Abstract
Glutathione peroxidase (GPX) plays a pivotal role in the protection of cells against oxidative damage. The green alga Chlamydomonas reinhardtii expresses both selenocysteine-containing GPX and the non-selenium GPX homolog (GPXH). We previously reported that supplementation of selenium to algal culture induces GPXH to exhibit GPX activity. Here we investigated the incorporation of selenium into GPXH and its causal relationship with the upregulation of the enzymatic activity. GPXH was purified from algal cells grown with selenium and proteolytically digested into four fragments. Selenium content analysis for these proteolytic fragments confirmed that GPXH-incorporated selenium is predominantly enriched in a fragment that carries the putative catalytic residue Cys-38. We next constructed three kinds of engineered GPXH proteins by substituting Ser for one of three Cys residues in native GPXH, Cys-38, -66, and -84, using a bacterial overexpression system, resulting in Cys38Ser, Cys66Ser, and Cys84Ser derivatives, respectively. Of these, the Cys66Ser and Cys84Ser derivatives exhibited the same level of selenium-dependent GPX activity as the normal recombinant GPXH, whereas the Cys38Ser mutant GPXH not only lost its activity completely but also demonstrated severely impaired incorporation of selenium. These findings strongly suggest that selenium is post-translationally assimilated into the Cys-38 of the GPXH protein, thereby enhancing its enzymatic activity.