Activity Identification And Protein Crystallization Of Potato Glutathione Synthesis-related Enzymes:StGSH1 And StGSH2

Potato as one of important staple crops,its growth and development are harmed by active oxygen species(ROS)which produced from bio/abio-stresses.Glutathione(GSH)plays an important role in the stress response to ROS.Glutamyl cysteine Ligase(GSH1)and Glutathione Synthase(GSH2)are two key enzymes in GSH biosynthesis.In this paper,physical properties and structure characters of StGSH1 and StGSH2 proteins were predicted;then these two proteins were expressed by prokaryotic protein expression system and purified by ion-exchange chromatography and size exclusion chromatography;after,the substrate specificity,optimum temperature and optimum p H value of StGSH1 and StGSH2 were explored;then the crystalization condition of StGSH2 protein were screened,and the preliminary StGSH1 crystal structure was done.These results will pave a foundation for further biological function and structural analysis of StGSH1 and StGSH2.The main results are showed as follows:1)Optimized the StGSH1 gene codon,constructed prokaryotic expression system,isolated StGSH1 protein by column affinity chromatography,anion exchange chromatography and size exclusion chromatography,and identified this protein exist in the form of monomer and dimer.Same work was done on StGSH2,identified this protein exist as monomer and could not form dimer or polymer.2)For substrate specificity StGSH1,it’s affinity with cysteine was the highest,followed by ATP and glutamate,showed that StGSH1 protein had glutamyl-cysteine ligase activity.In addition,the optimum reaction temperature and optimum reaction p H value of StGSH1 in vitro were 37℃ and 8.3)For substrate specificity of StGSH2,it’s affinity with γ-glutamyl cysteine(γ-EC)was the highest,followed by ATP and glycine,showed that StGSH1 had glutathione synthetase activity.In addition,the optimum reaction temperature and optimum reaction p H value of StGSH2 in vitro were 37℃ and 8.4)The crystallization condition of StGSH1 protein were screened,results showed that the monomer form of StGSH1 was easy to crystallize,while the dimer form was difficult.Final optimization conditions were 0.17 M ammonium acetate plus 22%(w/v)PEG3350 and 0.1 M Bis-Tris(p H=5.5).Also,preliminary crystallization condition of StGSH2 protein were screened,and related work is still under way.5)The preliminary analysis of the crystal structure of StGSH1 protein has been completed.