Structure-Guided Activity Enhancement and Catalytic Mechanism of Yeast Grx8

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• 作者：YaJun Tang ; Jiahai Zhang ; Jiang Yu ; Ling Xu ; Jihui Wu ; Cong-Zhao Zhou ; Yunyu Shi
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：April 8, 2014
• 年：2014
• 卷：53
• 期：13
• 页码：2185-2196
• 全文大小：656K
• 年卷期：v.53,no.13(April 8, 2014)
• ISSN：1520-4995

文摘

Glutaredoxins (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 (Cys³³-Pro³⁴-Asp³⁵-Cys³⁶) 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 pK_a 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鈥損ong mechanism of catalysis. All together, these findings provide structural insights into the catalytic mechanism of dithiol Grxs.