Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase

• Published in: Nature (London) 2010-11, Vol.468 (7321), p.330-333
• Main Authors: Dai, Qing, Jian, Xing, Hou, Guanhua, He, Chuan, Yi, Chengqi, Cui, Qiang, Jia, Guifang, Zheng, Guanqun, Yang, Cai-Guang, Zhang, Wen
• Format: Article
• Language: English
• Subjects: 631/45/535; 631/92/607/1159; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; Anaerobic conditions; Analysis; Analytical, structural and metabolic biochemistry; Biochemistry; Biological and medical sciences; Catalysis; Chemical properties; Cross-Linking Reagents - chemistry; Crystal structure; Crystallization; Crystallography, X-Ray; Crystals; Data collection; Deoxyribonucleic acid; Dioxygenases - chemistry; Dioxygenases - metabolism; DNA; DNA - chemistry; DNA - metabolism; DNA binding proteins; DNA Repair; DNA Repair Enzymes - metabolism; E coli; Enzymes; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli - enzymology; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Genetic aspects; Humanities and Social Sciences; Humans; Hydrogen bonds; Iron; Iron - metabolism; Ketoglutaric Acids - metabolism; letter; Mass spectrometry; Methylation; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; Models, Molecular; multidisciplinary; Nucleic acids; Oxidation; Oxidation-Reduction; Oxidation-reduction reaction; Oxidoreductases; Physiological aspects; Purines; Science; Science (multidisciplinary); Static Electricity; Substrate Specificity
• ISSN: 0028-0836; 1476-4687
• 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,N6-etheno adenine ( A), 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 -ketoglutarate ( KG), to dioxygen initiates oxidation in crystallo. Glycol (from A) 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.