小立碗藓GPX家族的功能分化与催化特性研究

Functional Divergence and Catalytic Properties of Glutathione Peroxidase Family from Physcomitrella patens

谷胱甘肽过氧化物酶(GPX)在植物抵抗氧化胁迫中发挥重要作用。该研究从小立碗藓(Physcomitrella patens)基因组中挖掘到3个GPX基因,分别命名为PpGPX1、PpGPX2和PpGPX3。其中PpGPX1和PpGPX3只含有1个外显子,而PpGPX2含有6个外显子。表达模式分析发现PpGPX1和PpGPX2在检测的所有条件下均表达,而PpGPX3在检测的所有条件下均不表达。蛋白亚细胞定位分析发现,PpGPX1蛋白定位在细胞质,而PpGPX2蛋白定位在叶绿体。在大肠杆菌中表达并纯化了PpGPX1和PpGPX2蛋白,酶学性质分析发现,PpGPX1和PpGPX2蛋白均只能利用Trx电子供体系统,而不能利用GSH电子供体系统;PpGPX2蛋白对过氧化物底物的催化活性和催化效率均高于PpGPX1。基因结构、表达模式、亚细胞定位和蛋白酶学性质的差异预示小立碗藓GPX基因家族成员发生了功能分化,将PpGPX2蛋白的Pro158、Phe167和Phe172氨基酸残基均突变为Ala,发现突变体蛋白对底物催化活性降低,说明这3个氨基酸位点对PpGPX2蛋白具有重要催化活性。

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 analy- sis revealed that PpGPX1 and PpGPX2 were expressed in all tested conditions. However,PpGPX3 tran- scripts 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 chloro- plast, respectively. PpGPX1 and PpGPX2 were then over-expressed in E. coli and purified using a Ni Sepha- rose 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 exam- ined by site-directed mutagenesis. The mutant proteins showed decreased catalytic activity which indicated that these three residues were important for enzymatic activity.