Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

摘要

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology, including the oxidative demethy-lation of methylated nucleic acids and histories. Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids, whichare otherwise cytotoxic and/or muta-genic. 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 eA) and hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-meC) is also proposed, based on crystal-lographic 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.