Structural and Functional Characterization of Two Unusual Endonuclease III Enzymes from Deinococcus Radiodurans.

Abstract While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 ( Dr EndoIII1), DR0289 ( Dr EndoIII2) and DR0982 ( Dr EndoIII3). Here we have determined the crystal structures of Dr EndoIII1 and an N-terminally truncated form of Dr EndoIII3 ( Dr EndoIII3Δ76). We have also generated a homology model of Dr EndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus . However, both Dr EndoIII1 and Dr EndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of Dr EndoIII3 seems to be less accessible for DNA interactions, while in Dr EndoIII1 it seems to be more open. Analysis of the enzyme activities shows that Dr EndoIII2 is most similar to the previously studied enzymes, while Dr EndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. Dr EndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity.