Related Experiment Video

Updated: Jan 21, 2026

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides
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Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Published on: September 7, 2019

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Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage

Carl J Schiltz1, April Lee1, Edward A Partlow1

  • 1Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA.

Nucleic Acids Research
|August 11, 2019
PubMed

Insights

Overcoming lysogenization defect (OLD) proteins are novel nucleases with a unique structure. This study reveals their catalytic core and a two-metal ion mechanism essential for DNA cleavage.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Microbiology

Background:

  • Overcoming lysogenization defect (OLD) proteins are a family of uncharacterized nucleases found in bacteria, archaea, and viruses.
  • These enzymes possess an N-terminal ATPase domain and a C-terminal Toprim domain, common in DNA replication, recombination, and repair proteins.
  • The in vivo functions and structural details of OLD proteins are largely unknown.

Purpose of the Study:

  • To classify OLD proteins into distinct groups based on genetic and sequence data.
  • To determine the high-resolution crystal structures of the catalytic C-terminal regions of Class 2 OLD proteins from Burkholderia pseudomallei and Xanthamonas campestris.
  • To elucidate the structural basis for OLD protein nuclease activity and DNA binding.

Main Methods:

  • Bioinformatic analysis to identify and classify OLD protein families.
  • X-ray crystallography to determine the structures of OLD protein catalytic domains.
  • Biochemical analysis to investigate the active site and metal ion coordination.

Main Results:

  • Two distinct classes of OLD proteins were identified.
  • Crystal structures of the catalytic regions of Burkholderia pseudomallei and Xanthamonas campestris Class 2 OLD proteins were resolved at 2.24 Å and 1.86 Å, respectively.
  • The structures revealed a novel two-domain architecture (altered Toprim and a unique helical domain) coordinating two magnesium ions, indicative of a two-metal catalysis mechanism for DNA cleavage.
  • A distinct DNA-binding mode, different from other Toprim-containing proteins, was proposed.

Conclusions:

  • The study defines the fundamental structural properties of the OLD protein catalytic core.
  • A mechanism for nuclease activity involving two-metal ion catalysis and a novel DNA-binding mode is proposed.
  • These findings provide crucial insights into the function and mechanism of this uncharacterized nuclease family.

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