Engineering glutathione transferase to a novel glutathione peroxidase mimic with high catalytic efficiency. Incorporation of selenocysteine into a glutathione-binding scaffold using an auxotrophic expression system.
Glutathione peroxidase (GPx, EC 1.11.1.9) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several attempts have been made to imitate its function for mechanical study and for its pharmacological development as an antioxidant. By replacing the active site serine 9 with a cysteine and then substituting it with selenocysteine in a cysteine auxotrophic system, catalytically essential residue selenocysteine was bioincorporated into GSH-specific binding scaffold, and thus, glutathione S-transferase (GST, EC 2.5.1.18) from Lucilia cuprina was converted into a selenium-containing enzyme, seleno-LuGST1-1, by genetic engineering. Taking advantage of the important structure similarities between seleno-LuGST1-1 and naturally occurring GPx in the specific GSH binding sites and the geometric conformation for the active selenocysteine in their common GSH binding domain-adopted thioredoxin fold, the as-generated selenoenzyme displayed a significantly high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione, being comparable with those of natural GPxs. The catalytic behaviors of this engineered selenoenzyme were found to be similar to those of naturally occurring GPx. It exhibited pH and temperature-dependent catalytic activity and a typical ping-pong kinetic mechanism. Engineering GST into an efficient GPx-like biocatalyst provided new proof for the previous assumption that both GPx and GST were evolved from a common thioredoxin-like ancestor to accommodate different functions throughout evolution.
Yu
Hui-Jun
HJ
Key Laboratory for Supramolecular Structure and Materials of Ministry of Education, Jilin University, 10 Qianwei Road, Changchun 130012, People's Republic of China.
Liu
Jun-Qiu
JQ
Bock
August
A
Li
Jing
J
Luo
Gui-Min
GM
Shen
Jia-Cong
JC
D002384:Catalysis; D005978:Glutathione / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000096:biosynthesis*; D005982:Glutathione Transferase / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D015202:Protein Engineering; D017279:Selenocysteine / Q000378:metabolism*; D013696:Temperature
D017279:Selenocysteine; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; D005978:Glutathione