi Yi  1 , Guifang Jia, Guanhua Hou, Qing Dai, Wen Zhang, Guanqun Zheng, Xing Jian, Cai-Guang Yang, Qiang Cui, Chuan He
Affiliations

Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

Chengqi Yi et al. Nature. .

Abstract

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology, including the oxidative demethylation of methylated nucleic acids and histones. Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes. Here we present structural characterization of oxidation intermediates for these demethylases. Using a chemical cross-linking strategy, complexes of AlkB-double stranded DNA (dsDNA) containing 1,N(6)-etheno adenine (εA), N(3)-methyl thymine (3-meT) and N(3)-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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Figures

Figure 1
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.
Figure 2
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 2Fobs-Fcal maps are contoured at 1.0σ and the green Fobs-Fcal simulated-annealing omit maps were computed by removing extra atoms of intermediates (compared to the original substrates) and are contoured at 3.0σ. The extra atoms are shown in red. Hydrogen bonds are shown as dotted lines.
Figure 3
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, QM/MM calculated structure of 4. c, Optimized structure of model 3 from the oxidized 3-meC crystal. For both calculated structures, carbon atoms are colored in cyan, nitrogen in blue, oxygen in red, iron in pink, and hydrogen in grey. Red NBO charges are labelled for several base atoms and key distances are marked in black (Å).
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