title: Site-directed mutagenesis of manganese peroxidase from Phanerochaete chrysosporium in an in vitro expression system.
abstract: An enzymatically active fungal manganese peroxidase (MnP) from Phanerochaete chrysosporium was synthesized in an in vitro coupled transcription-translation system. The synthesized MnP had the expected molecular mass (43,000Da) and catalyzed the oxidation of 2,6-dimethoxyphenol (DMP) in the presence of hydrogen peroxide and Mn(II). A distal arginine residue (Arg 42) of the peroxide binding pocket and the potential N-glycosylation site (Asn 131) was site-directed mutagenized and corresponding mutant enzymes were also in vitro synthesized. Activities of the mutant enzymes towards 2,6-DMP were not significantly compromised although their dynamic characteristics were obviously different from the wild-type enzyme. The effect of the mutations was explained by using a computer-based three-dimensional modeling. These results demonstrated that in vitro expression of MnP provided a convenient and efficient system for characterization of fungal peroxidases.
authors.affiliation: The State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, Shandong, PR China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Zhang
authors.forename: Yuxiao
authors.initials: Y
authors.lastname: Wang
authors.forename: Lushan
authors.initials: L
authors.lastname: Wang
authors.forename: Guanjun
authors.initials: G
authors.lastname: Chen
authors.forename: Weifeng
authors.initials: W
authors.lastname: Liu
authors.forename: Peiji
authors.initials: P
authors.lastname: Gao
mesh_terms: D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003198:Computer Simulation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics; D011748:Pyrogallol / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011748:Pyrogallol; C010120:pyrogallol 1,3-dimethyl ether; D010544:Peroxidases; C051129:manganese peroxidase