Rational Design Of Artificial Dehaloperoxidases With High Efficiency Based On Myoglobin

Protein design,especially metalloproteins including heme proteins,has received much attention in the last few decades,which not only elucidates the structure and function relationship of natural enzymes,but also provides the ability to create artificial proteins with improved properties and functions.Nature has evolved a variety of approaches such as diverse post-translational modifications(PTMs)to regulate the protein structure and function.Inspired by nature,various approaches have been developed for functional protein design,including the design of a metal-binding site,introduction of an unnatural amino acid,and incorporation of a metal complex or cluster into native or de novo protein scaffolds.Despite these achievements,it is still of interest to develop other efficient methods for the design of functional metalloproteins.As a common PTM of Cys residues,a disulfide bond plays a crucial role in the stabilization of the tertiary structure of proteins.Moreover,the oxidation state of a disulfide/Cys has been shown to regulate both ligand binding and protein reactivity.we herein rationally introduced a pair of cysteines(V21C/V66C)into the scaffold of myoglobin(Mb),mimicking those in native Cytoglobin(Cgb).A de novo designed intramolecular disulfide bond in myoglobin,was confirmed by an X-ray structure for the first time and was demonstrated to regulate both the structure and function of this protein,which fulfills the design of an artificial dehaloperoxidase,with an activity exceeding that of a native enzyme.The design of functional metalloenzymes is attractive but few designs can exceed the catalytic rate of native enzymes.In order to create an effective artificial dehaloperoxidase(DHP),we redesigned the heme center of myoglobin(Mb)by introducing a distal Tyr43 and replacing the distal His64 with an Asp,which combines the structural features of chloroperoxidase(a distal Asp)with DHP(a distal Tyr).The rationally designed F43Y/H64 D Mb was shown to exhibit a remarkable dehalogenation activity,with a catalytic efficiency more than one thousand folds higher than that of a native DHP from A.ornata.To the best of our knowledge,this is the highest activity reported to date for an artificial DHP.Moreover,X-ray structures of F43Y/H64 D Mb and in complex with a typical substrate,2,4,6-trichlorophenol(TCP),revealed a distal substrate binding site and crucial roles of both distal Tyr43 and Asp64.This study not only provides insights into the structure and function relationship of native and artificial DHPs,but also suggests potential applications in bioremediation of toxic halophenols.