The Structure And Function Of Saccharomyces Cerevisiae Glutaredxoin Monothiol Grx5

1. The crystal structure research on the Saccharomyces cerevisiae glutaredxoin monothiol Grx5 and its mutants Cys60Ser and Cys117SerGlutaredoxins (Grxs) are small proteins that catalyze the reduction of protein disulfides or glutathione-protein-mixed disulfides using reduced glutathione (GSH) as the electron donor. Grxs can be categorized into dithiol Grxs with the characteristic CPY/FC motifs and monothiol Grxs with the CGFS motif. Compared to dithiol Grxs, monothiol Grxs have been not thoroughly studied and many questions needed to be clarified with regard to the catalytic mechanism and cellular functions. We solved the crystal structure of Grx5 from Saccharomyces cerevisiae at the resolution of 1.67 ?. The overall structure of Grx5 adopts a classic glutaredoxin-like fold. The spatial distance between the thiols of Cys60 and Cys117 is as far as ~14 ?, thus the region around the two cysteines should undergo a large conformational change to facilitate the disulfide bond formation. Structural comparison with glutathionylated yeast dithiol glutaredoxins Grx1 and Grx2 reveals some differences in GSH binding site, which may implies that Grx5 have not classic Grx enzyme activity as same as Grx1 and Grx2.2. The biochemical function research on the Saccharomyces cerevisiae glutaredxoin monothiol Grx5The full length Grx5 consists of 150 amino acids, and possesses a monothiol (CGFS) potential active site. It shows high sequence homology to the other monothiol glutaredoxins. In the previous study, the null GRX5 gene in vivo is essential. Grx5 is inactive in a standard glutaredoxin assay showing that the oxidized form of Grx5 could not reduced directly by GSH. However,our activity assay showed that oxidized Grx5 could be reduced by Grx2 and Trr2 respectively.Grx3 combined with Grx4 can bind Fra1 and Fra2 to form the Fra-Grx complex in the yeast cytoplasm. The complex could sense the signal from Fe-S cluster synthesis in mitochondria and transfer it to the cytoplasmic iron-sensitive transcription factor Aft1. Grx5 plays important role in the biosysthsis of Fe-S cluster in S. cerevisia mitochrondia as well. Based on the activity assay above, the stimulation of Grx5 Fe-S cluster binding reconstitution by Grx2 was performed under the anaerobic condition.