Structural basis for the substrate selectivity of Helicobacter pylori NucT nuclease activity

PLoS One. 2017 Dec 4;12(12):e0189049. doi: 10.1371/journal.pone.0189049. eCollection 2017.

Abstract

The Phospholipase D (PLD) superfamily of proteins includes a group of enzymes with nuclease activity on various nucleic acid substrates. Here, with the aim of better understanding the substrate specificity determinants in this subfamily, we have characterised the enzymatic activity and the crystal structure of NucT, a nuclease implicated in Helicobacter pylori purine salvage and natural transformation and compared them to those of its bacterial and mammalian homologues. NucT exhibits an endonuclease activity with a strong preference for single stranded nucleic acids substrates. We identified histidine124 as essential for the catalytic activity of the protein. Comparison of the NucT crystal structure at 1.58 Å resolution reported here with those of other members of the sub-family suggests that the specificity of NucT for single-stranded nucleic acids is provided by the width of a positively charged groove giving access to the catalytic site.

MeSH terms

  • Amino Acid Sequence
  • Crystallography, X-Ray
  • Endonucleases / chemistry
  • Endonucleases / metabolism*
  • Helicobacter pylori / enzymology*
  • Protein Conformation
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Endonucleases

Grant support

This work was supported by funds from the Commissariat à l’Energie Atomique, the Centre National de la Recherche Scientifique and Institut National pour la Santé et la recherché Médicale (UMR967), by the University Paris-Sud 11 (UMR8619), by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 and by a grant from the Indo-French Centre for Promotion of Advanced research (CEFIPRA) (grant number 5203-5 to JPR). LC and CC were supported by Ph.D fellowships from the French Ministry of Education and the DIM-Malinf, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.