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

Abstract

Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions.Achloroplasticmonothiol glutaredoxin,AtGRXS16 fromArabidopsis 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 regulationandDNAcleavagein 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-NTDwas 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 amanner of regulation for Grxs and provide insights into themechanistic link between redox regulation and DNA metabolism in chloroplasts.