title: Structure-guided activity enhancement and catalytic mechanism of yeast grx8.
abstract: 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 (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.
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China , Hefei, Anhui 230027, People's Republic of China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Tang
authors.forename: Jiahai
authors.initials: J
authors.lastname: Zhang
authors.forename: Jiang
authors.initials: J
authors.lastname: Yu
authors.forename: Ling
authors.initials: L
authors.lastname: Xu
authors.forename: Jihui
authors.initials: J
authors.lastname: Wu
authors.forename: Cong-Zhao
authors.initials: CZ
authors.lastname: Zhou
authors.forename: Yunyu
authors.initials: Y
authors.lastname: Shi
mesh_terms: D055162:Biocatalysis; D004789:Enzyme Activation; D054477:Glutaredoxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: C000588987:GRX8 protein, S cerevisiae; D054477:Glutaredoxins; D011994:Recombinant Proteins