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United States Department of Agriculture

Agricultural Research Service


Location: Children's Nutrition Research Center

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

item Liu, Xi
item Feng, Yingang
item Liu, Jian-zhong
item Chen, Yuling
item Pham, Khanh
item Deng, Haiteng
item Hirschi, Kendal
item Wang, Xinquan
item Cheng, Ning-hui

Submitted to: Proceedings of the National Academy of Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2012
Publication Date: 6/4/2013
Citation: Liu, X., Feng, Y., Liu, J., Chen, Y., Pham, K., Deng, H., Hirschi, K.D., Wang, X., Cheng, N. 2013. Structural insights into the N-terminal GIY-YIG endonuclease activity of "Arabidopsis" glutaredoxin AtGRXS16 in chloroplasts. Proceedings of the National Academy of Sciences. 110(23):9565-9570.

Interpretive Summary: A group of proteins, called Glutaredoxins (Grxs), are important for protecting the life of cells from damage caused by free radicals. However, how these proteins act in the cell and whether these proteins are directly involved in the process of damage repair are still largely unknown. In the present study, a new member of Grxs was identified that comprises two distinct functional regions, in which one region is similar to other members of Grxs, but another one is unique and its function is unknown. Using the structural biology and biochemical approaches, this new region of the protein was determined as an enzyme that could be crucial for repairs of damaged genetic material in the cell. This study also found a novel mechanism on how this protein is being regulated. Therefore, this study discovers a link between Grx-mediated function and DNA metabolism, and these findings provide insights for engineering protein function that will lead to a better solution to improve agricultural production.

Technical Abstract: 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 lambdaDNA 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.

Last Modified: 06/26/2017
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