Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

Abstract

The AlkB type proteins are demethylases that are thought to play a part in DNA repair by oxidatively removing methyl adducts on DNA, RNA and histones. Yi et al. have determined the structure of AlkB oxygenase crystallized in complex with various modified DNAs. By growing the crystals under anaerobic conditions and then exposing them to dioxygen to initiate oxidation, two different intermediates were trapped. A third type of intermediate was determined using additional computational analysis. These structures provide detailed mechanistic insight into how these enzymes perform oxidative demethylation. Mononuclear iron-containing oxygenases have many important roles in the cell, including the demethylation of DNA and histones. These authors crystallized the AlkB oxygenase in complex with various modified DNAs. By growing the crystals under anaerobic conditions and then exposing them to dioxygen to initiate oxidation, two different intermediates were trapped. A third type of intermediate was determined using additional computational analysis. These structures provide insight into how these enzymes perform oxidative demethylation. 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.