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

Shuang yong Xu, Alexandre P. Kuzin, Jayaraman Seetharaman, Alice Gutjahr, Siu Hong Chan, Yang Chen, Rong Xiao, Thomas B. Acton, Gaetano T. Montelione, Liang Tong

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Abstract

The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-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 Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of Gmet_0936 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, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 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. Gmet_0936 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 Gmet_0936 may be a DNA repair enzyme.

Original languageEnglish (US)
Article numbere72114
JournalPloS one
Volume8
Issue number9
DOIs
StatePublished - Sep 6 2013

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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