INTERACTION OF Leptospira interrogans FERREDOXIN-NADP(H) REDUCTASE WITH FERREDOXIN

ROSSI, M.A.; LOPEZ-RIVERO, A; CECCARELLI, E. A.; CATALANO DUPUY, D. L.

Rosario

Congreso; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Leptospira interrogans is a parasitic bacterium that infects humans and causes leptospirosis. L. interrogans contains a plastidic-type ferredoxin-NADP+ reductase (LepFNR) similar to the enzymes present in plant plastids and different from the bacterial-type FNRs. A unique feature found in LepFNR structure is a loop that ranges from I73 to R94. By structural modeling we noticed that this structure may interfere with the ferredoxin (Fd) binding. Also, a conserved lysine essential for productive binding of Fd to plant FNRs, corresponds to a threonine residue (T92) in LepFNR. This allow us to suggest that the binding of Fd LB107, identified as a substrate of LepFNR should take place by a different arrangement from the observed in plant enzymes. We designed and constructed different site-directed mutants of LepFNR altering the subdomain I73-R94. We succeeded in expressing and purifying the mutants proteins properly folded and with the FAD bound. We measured the diaphorase activity catalyzed by the LepFNR variants. We found that the substitutions did not lead to mayor changes in this activity and in the NADP(H) binding. We were also able to measure Fd-dependant cytochrome c reductase activity with the mutants. Our results indicate that the LepFNR subdomain I73-R94 participates in Fd binding and intervenes in substrate specificity.