Abstract:In plants,glutathione peroxidase (GPX) plays important roles in protecting cells against oxidative stress.In this study,three GPXs (PpGPX1/2/3) were identified from the Physcomitrella patens genome.PpGPX1 and PpGPX3 only had one exon,while PpGPX2 had six exons.Semiquantitative RT-PCR analysis revealed that PpGPX1 and PpGPX2 were expressed in all tested conditions.However,PpGPX3 transcripts were not detected in any tested conditions.By transiently expressing C-terminal eGFP-GPX fusions in Arabidopsis protoplasts,we found that PpGPX1 and PpGPX2 were localized to cytoplasm and chloroplast,respectively.PpGPX1 and PpGPX2 were then over-expressed in E.coli and purified using a Ni Sepharose High Performance column.Enzymatic analysis found that P.patens GPXs showed enzymatic activity towards peroxide substrates using Trx as electron donor,but not GSH.PpGPX2 showed higher catalytic activity and catalytic efficiency towards peroxide substrates than that of PpGPX1.The differences of gene structures,gene expression,protein subcellular localization and enzymatic characteristics among P.patens GPXs indicating functional divergence.The function of Pro158,Phe167 and Phe172 of PpGPX2 were examined by site-directed mutagenesis.The mutant proteins showed decreased catalytic activity which indicated that these three residues were important for enzymatic activity.