title: A peroxiredoxin cDNA from Taiwanofungus camphorata: role of Cys31 in dimerization.
abstract: Peroxiredoxins (Prxs) play important roles in antioxidant defense and redox signaling pathways. A Prx isozyme cDNA (TcPrx2, 745 bp, EF552425) was cloned from Taiwanofungus camphorata and its recombinant protein was overexpressed. The purified protein was shown to exist predominantly as a dimer by sodium dodecyl sulfate-polyacrylamide gel electrolysis in the absence of a reducing agent. The protein in its dimeric form showed no detectable Prx activity. However, the protein showed increased Prx activity with increasing dithiothreitol concentration which correlates with dissociation of the dimer into monomer. The TcPrx2 contains two Cys residues. The Cys(60) located in the conserved active site is the putative active peroxidatic Cys. The role of Cys(31) was investigated by site-directed mutagenesis. The C31S mutant (C(31) → S(31)) exists predominantly as a monomer with noticeable Prx activity. The Prx activity of the mutant was higher than that of the corresponding wild-type protein by nearly twofold at 12 μg/mL. The substrate preference of the mutant was H2O2 > cumene peroxide > t-butyl peroxide. The Michaelis constant (K M) value for H2O2 of the mutant was 0.11 mM. The mutant enzyme was active under a broad pH range from 6 to 10. The results suggest a role of Cys(31) in dimerization of the TcPrx2, a role which, at least in part, may be involved in determining the activity of Prx. The C(31) residue does not function as a resolving Cys and therefore the TcPrx2 must follow the reaction mechanism of 1-Cys Prx. This TcPrx2 represents a new isoform of Prx family.
authors.affiliation: Institute of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, 2 Pei-Ning Rd., Chi-lung, 202, Taiwan.
authors.forename: Chih-Yu
authors.initials: CY
authors.lastname: Huang
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Chen
authors.forename: Lisa
authors.initials: L
authors.lastname: Wen
authors.forename: Dey-Chyi
authors.initials: DC
authors.lastname: Sheu
authors.forename: Chi-Tsai
authors.initials: CT
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D054464:Peroxiredoxins / Q000737:chemistry / Q000235:genetics*; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D005656:Fungal Proteins; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; D003545:Cysteine