title: Structure determination and biochemical characterization of a putative HNH endonuclease from Geobacter metallireducens GS-15.
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.
authors.affiliation: New England Biolabs, Inc. Research Department, Ipswich, Massachusetts, United States of America.
authors.forename: Shuang-yong
authors.initials: SY
authors.lastname: Xu
authors.forename: Alexandre P
authors.initials: AP
authors.lastname: Kuzin
authors.forename: Jayaraman
authors.initials: J
authors.lastname: Seetharaman
authors.forename: Alice
authors.initials: A
authors.lastname: Gutjahr
authors.forename: Siu-Hong
authors.initials: SH
authors.lastname: Chan
authors.forename: Yang
authors.initials: Y
authors.lastname: Chen
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Acton
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D053837:DNA Cleavage; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000235:genetics; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics; D045302:Geobacter / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011089:Polydeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011089:Polydeoxyribonucleotides; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D045643:DNA Repair Enzymes
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