Structure/function analysis of two mutated Endonuclease III enzymes from the extremophile Deinococcus radiodurans

Deinococcus radiodurans is an extremely radiation and desiccation resistant bacterium which can withstand 200 times higher doses of ionizing irradiation than other bacteria without losing viability. The resistance mechanism is not known, but an efficient DNA repair machinery is considered to play a key role in it. Endonuclease III (EndoIII) is an ubiquitious bifunctional DNA glycosylase that belongs to the helix-hairpin-helix family of DNA glycosylases, with an [4Fe-4S] cluster. It has specificity for a broad range of oxidized pyrimidines lesions, removing numerous forms of damaged bases from DNA. We have previously performed structure/function analysis of three EndoIII enzymes from D. radiodurans, EndoIII1, 2 and 3, which showed that EndoIII1 and 3 possesses unusual properties compared to previously studied EndoIII enzymes. The structural analysis revealed that some specific amino acid substitutions close to the active site could explain the observed alternative activities. Here we report the construction, crystallization and characterization of these mutants. We have also analyzed the effect of the mutated amino acids on the redox properties of the enzyme.