A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a mole-cular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys¹¹² between the active Cys⁵¹ and Cys¹⁷¹ residues. In the present study, additional Cys residues (Cys³¹, Cys¹¹², and Cys¹⁹²) were added to PpPrx variants to improve their enzymatic func-tion. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioen-gineering systems and industries, such as to develop or-ganisms that are more resistant to extreme environments.

Keywords: dual function engineering, dual functions, extreme stress, peroxiredoxin, site-directed mutagenesis