Mechanism and Catalytic Site Atlas

The Escherichia coli Ada protein is involved in the repair of alkylated DNA. It repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteines. Since it reacts stoichiometrically with its substrate rather than catalytically it is referred to as a suicide enzyme. However, when it is methylated, it gains the ability to bind in a sequence-specific manner to DNA, and it can then activate the transcription of a methylation-resistance regulon.

This enzyme consists of two domains. The 20-kDa N-terminal domain (N-Ada20) repairs the S-diastereomer of methyl phosphotriesters and in the methylated form binds DNA sequence-specifically, while the 19-kDa C-terminal domain (C-Ada 19) repairs the mutagenic lesion 06-methylguanine by direct methyl transfer to Cys-321. Therefore, each domain has a specific catlytic function. The resting state of the enzyme has the thiolate form of Cys38 coordinated to the zinc N-Ada centre.

The N Ada metal coordination sphere sits at the bottom of a well in the active site. The active site environment is specific enough in shape and size to allow the methyl to approach from the Si diastereomeric face of the methylated phosphate DNA backbone group [PMID:8260490, PMID:11284682]. Spectroscopic studies have shown the sulfur to remain coordinated to the zinc centre once the reaction has occurred, even though the ability of the ligand to coordinate is reduced by the presence of the methyl group [PMID:9383376].

The presence of a methyl group within the Zn coordination sphere changes the tertiary structure of the enzyme sufficiently to reveal a specific DNA binding region in the N terminal region. The protein is now activated to bind onto, and increase transcription of the methylation regulon, coding for proteins involved in DNA methylation repair. The methylated form of Ada acts as a chemo-sensor for potentially mutagenic methylation within the cell [PMID:16464003].