The search for a perfect model to mimic the properties of the selenoenzyme glutathione peroxidase (GPx) has inspired great interest. Rational design and redesign of the structure-function relationship has become an indispensable technique. In this report, the active site of selenosubtilisin was successfully rebuilt by transferring the catalytically essential residue selenocysteine (Sec) to the edge of the substrate-binding pocket of the enzyme by artificial manipulation. Founding on computer-aided molecular simulation, the amino acid residue at position 63 (Ser in the wild-type enzyme) was selectively replaced with Sec using a cysteine auxotrophic expression system. The novel seleno(63)-subtilisin E gave a prominent 100-fold higher efficiency than the original seleno(221)-subtilisin E for GPx activity. Moreover, this seleno(63)-subtilisin E also had efficient hydrolase activity. (C) 2017 Elsevier Inc. All rights reserved.