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 redoxmetabolic pathways. An aromatic residue (Y308 in pea FNR)occupies the NADP(H) binding site; which has to be displaced forthe substrate to interact with the active site. In bacterial FNRs, theYresidue is followed by aWwhich may restricts theYdisplacement.Moreover, in plastidic FNRs, a loop maintains the FAD in anextended conformation. The absence of this loop in bacterial FNRs,combined with the lack of movement of the Y residue, could beresponsible for the low catalytic efficiency of this type ofreductases. In order to shed light into this mechanism, weengineered a plastidic FNR variant. By means of site-directedmutagenesis we deleted the loop and/or added aWresidue next toY308 in pea FNR. Interestingly, the deletion of the loop did notproduce a soluble FNR. Nevertheless, the addition of aWresiduecompletely suppressed this effect. This mutant FNR was purified,characterized and their kinetic parameters determined.Surprisingly, these parameters did not differ significantly fromthose of the wild type pea FNR. Taken together, these observationsled us to conclude that the modifications introduced in pea FNRmay not be essential for catalytic efficiency and that the terminalaromatic amino acid in bacterial FNRs are mainly involved in thestructural integrity of the enzyme.