Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase.
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.
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Chengqi
C
Yi
Guifang
G
Jia
Guanhua
G
Hou
Qing
Q
Dai
Wen
W
Zhang
Guanqun
G
Zheng
Xing
X
Jian
Cai-Guang
CG
Yang
Qiang
Q
Cui
Chuan
C
He
D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D008745:Methylation; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D055672:Static Electricity; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D004247:DNA; D007501:Iron; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C486439:ALKBH3 protein, human; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; C466427:AlkB protein, E coli; C486440:ALKBH2 protein, human; D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D045643:DNA Repair Enzymes