title: Development of the radical-stable Coprinus cinereus peroxidase (CiP) by blocking the radical attack.
abstract: Despite the potential use of peroxidases as industrial biocatalysts, their practical application is often impeded due to suicide inactivation by radicals generated in oxidative reactions. Using a peroxidase from Coprinus cinereus (CiP) as a model enzyme, we revealed a dominant factor for peroxidase inactivation during phenol oxidation, and we engineered radical-stable mutants by site-directed mutagenesis of an amino acid residue susceptible to modification by phenoxyl radical. Mass spectrometry analysis of inactivated CiP identified an adduct between F230 and a phenoxyl radical, and subsequently, the F230 residue was mutated to amino acids that resisted radical coupling. Of the F230 mutants, the F230A mutant showed the highest stability against radical inactivation, retaining 80% of its initial activity, while the wild-type protein was almost completely inactivated. The F230A mutant also exhibited a 16-fold higher turnover of the phenol substrate compared with the wild-type enzyme. Furthermore, the F230A mutant was stable during the oxidation of other phenolic compounds, including m-cresol and 3-methoxyphenol. No structural changes were observed by UV-vis and CD spectra of CiP after radical coupling, implying that the F230-phenol radical adduct inactivated CiP by blocking substrate access to the active site. Our novel strategy can be used to improve the stability of other peroxidases inactivated by radicals.
authors.affiliation: Interdisciplinary Program of Bioengineering, School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, South Korea; Korea Research Institute of Chemical Technology, Daejeon 305-600, South Korea.
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, South Korea; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3ES, Canada.
authors.forename: Jeong Chan
authors.initials: JC
authors.lastname: Joo
authors.affiliation: Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, South Korea.
authors.forename: Han Sang
authors.initials: HS
authors.lastname: Kim
authors.affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 500-712, South Korea.
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
authors.affiliation: Korea Research Institute of Chemical Technology, Daejeon 305-600, South Korea.
authors.forename: Bong Keun
authors.initials: BK
authors.lastname: Song
authors.affiliation: Interdisciplinary Program of Bioengineering, School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, South Korea. Electronic address: yjyoo@snu.ac.kr.
authors.forename: Young Je
authors.initials: YJ
authors.lastname: Yoo
authors.affiliation: Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, South Korea. Electronic address: metalkim@kw.ac.kr.
authors.forename: Yong Hwan
authors.initials: YH
authors.lastname: Kim
mesh_terms: D003302:Coprinus / Q000378:metabolism*; D005656:Fungal Proteins; D013058:Mass Spectrometry; D009195:Peroxidase / Q000378:metabolism*; D015202:Protein Engineering
chemical_list: D005656:Fungal Proteins; D009195:Peroxidase
keywords: Mass spectrometry; Peroxidase inactivation; Protein engineering; Radical coupling; Radical-stable peroxidase