Catalytic mechanism of the zinc-dependent MutL endonuclease reaction

DNA mismatch repair endonuclease MutL binds two zinc ions. However, the endonuclease activity of MutL is drastically enhanced by other divalent metals such as manganese, implying that MutL binds another catalytic metal at some site other than the zinc-binding sites. Here, we solved the crystal structure of the endonuclease domain of Aquifex aeolicus MutL in the manganeseor cadmium-bound form, revealing that these metals compete with zinc at the same sites. Mass spectrometry revealed that the MutL yielded 5'-phosphate and 3'-OH products, which is characteristic of the two-metal-ion mechanism. Crystallographic analyses also showed that the position and flexibility of a highly conserved Arg of A. aeolicus MutL altered depending on the presence of zinc/manganese or the specific inhibitor cadmium. Site-directed mutagenesis revealed that the Arg was critical for the catalysis. We propose that zinc ion and its binding sites are physiologically of catalytic importance and that the two-metalion mechanism works in the reaction, where the Arg plays a catalytic role. Our results also provide a mechanistic insight into the inhibitory effect of a mutagen/carcinogen, cadmium, on MutL.

Automated summary

The endonuclease activity of MutL, a DNA mismatch repair enzyme, is enhanced by other divalent metals such as manganese, suggesting the presence of another catalytic metal. The crystal structure of Aquifex aeolicus MutL revealed that manganese and cadmium compete with zinc for binding at the same sites. Mass spectrometry analysis confirmed the presence of two-metal-ion mechanism and site-directed mutagenesis highlighted the importance of a conserved arginine residue for catalysis. These findings shed light on the role of zinc ions and the inhibitory effect of cadmium on MutL.