1,N6-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.
Atrial natriuretic peptide (ANP) is a cardiac hormone released by the atrium in response to stretching forces. ANP, which maintains cardiovascular homeostasis via its diuretic, natriuretic and hypotensive effects, acts through its receptor, GC-A. In this issue of the Biochemical Journal, Miura et al. (pages 1897–1918) established mouse immortalized endothelial cells stably expressing GC-A (SVEC/GC-A) and found that ANP promotes cell spreading and endothelial barrier function via regulation of CCM2, an adaptor protein involved in the pathogenesis of cerebral cavernous malformations, a neurovascular disease characterized by dysfunction of the endothelial barrier. The image shows ANP promotes co-localization of GC-A (green) and CCM2 (red) at membrane ruffles in SVEC/GC-A. Image kindly provided by Koichi Miura
1,N6-α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase
Małgorzata Dylewska, Jarosław T. Kuśmierek, Tomasz Pilżys, Jarosław Poznański, Agnieszka M. Maciejewska; 1,N6-α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase. Biochem J 1 June 2017; 474 (11): 1837–1852. doi: https://doi.org/10.1042/BCJ20161008
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