title: Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities.
abstract: Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70°C for 10min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70°C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions.
authors.affiliation: Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Piriya
authors.initials: P
authors.lastname: Luangwattananun
authors.affiliation: Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Warawan
authors.initials: W
authors.lastname: Eiamphungporn
authors.affiliation: Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Napat
authors.initials: N
authors.lastname: Songtawee
authors.affiliation: Pure and Applied Biochemistry, Department of Chemistry, Lund University, Lund SE-221 00, Sweden.
authors.forename: Leif
authors.initials: L
authors.lastname: Bülow
authors.affiliation: Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Chartchalerm
authors.initials: C
authors.lastname: Isarankura Na Ayudhya
authors.affiliation: Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Virapong
authors.initials: V
authors.lastname: Prachayasittikul
authors.affiliation: Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Electronic address: sakda.yai@mahidol.ac.th.
authors.forename: Sakda
authors.initials: S
authors.lastname: Yainoy
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology*; D002374:Catalase / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D002465:Cell Movement / Q000187:drug effects; D006207:Half-Life; D051379:Mice; D008970:Molecular Weight; D018384:Oxidative Stress / Q000187:drug effects; D010269:Paraquat / Q000009:adverse effects; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000235:genetics*
chemical_list: D000975:Antioxidants; D011994:Recombinant Proteins; D002374:Catalase; D013482:Superoxide Dismutase; D010269:Paraquat
keywords: Catalase; CuZnSOD; HIV-1 TAT; Protein engineering