title: Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.
abstract: Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack.
authors.affiliation: Interdisciplinary Program of Bioengineering, School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, Korea; Korea Research Institute of Chemical Technology, Daejeon, 305-600, Korea.
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Kim
authors.forename: Jeong Chan
authors.initials: JC
authors.lastname: Joo
authors.forename: Bong Keun
authors.initials: BK
authors.lastname: Song
authors.forename: Young Je
authors.initials: YJ
authors.lastname: Yoo
authors.forename: Yong Hwan
authors.initials: YH
authors.lastname: Kim
mesh_terms: D019943:Amino Acid Substitution; D002942:Circular Dichroism; D004795:Enzyme Stability; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase
keywords: horseradish peroxidase; mass spectrometry; radical coupling; radical stability; site-directed mutagenesis