Model for the binding of the putative tetrahedral intermediate in the active site of 8-oxoguanine deaminase.
Formation of 8-oxoguanine (8-oxoG) is the most common form of DNA damage and is caused by reactive oxygen species oxidizing the DNA base guanine. If this damage is not repaired, it can result in mismatched DNA, as 8-oxoG pairs with adenosine, rather than its customary cytosine.
This DNA lesion can be removed by 8-oxoguanine-DNA glycosylase, but how free 8-oxoG is metabolized has been something of a puzzle. Guanine in most organisms is deaminated to xanthine and then oxidized to uric acid, but none of the known guanine deaminases work on 8-oxoG. So what does happen?
Richard Hall from Texas A & M University and colleagues in collaboration with PSI NYSGXRC systematically searched for the enzyme that could deaminate 8-oxoG. They examined the structures of all the guanine, cytosine and adenosine deaminases, all members of the amidohydrolase (AHS) superfamily, and noticed a conserved HxxE motif near the C-terminus of the β-strand 5 within a (β/α)8 structural fold. In addition, the guanine deaminases had a conserved catalytic triad.
Protein sequence comparisons revealed a subcluster of around 200 AHS enzymes with a similar motif and the conserved catalytic triad. The team chose Pa0142 from Pseudomonas aeruginosa PA01 to express and purify and then test for deamination activity on aromatic bases. This enzyme showed activity for 8-oxoG, guanine, isocytosine and ammeline, which was something of a surprise as other guanine deaminases studied so far do not possess both guanine and 8-oxoG deaminase activity.
Pa0142 would not crystallize, but a homologous protein that is 43% identical to Pa0142 did crystallize and the structure was solved at a resolution of 2.2 Å. The homolog was found to be a more active enzyme that Pa0142 for guanine, isocytosine and 8-oxoG and the structure revealed a standard metal ligation scheme seen with other guanine deaminases but two residues in the binding pocket for guanine/xanthine that are conserved in other guanine deaminases are mutated in this new protein.
This approach of looking for functional motifs in primary protein sequences and then characterizing the likely proteins has resulted in identification of the first 8-oxoG deaminase.