1,N6-α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase.
PBN-AR
Instytucja
Instytut Biochemii i Biofizyki Polskiej Akademii Nauk
Informacje podstawowe
Główny język publikacji
EN
Czasopismo
BIOCHEMICAL JOURNAL (35pkt w roku publikacji)
ISSN
0264-6021
EISSN
1470-8728
Wydawca
PORTLAND PRESS LTD
DOI
URL
Rok publikacji
2017
Numer zeszytu
11
Strony od-do
1837-1852
Numer tomu
474
Identyfikator DOI
Liczba arkuszy
Słowa kluczowe
EN
AlkB dioxygenase;
DNA repair;
acrolein;
mutagenesis;
substrate binding
Streszczenia
Język
EN
Treść
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.
Inne
System-identifier
PX-591efa9fd5de24e14304fa0c
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