1,N6-α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase

Abstract

SummaryCombination of in vivo, in vitro and in silico studies shows that acrolein adduct to adenine is mutagenic and is effectively repaired by AlkB dioxygenase. Cationic form of R isomer of HPA is strongly favored in AlkB active centre. -α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein -an environmental pollutant and endocellular oxidative stress product. E. 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 determinated. 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 one 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 2

ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy

“…The assay was developed previously to monitor ligand binding to AlkB 36 . Fluorescence experiments were performed using a Varian Cary Eclipse spectrofluorimeter.…”