Swapping structural-functional determinants between bacterial and plant Ferredoxin NADP+ Reductases

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

Villa Carlos Paz - Córdoba - Argentina

Congreso; SAIB 44th Annual Meeting; 2008

Argentine Society for Biochemistry and Molecular Biology Research

FNRs are flavoenzymes that participe in a broad range of redox metabolic pathways. It has been hypothesized that Y308 (in pea FNR) must be displaced for the substrate to interact with the active site. In some bacterial FNRs (i.e. Escherichia coli), a W follows the Y residue, probably restricting its displacement. Moreover, in plastidic FNRs, a loop maintains the FAD in an extended conformation. The absence of this loop in bacterial FNR, and the lack of movement of the Y residue, have been pointed to be responsible for the low catalytic efficiency of this type of reductases. In order to shed light into this mechanism, we engineered a set of plastidic and bacterial FNRs in sakes of exchanging structural characteristics among them. By means of site-directed mutagenesis we deleted the loop and/or added a W residue next to Y308. Surprinsingly, the kinetic behavior did not differ significantly from the wild type enzime. In E. coli FNR we included the loop and deleted the terminal W obtaining a mutant enzyme which showed similar kinetics parameters to those of pea FNR. The measurements of FAD Kd revealed a decrease in FAD affinity in the FNRs that lacked the loop. Taken together, these observations leaded us to conclude that the loop in pea FNR may not be essential for high catalytic efficiency and that it is involved in FAD affinity improving the structural stability of the enzyme.