title: Engineered selenium-containing glutaredoxin displays strong glutathione peroxidase activity rivaling natural enzyme.
abstract: 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.
authors.affiliation: State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ge
authors.forename: Zhenhui
authors.initials: Z
authors.lastname: Qi
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiaoman
authors.initials: X
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Jiayun
authors.initials: J
authors.lastname: Xu
authors.forename: Junqiu
authors.initials: J
authors.lastname: Liu
authors.forename: Jiacong
authors.initials: J
authors.lastname: Shen
mesh_terms: 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
chemical_list: D054477:Glutaredoxins; D017279:Selenocysteine; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; D012643:Selenium