Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage.
Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 A resolution and analyse key site-directed mutants. The hABH3 structure reveals the beta-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His1-X-Asp/Glu-X(n)-His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.
Sundheim
Ottar
O
Department of Cancer Research and Molecular Medicine, NTNU, Trondheim, Norway.
Vågbø
Cathrine B
CB
Bjørås
Magnar
M
Sousa
Mirta M L
MM
Talstad
Vivi
V
Aas
Per A
PA
Drabløs
Finn
F
Krokan
Hans E
HE
Tainer
John A
JA
Slupphaug
Geir
G
D000071498:AlkB Homolog 1, Histone H2a Dioxygenase; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004249:DNA Damage / Q000502:physiology*; D019175:DNA Methylation; D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D049308:Dioxygenases; D006801:Humans; D007656:Ketoglutaric Acids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D012313:RNA / Q000378:metabolism*
D004268:DNA-Binding Proteins; D007656:Ketoglutaric Acids; D012313:RNA; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C486439:ALKBH3 protein, human; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; C499236:ALKBH1 protein, human; D000071498:AlkB Homolog 1, Histone H2a Dioxygenase; D045643:DNA Repair Enzymes