Application Of Neuroglobin-based Peroxidases In The Synthesis Of Indigo And Its Derivatives

Protein engineering provides a powerful method for designing functional enzymes,such as heme enzymes,by modifying the heme active site,introducing non-natural amino acids into intrinsic or novel protein scaffolds,and replacing heme with non-natural metal cofactors.For example,one of the member of the globin family,neuroglobin（Ngb）,has been converted into a highly efficient nitrite reductase and multifunctional peroxidase by rational modification of the hemoglobin active site.The old dye indigo is still demanded worldwide,whereas chemical synthesis of indigo may cause environmental problems.lternatively,the biosynthesis of indigo is an eco-friendly approach.In this study,by protein design and engineering,we showed that human neuroglobin（Ngb）can be converted into an enzyme capable of efficient synthesis of indigo from indole.Three mutations were rationally designed to stabilize the protein（A15C）,enhance H₂O₂ activation（H64D）,and facilitate electron transfer（F49Y）,respectively.By examining the X-ray crystal structure of human Ngb,we noticed that Phe49 is a suitable mutation target,which is located near the heme active site and close to the protein surface.Therefore,we constructed a triplet mutant of A15C/H64D/F49Y Ngb by replacing Phe49 with Tyr49,which has a hydroxyl difference and causes minimal structural perturbation.Sidechains of amino acids,such as Tyr,can also be used to adjust the binding of indole to the active site of hemoglobin through hydrogen bonding and hydrophobic interactions.Kinetic studies and product analysis revealed that the triple mutant A15C/H64D/F49Y Ngb exhibits an activity much higher than those of the engineered myoglobin mutants,without the need of expensive cofactors required for cytochromes P450.This enzyme can also efficiently catalyze the coupling oxidation of indole derivatives containing–Cl/–Br/–NO₂ groups,producing indigo derivative dyes,with a yield up to～90%and chemoselectivity up to～97%,respectively.Moreover,these synthesized dyes were successfully applied to dye cotton textiles with uniform colors such as blue,red,yellow,purple,and even green.Thus,we expect that the engineered enzyme of Ngb will have practical applications in the textile dyeing industry.