The Solution Structure And Catalytic Mechanism Study Of Saccharomyces Cerevisiae Grx8

Glutaredoxins (Grxs) are oxidoreductases that widely spread in all kingdoms of life. The yeast Saccharomyces cerevisiae encodes eight Grxs, among which Grx8 shares a sequence identity of 30% and 23% to the typical dithiol Grx1 and Grx2, respectively, but exhibits a much lower GSH-dependent oxidoreductase activity. To elucidate the catalytic mechanism, we solved the solution structure of Grx8, which displays a typical Grx fold. Structural analysis indicated that Grx8 possesses a negatively-charged CXXC motif (Cys33-Pro34-Asp35-Cys36) and a GSH recognition site, distinct from Grx1 and Grx2. Subsequent structure-guided site mutations revealed that the single mutant D35Y and double mutant N80T/L81V gained an elevation of activity to 10- and 11-fold, respectively; and moreover, the triple mutant D35Y/N80T/L81V has an increase of activity up to 44-fold, which is comparable to that of a canonical Grx. Biochemical analysis suggested that the increase of catalytic efficiency is resulted from a decreased pKa value of catalytic cysteine Cys33 and/or enhancement of the putative GSH recognition site. Moreover, NMR chemical shift perturbation analysis combined with GSH analog inhibition assays enabled us to elucidate that the wild-type Grx8 and all mutants adopt a ping-pang mechanism of catalysis. All together, these findings provided the structural insights into the catalytic mechanism of dithiol Grxs.