2m80
Solution structure of yeast dithiol glutaredoxin Grx8Solution structure of yeast dithiol glutaredoxin Grx8
Structural highlights
FunctionGLRX8_YEAST Glutathione-dependent oxidoreductase with lower activity compared to the other members of the glutaredoxin family. The disulfide bond functions as an electron carrier in the glutathione-dependent synthesis of deoxyribonucleotides by the enzyme ribonucleotide reductase.[1] [2] Publication Abstract from PubMedGlutaredoxins (Grxs) are wide-spread oxidoreductases that are found in all kingdoms of life. The yeast Saccharomyces cerevisiae encodes eight Grxs, among which, Grx8 shares a sequence identity of 30 and 23% with typical dithiol Grx1 and Grx2, respectively, but it exhibits a much lower GSH-dependent oxidoreductase activity. To elucidate its 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 (Cys(33)-Pro(34)-Asp(35)-Cys(36)) and a GSH-recognition site, which are distinct from Grx1 and Grx2. Subsequent structure-guided site mutations revealed that the D35Y single mutant and N80T/L81V double mutant possess increased activity of 10- and 11-fold, respectively; moreover, the D35Y/N80T/L81V triple mutant has increased activity of up to 44-fold, which is comparable to that of canonical Grx. Biochemical analyses suggested that the increase in catalytic efficiency resulted from a decreased pKa value of catalytic cysteine Cys33 and/or enhancement of the putative GSH-recognition site. Moreover, NMR chemical shift perturbation analyses combined with GSH analogue inhibition assays enabled us to elucidate that wild-type Grx8 and all mutants adopt a ping-pong mechanism of catalysis. All together, these findings provide structural insights into the catalytic mechanism of dithiol Grxs. Structure-guided activity enhancement and catalytic mechanism of yeast grx8.,Tang Y, Zhang J, Yu J, Xu L, Wu J, Zhou CZ, Shi Y Biochemistry. 2014 Apr 8;53(13):2185-96. doi: 10.1021/bi401293s. Epub 2014 Mar, 25. PMID:24611845[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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