Structure - function analysis of Arabidopsis thaliana CC-type glutaredoxins ROXY1 and ROXY9
Biochemistry
Final Report Abstract
Having established a protocol that allows expression and purification of a member of the land plant-specific family of class III glutaredoxins (GRXs), namely Arabidopsis thaliana ROXY9, we were able to ask the question whether ROXY9 has maintained the functions of the evolutionary older canonical GRXs. These function either as oxidoreductases within the antioxidative repair system (class I) or as iron sulfur (FeS) cluster binding proteins (class II). In contrast to class I GRXs, ROXY9 is inactive as an oxidoreductase on widely used glutathionylated model substrates like bis(2-hydroxyethyl)disulfide (HED), glutathionylated cysteine and roGFP. Since ROXY9 physically and genetically interacts with TGACG-binding transcription factor TGA1, we asked whether TGA1 might be a substrate for ROXY9. However, ROXY9 does not accelerate the glutathionylation of TGA1 under oxidative conditions. A prerequisite for the enzymatic activity of class I GRXs is the reversible glutathionylation of the first cysteine of the active site. Importantly, glutathionylation of this putative catalytically active cysteine in the CCLC motif of ROXY9 occurs only under highly oxidizing conditions established by the GSH/glutathione disulfide (GSSG) redox couple, while class I GRXs are oxidized to the glutathionylated and the disulfide containing protein species even at high GSH/GSSG ratios. We conclude that structural alterations in the glutathione binding site in ROXY9 lead to an altered glutathione binding mode which explains that glutathionylation is energetically unfavourable. This might have evolved to avoid overlapping functions with class I GRXs and raises questions of whether ROXY9 regulates TGA substrates through redox regulation. Consistently, complementation experiments with ROXY9 encoding a C/S exchange of the putative catalytically active cysteine revealed that this cysteine is not required for the function of ROXY9 as an activator of TGA1-regulated gene expression in roots. Further biochemical analysis suggests that ROXY9 dimerizes upon FeS cluster binding in reconstitution experiments under anaerobic conditions, but we failed to identify the cluster by spectroscopic analysis. Since the cysteines of the active site are not important for the in vivo activity of ROXY9, it is unlikely that binding of an FeS cluster plays a role for its function.
Publications
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Functional characterization of Arabidopsis thaliana CC-type glutaredoxin ROXY9. University Goettingen Repository.
Mrozek, Pascal
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Molecular basis for the enzymatic inactivity of class III glutaredoxin ROXY9 on standard glutathionylated substrates. Nature Communications, 16(1).
Mrozek, Pascal; Grunewald, Stephan; Treffon, Katrin; Poschmann, Gereon; Rabe von Pappenheim, Fabian; Tittmann, Kai & Gatz, Christiane
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Redox-inactive CC-type glutaredoxins interfere with TGA transcription factor–dependent repression of target promoters in roots. The Plant Cell, 37(3).
Thurow, Corinna; Pelizaeus, Anja Maren; Mrozek, Pascal; Hoßbach, Ben Moritz; Budimir, Jelena; Schmitt, Kerstin; Valerius, Oliver; Braus, Gerhard & Gatz, Christiane
