Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Nature. 2010 Nov 11;468(7321):330-3. doi: 10.1038/nature09497.
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.
单核含铁氧还蛋白在生物学中进行多种氧化功能，包括甲基化核酸和组蛋白的氧化去甲基化。大肠杆菌 AlkB 是第一个被发现修复甲基化核酸的此类酶，否则这些核酸具有细胞毒性和/或致突变性。已知 AlkB 人类同源物在各种过程中发挥关键作用。在这里，我们展示了这些去甲基酶的氧化中间体的结构特征。使用化学交联策略，分别稳定并结晶了含有 1,N(6)-乙撑腺嘌呤（εA）、N(3)-甲基胸腺嘧啶（3-meT）和 N(3)-甲基胞嘧啶（3-meC）的 AlkB-双链 DNA（dsDNA）的复合物。将这些在含有铁(II)和α-酮戊二酸（αKG）的厌氧条件下生长的晶体暴露于氧气中，可在晶体中引发氧化。捕获了乙二醇（来自 εA）和半亚胺（来自 3-meT）中间体；基于晶体学观察和计算分析，还提出了一种两性离子中间体（来自 3-meC）。这些前所未有的中间体的观察为这些去甲基酶的氧化去甲基化机制提供了直接支持。该研究还描绘了一种普遍的机制观点，说明如何从不同的生物底物中氧化去除甲基基团。
