Structural insights into the N-terminal GIY-YIG endonuclease activity of Arabidopsis glutaredoxin AtGRXS16 in chloroplasts

Proc Natl Acad Sci U S A. 2013 Jun 4;110(23):9565-70. doi: 10.1073/pnas.1306899110. Epub 2013 May 20.


Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from Arabidopsis thaliana, comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endonuclease motif and a C-terminal Grx module, to coordinate redox regulation and DNA cleavage in chloroplasts. Structural determination of AtGRXS16-NTD showed that it possesses a GIY-YIG endonuclease fold, but the critical residues for the nuclease activity are different from typical GIY-YIG endonucleases. AtGRXS16-NTD was able to cleave λDNA and chloroplast genomic DNA, and the nuclease activity was significantly reduced in AtGRXS16. Functional analysis indicated that AtGRXS16-NTD could inhibit the ability of AtGRXS16 to suppress the sensitivity of yeast grx5 cells to oxidative stress; however, the C-terminal Grx domain itself and AtGRXS16 with a Cys123Ser mutation were active in these cells and able to functionally complement a Grx5 deficiency in yeast. Furthermore, the two functional domains were shown to be negatively regulated through the formation of an intramolecular disulfide bond. These findings unravel a manner of regulation for Grxs and provide insights into the mechanistic link between redox regulation and DNA metabolism in chloroplasts.

Keywords: nuclear magnetic resonance; reactive oxygen species.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs / genetics
  • Antiporters
  • Arabidopsis / enzymology*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Carrier Proteins / metabolism*
  • Chloroplasts / enzymology*
  • Chromatography, Gel
  • DNA / metabolism*
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Glutaredoxins / metabolism*
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Mutation, Missense / genetics
  • Oxidation-Reduction
  • Protein Folding
  • Protein Structure, Tertiary
  • Reactive Oxygen Species / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism
  • Spectrophotometry, Ultraviolet
  • Yeasts


  • Antiporters
  • Arabidopsis Proteins
  • CXIP1 protein, Arabidopsis
  • Carrier Proteins
  • Glutaredoxins
  • Grx5 protein, S cerevisiae
  • Reactive Oxygen Species
  • Saccharomyces cerevisiae Proteins
  • DNA
  • Endonucleases
  • GRXS16 protein, Arabidopsis

Associated data

  • PDB/2LWF