| Glutathione peroxidase (GPX) is a key enzyme in reactive oxygen species-scavenging system, and plays important roles in plant resistance to abiotic and biotic stresses. Previous studies of GPX mainly focused on angiosperms, but the studies of GPXs in bryophytes was rare. In this study, three GPXs (PpGPX1/2/3) were identified from the Physcomitrella. patens genome. The gene structure, phylogeny, gene expression, subcellular localization, protein structure and biochemical function of the PpGPXs were investigated. The main results are as follows:1. PpGPX1, PpGPX2, and PpGPX3 genes encode proteins of 170,247 and 184 amino acids, with predicted molecular weight of 19.1,26.8 and 20.8 kDa, respectively. PpGPX1 and PpGPX3 contains one exon, respectively, and PpGPX2 contains six exons.2. Land plant GPXs shared high sequence similarity ranging from 39.6%-99.3% resulted from protein sequence similarity analysis. The Physcomitrella GPXs grouped together in the phylogenetic tree indicated by phylogenetic analysis, suggesting that the Physcomitrella GPX gene family was existed in the early time of land plants differentiation.3. The gene expression pattern of PpGPX1, PpGPX2, and PpGPX3 under normal growth conditions and under different stress conditions were analyzed by RT-PCR. The results showed that PpGPX1 and PpGPX2 were expressed in normal growth and stress conditions, and PpGPX3 was not expressed in all samples. So, PpGPX3 may be not expressed, or may be expressed in specific developmental stages or conditions.4. PpGPXl and PpGPX2 was ligated with green fluorescent protein gene, and transiently expressed in Arabidopsis protoplast, respectively. Protein subcellular localization analysis showed that PpGPXl was located in cytoplasm, and PpGPX2 was located in chloroplast.5. The three dimensional structures of the PpGPX1 and PpGPX2 proteins were modeled. Both PpGPXl and PpGPX2 had typical GPX protein structure, containing 4 α-helix and 6β-sheet. Protein structure superposition analysis found that PpGPX1 and PpGPX2 had similar protein structure. Except minor difference in loop region, other parts overlapped well.6. PpGPX1 and PpGPX2 protein was overexpressed in E.coli, and was purified. The enzymatic activities of the two proteins were assayed. The results showed that Physcomitrella GPXs reduced the peroxide substrates using Trx electron donor system. The catalytic activity to H2O2, COOH and tBOOH, and the affinity and catalytic efficienty of PpGPX2 to Trx was higher than PpGPXl, suggesting protein functional divergence of Physcomitrella GPXs.7. Prol 58, Phe167, and Phe172 of PpGPX2 protein was respectively mutated into Ala by site-directed mutation. The peroxidase activity and kinetic constants of the three mutants were detected. The results showed that the activities of the three mutant proteins to the three peroxide substrates decreased compared with the wild type PpGPX2 protein. Among the three mutants, the catalytic activity of PpGPX2 (F172A) to the substrates decreased most; with the kcat/Km values of PpGPX2 (F172A) mutant protein on Trx and H2O2 decreased to 15.80% and 4.69% of the wild type, respectively. This suggested that the three sites, especially F172 played an important role in the catalytic function of Physcomitrella. GPX protein.By integrating sequence analysis, phylogeny, gene expression, protein subcellular localization, protein structure and enzymatic function analysis, we investigated the functional divergence and catalytic properties of Physcomitrella GPX gene family. The results showed functional divergence existed among Physcomitrella GPXs. We also identified the key amino acidsof GPX catalytic function by protein site-directed mutation... |
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