Swapping structural-functional determinants between bacterial and plant Ferredoxin NADP(H) Reductases

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

Villa Carlos Paz, Córdoba

Congreso; XLIV Reunión Anual de la Sociedad Argentina de investigación en Bioquímica y Biología Molecular (SAIB); 2008

Sociedad Argentina de investigación en Bioquímica y Biología Molecular (SAIB)

FNRs are flavoenzymes that participate in a broad range of redoxmetabolic pathways. It has been hypothesized that Y308 (in peaFNR) must be displaced for the substrate to interact with the activesite. In some bacterial FNRs (i.e. Escherichia coli), a W follows theY residue, probably restricting its displacement. Moreover, inplastidic FNRs, a loop maintains the FAD in an extendedconformation. The absence of this loop in bacterial FNRs and, thelack of movement of the Y residue, have been pointed to beresponsible for the low catalytic efficiency of this type ofreductases. In order to shed light into this mechanism, weengineered a set of plastidic and bacterial FNRs in sakes ofexchanging structural characteristics among them. By means ofsite-directed mutagenesis we deleted the loop and/or added a Wresidue next to Y308. Surprisingly, the kinetic behavior did notdiffer significantly from the wild type enzyme. In E. coli FNR weincluded the loop and deleted the terminal W obtaining a mutantenzyme which showed similar kinetics parameters to those of peaFNR. The measurements of FAD Kd revealed a decrease in FADaffinity in the FNRs that lacked the loop. Taken together, theseobservations leaded us to conclude that the loop in pea FNR maynot be essential for high catalytic efficiency and that it is involved inFAD affinity improving the structural stability of the enzyme.