Conversion of Human Neuroglobin into a Multifunctional Peroxidase by Rational Design

Abstract

Protein design has received much attention in the last decades. With an additional disulfide bond to enhance the protein stability, human A15C neuroglobin (Ngb) is an ideal protein scaffold for heme enzyme design. In this study, we rationally converted A15C Ngb into a multifunctional peroxidase by replacing the heme axial His64 with an Asp residue, where Asp64 and the native Lys67 at the heme distal site were proposed to act as an acid–base catalytic couple for H2O2 activation. Kinetic studies showed that the catalytic efficiency of A15C/H64D Ngb was much higher (∼50–80-fold) than that of native dehaloperoxidase, which even exceeds (∼3-fold) that of the most efficient native horseradish peroxidase. Moreover, the dye-decolorizing peroxidase activity was also comparable to that of some native enzymes. Electron paramagnetic resonance, molecular docking, and isothermal titration calorimetry studies provided valuable information for the substrate–protein interactions. Therefore, this study presents the rational design of an efficient multifunctional peroxidase based on Ngb with potential applications such as in bioremediation for environmental sustainability.

Design and Engineering of an Efficient Peroxidase Using Myoglobin for Dye Decolorization and Lignin Bioconversion

“…Based on the above HPLC and ESI-MS results, we proposed a generation route of the reaction products, which may involve the generation of reactive radical species upon the activation of H 2 O 2 by the heme enzymes [ 13 , 19 , 30 , 46 , 47 ], resulting in various types of bond cleavages. As shown in Figure 9 , for the oxidative cleavage, the monomers can hardly exist alone, and they might be recombined into new species.…”

“…In addition to chemical and physical methods, biotechnological treatments use both native and artificial metalloenzymes such as heme enzymes and others [ 8 , 9 , 10 , 11 , 12 , 13 ]. Comparatively, the biologically catalytic process often occurs under mild reaction conditions.…”

Biotransformation of Lignin by an Artificial Heme Enzyme Designed in Myoglobin With a Covalently Linked Heme Group

“…The molar concentration of Kraft lignin was calculated using an average molecular mass of 10,000 Da. The observed rate constants (k obs ) were calculated by fitting the absorbance at 465 nm versus time to a singleexponential decay function (Chen et al, 2021).…”

Design and engineering of neuroglobin to catalyze the synthesis of indigo and derivatives for textile dyeing

“…[16][17][18] Moreover, the other member of globins, neuroglobin (Ngb), has also been converted into efficient nitrite reductase and multifunctional peroxidase, by rational modification of the heme active site. 19,20 Indigo is an old blue textile dye extracted from plants and is still demanded worldwide, and synthetic indigo is widely used for dyeing cotton and jeans. 21 Meanwhile, the chemical synthesis of indigo requires organic solvents and causes environmental problems such as the release of toxic wastes.…”

“…1A), 41 whereas the replacement of His64 with an aspartate (H64D mutation) converts it into a multifunctional peroxidase, with both dehaloperoxidase and dye-decolorizing peroxidase activities. 20 Human Ngb contains three Cys residues and two of them (Cys46 and Cys55) form an intramolecular disulfide bond. The protein stability can be enhanced dramatically (thermal stability >100 °C) by introducing a Cys at position 15 (A15C mutation) to form a de novo disulfide bond with the third Cys120 (Fig.…”