Assessment of NADP(H) dependent oxido-reductases of soxRS regulon in Escherichia coli.

GIRÓ MARIANA; RODRIGUEZ RAMIRO; CARRILLO NÉSTOR; KRAPP ADRIANA

Sociedad de Biología de Rosario

Congreso; VI Congreso - XXIV Reunión anual de la sociedad de Biología de Rosario; 2004

sociedad de Biología de Rosario.

The soxRS regulon modulates a global response to oxidative stress imposed by superoxide (.O2-) and nitric oxide. This defensive system has several facets, encoding products with scavengers and repair specific activities, replacing unstable enzymes, etc. Among the identified members of the soxRS response are two NADP(H)  dependent oxidoreductases, Glucose 6 phosphate dehydrogenase (G6PD) and Ferredoxin NADP(H) reductase encoded by zwf and fpr genes, respectively. G6PD, catalyses the first step in the oxidative branch of the pentose phosphate pathway, wich generates NADPH for reductive biosynthesis. FPR mediates the electron transfer from NADPH to ferredoxin or flavodoxins providing low potential carriers for several oxido-reductive pathways. Mutant bacteria in zwf and fpr are susceptible to oxidative stress. In this context G6PD mantains a high ratio of NADPH/NADP+, under oxidative conditions that deplete NADPH. The antioxidant roll  played by FPR is less well understood. One  hypotheses suggests that FPR catalyzes the oxidation of NADPH using different oxidants, taking advantage of the promiscuity on its side acceptor, avoiding that this metabolito  accumulated in high levels promoves the generation of hidroxilo radical by reduction transition metal. To investigate the relative contributions of G6PD and FPR as member of  soxRS regulon we characterised the time course of their induction during a oxidative stress and the overproduction of G6PD in E. coli.   By means of western blot and measures of activity we observed a fast induction of G6PD  during an  oxidative stress with metil viológeno (generator of O2 -). FPR increase  is observed later and surpassed the induction relative to the one of G6PD.  On the other hand, using a gene reporter soxS'lacZ we analyzed the state of activation of the soxS gene in a G6PD overproducing strain. These observations indicate  that in this strain exists smaller activation of soxS, in agree with a greater fraction of NADP in the reduced state.  The obtained results confirm the function suggested for G6PD, a participation of dupla NADPH/NADP+ in the activation of soxRS regulon and support the hypothesis of a FPR roll  in controlling the accumulation of NADPH.