1. 1,N 6 -α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase.
- Author
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Dylewska M, Kuśmierek JT, Pilżys T, Poznański J, and Maciejewska AM
- Subjects
- Adenine chemistry, Adenine metabolism, Adenine toxicity, AlkB Enzymes chemistry, AlkB Enzymes genetics, Binding Sites, Biocatalysis, Carcinogens, Environmental chemistry, Carcinogens, Environmental toxicity, DNA Adducts chemistry, DNA Adducts toxicity, DNA, Bacterial chemistry, DNA, Bacterial drug effects, DNA, Bacterial metabolism, Enzyme Stability, Escherichia coli drug effects, Escherichia coli growth & development, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Hydroxylation, Molecular Conformation, Molecular Dynamics Simulation, Mutagenesis drug effects, Mutagens chemistry, Mutagens toxicity, Oxidation-Reduction, Protein Conformation, Quantum Theory, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Stereoisomerism, Substrate Specificity, Adenine analogs & derivatives, AlkB Enzymes metabolism, Carcinogens, Environmental metabolism, DNA Adducts metabolism, DNA Repair, Escherichia coli Proteins metabolism, Models, Molecular, Mutagens metabolism
- Abstract
1,N
6 -α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein - an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A → C and A → T transversions and, less frequently, A → G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)- Published
- 2017
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