Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2.
The manganese peroxidase produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+), is easily inactivated by the hydrogen peroxide (H2O2) presented in the reaction. We attempted to increase H2O2 resistance by the conformational stabilization around the H2O2-binding pocket. Based on its structural model, engineering of oxidizable Met273 located near the pocket to a non-oxidizable Leu showed a great improvement. Furthermore, after treatment at 1 mM H2O2 where the wild-type is completely inactivated, full activity can be retained by engineering the Asn81, which might have conformational changes due to the environment of the pocket, to a non-bulky and non-oxidizable Ser.
Miyazaki
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C
Toyota Central R&D Labs., 41-1, Yokomichi, Aichi 480-1192, Nagakute, Japan.
Takahashi
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H
D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D020075:Phanerochaete / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D008715:Methionine; D006861:Hydrogen Peroxide; D010544:Peroxidases; C051129:manganese peroxidase; D010100:Oxygen