Structure Determination and Biochemical Characterization of a Putative HNH Endonuclease from Geobacter metallireducens GS-15: e72114

The crystal structure of a putative HNH endonuclease, GmetUL0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Aa resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel beta -sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of GmetUL0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of GmetUL0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, GmetUL0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified GmetUL0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. GmetUL0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn2+-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting GmetUL0936 may be a DNA repair enzyme.