Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Methylating agents are common environmental compounds and key in cancer therapy.
• 1-methyladenine and 3-methylcytosine lesions in DNA/RNA cause cytotoxicity from these agents.
• Human ABH3 (hABH3) and E. coli AlkB enzymes directly reverse these DNA/RNA lesions.

Purpose of the Study:

• To determine the structure of the hABH3 catalytic core.
• To analyze key site-directed mutants of hABH3.
• To understand the mechanism of DNA/RNA repair by hABH3.

Main Methods:

• X-ray crystallography at 1.5 A resolution.
• Analysis of site-directed mutants.
• Biochemical assays to study enzyme activity.

Main Results:

• The structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) was determined.
• hABH3 belongs to the Fe(II)/2OG-dependent dioxygenase superfamily, utilizing a beta-strand jelly-roll fold.
• A distinct DNA/RNA binding groove and a flexible hairpin involved in nucleotide flipping and DNA discrimination were identified.
• Self-hydroxylation of an active site leucine was observed, potentially preventing radical generation.

Conclusions:

• The study establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily.
• Key catalytic residues and a flexible hairpin involved in DNA/RNA binding and discrimination were uncovered.
• The findings provide insights into the mechanism of DNA/RNA repair and potential protective mechanisms against oxidative damage.