A de novo binuclear zinc enzyme with DNA cleavage activity

Abstract

Metallohydrolases are broadly used throughout biology, often to catalyze the degradation of macromolecules such as DNA and proteins. Many of these enzymes function with zinc in their active site, and an important subset of these enzymes utilize a binuclear zinc active site. Mimics of these enzymes have been developed, some of which catalyze the digestion of DNA. However, the majority of the mimics that utilize zinc are small molecules, and most are mononuclear. Herein, we report DNA cleavage activity by the de novo designed Due Ferri single-chain (DFsc) protein containing a binuclear zinc active site. This binuclear zinc–protein complex is able to digest plasmid DNA at rates up to 50 ng/h, and these cleavage rates are affected by changes to amino acid residues near the zinc-binding site. These results indicate that the DFsc scaffold is a good model system to carry out careful structure–function relationship studies to understand key structural features that influence reactivity in natural binuclear zinc hydrolases, as it is the first report of a binuclear model system in a protein scaffold.

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Acknowledgements

This work was supported by a National Science Foundation Major Research Instrumentation grant (1725534). OMP and KMB were supported in part by the Mindlin Foundation (MF18-UMR31). AP, OMP, and KMB were supported, in part, by the Cross-Disciplinary Science Institute at Gettysburg College (X-SIG). KMB thanks Zakiya Whatley and Shelli Frey for helpful conversations. AP and KMB thank Micaylah Bowers and Meredith Brown for support and assistance in experiment preparations.

Funding

This work was supported by a National Science Foundation Major Research Instrumentation grant (1725534). OMP and KMB were supported in part by the Mindlin Foundation (MF18-UMR31). AP, OMP, and KMB were supported, in part, by the Cross-Disciplinary Science Institute at Gettysburg College (X-SIG).

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Correspondence to Katherine M. Buettner.

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Paredes, A., Peduzzi, O.M., Reig, A.J. et al. A de novo binuclear zinc enzyme with DNA cleavage activity. J Biol Inorg Chem (2021). https://doi.org/10.1007/s00775-020-01845-5

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Keywords

  • Metallohydrolase
  • Binuclear zinc hydrolase
  • DNA cleavage