Researching Amino Acids In The Heme Distal Pocket Impact On Peroxidase Activity Of Myoglobin

Protein design,especially for functional enzyme design,has made tremendous progress during the past decade.In addition to shedding light on the structure and function relationship of natural enzymes,protein design enables us to create artificial enzymes for potential applications.Meanwhile,designer enzymes with activities comparable to those of natural enzymes are still limited.It is challenging to achieve a catalytic efficiency exceeding those of native enzymes,which requires not only a full understanding of the structure and function relationship of natural enzymes but also the development of new strategies for enzyme design.Functional enzyme design has made tremendous progress,but designer enzymes with activities comparable to those of natural enzymes are still limited.In this study,we rationally engineered a functional peroxidase with a catalytic binding site of guaiacol in a model protein,myoglobin(Mb),by replacing Phe46 with a serine(F46S mutation),together with a distal Tyr in the heme pocket(F43Y mutation).The double mutant F43Y/F46 S Mb exhibited an overall catalytic efficiency that exceeds most natural peroxidases and is similar to the most efficient horseradish peroxidase.The catalytic substrate binding site was further confirmed by X-ray crystallography,EPR spectroscopy,and inhibition studies,as well as molecular docking simulations.Remarkably,this study reports an artificial peroxidase with an engineered catalytic binding site whose structure was solved both in the absence and presence of the substrate.Approaches to degradation of industrial dyes are desirable,of which bioremediation is more favorable.In addition to the use of native enzymes,rational design of artificial enzymes provides an alternative approach.Meanwhile,few designs can achieve a catalytic activity comparable to that of native enzymes.We have previously designed two generations of artificial dye-decolorizing peroxidases(DyPs)in myoglobin(Mb)by introduction of Tyr43 and Trp138 in the heme pocket;however,the activity is moderate.To improve the activity of the artificial DyP,we herein designed a third generation by introduction of an additional Trp(P88W)to the protein surface,named F43Y/F138W/P88 W Mb.The third generation of artificial DyP was shown to exhibit a catalytic efficiency exceeding that of various native DyPs and comparable to that of the most efficient native DyPs.Titration of reactive blue 19(RB19)and molecular docking studies revealed crucial roles of Trp88 in substrate binding and oxidation,which acts as a catalytic site.This study not only provides clues for heme protein design but also suggests that the artificial DyP has potential applications for bioremediation in the future.