Structural interconversion between Plant and Bacterial Ferredoxin-NADP(H) Reductases

MUSUMECI, M. M.; CECCARELLI, E. A.

Mar del Plata, Argentina.

Congreso; XLIII Reunión Anual de la Sociedad Argentina de investigación en Bioquímica y Biología Molecular (SAIB); 2007

Sociedad Argentina de investigación en Bioquímica y Biología Molecular

FNRs are flavoenzymes that participate in a broad range of redox metabolic pathways. An aromatic residue (Y308 in pea FNR) occupies the NADP(H) binding site; which has to be displaced for the substrate to interact with the active site. In bacterial FNRs, theY residue is followed by aWwhich may restricts theYdisplacement. Moreover, in plastidic FNRs, a loop maintains the FAD in an extended conformation. The absence of this loop in bacterial FNRs, combined with the lack of movement of the Y residue, could be responsible for the low catalytic efficiency of this type of reductases. In order to shed light into this mechanism, we engineered a plastidic FNR variant. By means of site-directed mutagenesis we deleted the loop and/or added aWresidue next to Y308 in pea FNR. Interestingly, the deletion of the loop did not produce a soluble FNR. Nevertheless, the addition of aWresidue completely suppressed this effect. This mutant FNR was purified, characterized and their kinetic parameters determined. Surprisingly, these parameters did not differ significantly from those of the wild type pea FNR. Taken together, these observations led us to conclude that the modifications introduced in pea FNR may not be essential for catalytic efficiency and that the terminal aromatic amino acid in bacterial FNRs are mainly involved in the structural integrity of the enzyme.