Recombinant Expression And Study On The Phosphatase Activity Site Of Peroxidase From Proso Millet

Peroxidase(POD,EC 1.11.1.7)is a kind of oxidoreductase widely existing in biology The heme group of natural peroxidase is usually ferriporphyrin IX,which contains four pyrrole rings connected with Fe(?).H2O2 can interact with heme,the active center of peroxidase,and activate peroxidase as the reaction intermediate to mediate the redox reaction.The class ? peroxidase catalase superfamily widely exists in plants,which is usually secreted into cell walls,extracellular bodies or vacuoles,and plays a role in the oxidation of lignin precursors,auxin and secondary metabolites.Protein phosphatase is a kind of dephosphorylation group which can dephosphorylate the phosphorylated substance Protein phosphatase and protein kinase play an irreplaceable role in biological activities,such as cell division,signal transduction,RNA splicing,DNA repair,expression of proto oncogenes and tumor suppressor genesProso millet peroxidase(PmPOD)is a kind of plant peroxidase extracted from millet Its typical feature is that it contains heme cofactor.PmPOD not only has peroxidase activity dependent on heme cofactor,but also has phosphatase activity independent of heme cofactor.In vitro,PmPOD can exert its phosphatase activity.For example,PmPOD can break the phosphodiester bond of DNA or the phosphodiester bond of deoxyribonucleoside-5 '-monophosphates(dNMPs).The results of previous experiments show that Mg2+plays an important role in the hydrolysis of phosphate group in the substrate of PmPOD.However,the mechanism of Mg2+enhancing the activity of PmPOD phosphatase and the active site of PmPOD exerting phosphatase are not clear.Therefore,this paper will study from the following three aspects:1.Firstly,the effect and mechanism of Mg2+on the hydrolysis of dNMPs by phosphatase activity in PmPOD were explored.Through experimental research,the results show that during the binding process of PmPOD and substrate,Mg2+and dNMPs first combine to form a reaction complex,and this complex is then combined with PmPOD to hydrolyze the phosphate group.Through fluorescence spectrum analysis,Mg2+can change the quenching mode of PmPOD fluorophore from dynamic to static.In addition,Mg2+can increase the binding constant Ka of the reaction between PmPOD and dNMPs by about 2 to 10 times and the hydrolysis rate V by about 3 to 13 times(compared with the absence of Mg2+).Among them,Ka and V of dCMP are the largest,while Ka and V of dAMP are the smallest;2.The gene sequence corresponding to the predicted phosphatase active site fragment(147-296 aa)in PmPOD was constructed on the expression vector pET-32a for prokaryotic expression.The results of affinity purification,refolding and plasmid cleavage showed that the recombinant truncated proso millet POD(tPmPOD)was obtained;3.Five mutant proteins were obtained by mutating the conserved amino acids His191,Cys192,Arg198,Cys219 which formed disulfide bond with the active site Cys192 and Asp225 which had acid-base catalysis into nonpolar amino acid A(alanine).High performance liquid chromatography and fluorescence spectroscopy were used to study the active sites of PmPOD.The results showed that the binding constant and reaction rate of the mutant protein phosphatase activity decreased 1.5-3.7 times and 2.2-14.8 times,respectively,compared with tPmPOD.Among them,the mutant tPmPOD-C192A had the lowest phosphatase activity,and its binding constant and hydrolysis rate decreased by 3.7 and 14.8 times respectively in the process of substrate hydrolysis.These five sites all play an important role in the activity of PmPOD phosphatase.Among them,Cys192 is the nucleophilic group,and Cys219 forms a disulfide bond with Cys192 to protect the stability of its sulfhydryl group,which is very important for the activity of PmPOD phosphatase.