Rational Design Of An Artificial Nuclease And Peroxidase Based On Myoglobin

Heme enzymes are a large class of metalloenzymes,which contain one or multiple heme groups and play vital catalytic roles in biological systems.To date,many strategies have been developed for design of functional heme enzymes,such as redesign of the heme active metal site by introduction of non-heme metal ions or metal clusters,and by incorporation of unnatural amino acids and non-native cofactors,etc.Design of artificial nucleases is essential in biotechnology and biomedicine,whereas few artificial nucleases can both cleave and degrade DNA molecules.Heme proteins are potential enzymes for DNA cleavage.Using a small heme protein,myoglobin(Mb),as a model protein,we engineered a metal-binding motif of[1-His-1-Glu](native His64 and mutated Glu29)in the heme distal site.The single mutant of L29E Mb was capable of not only efficient DNA cleavage but also DNA degradation upon Mg²⁺binding to the heme distal site,as shown by an X-ray crystal structure of the Mg²⁺-L29E Mb complex.Molecular docking of the protein-DNA complex revealed multiple hydrogen-bonding interactions at their interfaces,involving both minor and major grooves of DNA.Moreover,both the distal Arg45 and the ligand Glu29 were identified as critical residues for the nuclease activity.This study reports the structure of a water-bridged hetero-dinuclear center of Mg-heme(Mg²⁺-H₂O-Fe³⁺),showing a similar function as the homo-dinuclear center(Mg _A²⁺-H₂O-Mg _B²⁺)in natural nuclease,which indicates that the Mg²⁺-L29E Mb complex is an effective artificial nuclease.Inspired by the structural feature of native peroxidases,an artificial peroxidase was rationally designed in F43Y myoglobin with a Tyr-heme cross-link by further introduction of key residues,including both a distal Arg and a Trp close to the heme group,which exhibits an enhanced peroxidase activity similar to the most efficient native horseradish peroxidase.This study provides a simple approach for design of artificial heme enzymes by the combination of catalytic elements of native enzymes with the post-translational modification of heme proteins.