Tolerance of Escherichia coli cells transformed with chloroplast 2-Cys peroxiredoxin to oxidative stress

ARÁN, MARTÍN; SENN, ALEJANDRO M.; CAPORALETTI, D. AND WOLOSIUK, RICARDO A.

San Carlos de Bariloche, Argentina.

Simposio; XXXIX SAIB ANNUAL MEETING; 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

2-Cys peroxiredoxin (2-Cys Prx) is a ubiquitous family of peroxidases that are devoid of the prosthetic heme group. Two highly conserved cysteines play an important role in modulating the concentration of hydrogen peroxide, thereby control signal transduction and impart tolerance to oxidative stress. To characterize the contribution of 2-Cys Prx to chloroplast metabolism, we cloned and expressed the mature form from rapeseed leaves in pET 22b(+) and subsequently studied the functional characteristics. Next, we analyzed, as a functional assay for undertaking studies of directed evolution, whether this recombinant protein bestows the capacity to tolerate oxidative stress in heterologous systems. Therefore, E.coli cells were transformed with the abovementioned plasmid and subsequently grew in media containing a variety of oxidants. Relative to cells transformed with the vector bearing chloroplast fructose-1,6- bisphosphatase (control), counterparts carrying the plasmid that codes for 2-Cys Prx tolerated higher concentrations of hydroperoxides as well as chemical oxidants. At variance, both strains elicited similar response to chemicals that deprive the intracellular concentrations of thiols, diamide and diethylmaleate. These results not only suggested that chloroplast 2-Cys Prx functions in the defense system against oxidation in E.coli cells, likely as electron donor to hydrogen peroxide, but also provided a protocol to estimate the functional capacity of chloroplast 2-Cys Prx