Engineered selenium-containing glutaredoxin displays strong glutathione peroxidase activity rivaling natural enzyme.
Insertion of selenocysteine (Sec) into protein scaffolds provides an opportunity for designing enzymes with improved and unusual catalytic properties. The use of a common thioredoxin fold with a high affinity for glutathione in glutaredoxin (Grx) and glutathione peroxidase (GPx) suggests a possibility of engineering Grx into GPx and vice versa. Here, we engineered a Grx domain of mouse thioredoxin/glutathione reductase (TGR) into a selenium-containing enzyme by substituting the active site cysteine (Cys) with selenocysteine (Sec) in a Cys auxotrophic system. The resulting selenoenzyme displayed an unusually high GPx catalytic activity rivaling that of several native GPxs. The engineered seleno-Grx was characterized by mass spectrometry and kinetic analyses. It showed a typical ping-pong kinetic mechanism, and its catalytic properties were similar to those of naturally occurring GPxs. For example, its second rate constant (k(cat)/K(mH2O2)) was as high as 1.55x10(7) M(-1) min(-1). It appears that glutathione-dependent Grx, GPx and glutathione transferase (GST) evolved from a common thioredoxin-like ancestor to accommodate related glutathione-dependent functions and can be interconverted by targeted Sec insertion.
Ge
Yan
Y
State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012, People's Republic of China.
Qi
Zhenhui
Z
Wang
Ying
Y
Liu
Xiaoman
X
Li
Jing
J
Xu
Jiayun
J
Liu
Junqiu
J
Shen
Jiacong
J
D000818:Animals; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D054477:Glutaredoxins / Q000737:chemistry / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012643:Selenium / Q000737:chemistry / Q000378:metabolism*; D017279:Selenocysteine / Q000737:chemistry / Q000378:metabolism
D054477:Glutaredoxins; D017279:Selenocysteine; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; D012643:Selenium