Construction And Properties Of Cleavage DNA Shear Enzyme Based On Myoglobin

Many natural proteins have significant adaptability to the changes,and the modification of the microenvironment of the active sites can lead to a wider chemical transformation of the natural protein skeleton.Therefore,protein redesign is one of the most effective methods in artificial metal enzyme design and engineering.Recombinant metalloprotein is an effective means to explore how the structure around the metal center affects its function.In addition,these biological models provide an opportunity to develop new catalysts.Studies on the interaction of protein with DNA help to understand the relationship between protein structure and artificial nuclease function from the molecular level,and can provide practical clues for the creation of nuclease with advanced functions and potential application values.Using zinc protoporphyrin(Zn PP)as the raw material,we have replaced the natural heme in myoglobin(Mb),and introduced a distal histidine into the active site,and designed a channel to the active center.Three recombinant myoglobin mutants,Zn PP-F43 H,Zn PP-H64 A Mb,Zn PP-F43H/H64 A Mb were obtained.Myoglobin with redesigned active center was constructed and compared with Mbs under the same conditions by gel electrophoresis.The results showed that the recombinant protein enhibited a new photoinduced DNA cleavage activity and was a photoinduced artificial nuclease.The spin trapping experiments showed that the break of double-stranded DNA is mainly attributed to the·OH radical induced by light,while 1O2 has little contribution to the breakage of double-stranded DNA.In this study,DMPO was used as a trapping agent,and ·OHs were detected in photoactivated Zn PP-Mbs for the first time.At the same time,we used myoglobin as the framework of protein molecule,introduced glutamic acid around the heme of active center,added Mn?or Co? ions to form metal ion-protein complex,and construct artificial metal enzymes with catalytic function.These are new artificial metalloprotein hydrolases.Compared with wild-type Mb or single L29 E Mb protein,L29 E Mb+MnII(or CoII)metalloprotease showed better hydrolysis and cleavage ability to DNA,and its catalytic efficiency was greatly improved,and its catalytic function was not disturbed by O2 in air.