Study On The Phosphatase Activity Of Peroxidase From Proso Millet

Peroxidase(POD)is a type of oxidoreductase that is widely found in bacteria,fungi,plants and animals,which utilize hydrogen peroxide to mediate the oxidation of various inorganic and organic substrates.Most of the POD are a heme protein that consists of single polypeptide chain and Fe(III)-protoporphyrin IX prosthetic group,the apoprotein molecule must bind to heme to form a holoenzyme,and the heme prosthetic group is the carrier of POD electron transfer.Class III POD is a secreted peroxidase derived from higher plants.It is synthesized in cytoplasm and can be transferred to cell wall or vacuole,participating in a variety of physiological functions,such as the removal of toxic peroxide in organism,the synthesis of cell wall and wound healing,auxin synthesis and metabolism.Other heme-containing prosthetic proteins(myoglobin,cytochrome c,etc.)can oxidatively cleave DNA in the presence of H2O2.Pm POD is a cationic peroxidase containing a heme auxiliary group that is derived from millet,which can induce necrotic apoptosis in colorectal cancer cells.Whether Pm POD-induced necrotic apoptosis is associated with Pm POD disruption of DNA.Therefore,this study will focus on the cleavage and mechanism of DNA by Pm POD.In this study,agarose gel electrophoresis was used to investigate whether Pm POD could cleavage DNA;by using p BR322,p UC18 and p GEM-T as substrates,combined with DTT,Na N3,and divalent metal particles.The molecular mechanism of Pm POD cleavage of DNA was investigated by means of linkage and transformation experiments and spectral scanning,it was confirmed that Pm POD-induced DNA fragmentation is hydrolytic cleavage or oxidative cleavage;furthermore,using d NMPs as substrate,the hydrolysis of d NMPs by Pm POD was analyzed by HPLC.Based on the above studies,the optimum p H values of Pm POD peroxidase and phosphatase enzyme activities were determined respectively.Finally,the three-dimensional structure of Pm POD was analyzed by X-ray crystallography.Based on the amino acid sequence of Pm POD,the actived site of phosphatase activityof Pm POD was analyzed.The experimental results show that,within a certain time and concentration range,Pm POD cleaves the supercoiled circular DNA into nicked and linear DNA,through a cleavage mechanism similar to natural endonuclease,while at high concentration of Pm POD and long-term effects,The plasmid DNA can be completely degraded.The connection and transformation experiments show that the Pm POD can break the plasmid DNA in the way of hydrolytic cleavage,and the inhibition and spectral scanning experiments show that the active oxygen(ROS)and the iron disulfide IX part do not participate in the Pm POD-mediated DNA cleavage,indicating that the Pm POD is different from other reported hemin proteins(myoglobin,etc.),which cleavage DNA by hydrolysis cleavage rather than oxidative cleavage mechanism.The Divalent metal ion experiments have shown that,similar to other endonucleases,Mg2+ significantly enhances the DNA cleavage activity of Pm POD.Further studies have shown that Pm POD can hydrolyze the phosphate of d NMPs,indicating that Pm POD belongs to a phosphatase.Subsequently,Pm POD was compared with horseradish peroxidase(HRP),and it was found that the phosphatase activity of Pm POD was much higher than HRP,but the peroxidase activity was lower than HRP.In this study,the three-dimensional structure of Pm POD was obtained by X-ray crystal diffraction technology.Through the comparative analysis of the amino acid sequences of Pm POD and other food crop sources POD,it was revealed that the Pm POD sequence contains a conserved sequence of HCXXXXXR,which is similar to the active sites of the reported dual-specific phosphatases(DUSPs).This study provides a powerful support for the mechanism of DNA cleavage by this kind of peroxidase,and provides a reference value for further study on its enzymatic research.