Structural determinants for the high catalytic efficiency of plastidic Ferredoxin-NADP+ reductases

MUSUMECI M. A.; BOTTI H.; BUSCHIAZZO A.; CECCARELLI E.A.

Tucumán, Argentina.

Congreso; SAIB 45th Annual Meeting; 2009

Sociedad Argentina de Investigación Bioquímica y Biología Moleculat (SAIB)

The goal of this work was to search for the structural characteristic that endows high catalytic efficiency to the plastidic ferredoxin-NADP+ reductases (FNRs). This features may be absents in the low catalytic efficiency bacterial FNRs (i.e. E. coli). The conformation adopted for the FAD prosthetic group (extended in plastidic and contracted in bacterial FNRs) and the movement of C-terminal tyrosine both may be responsible for the high catalytic efficiency. Thus, we engineered FNR chimeras of pea FNR containing structural features of the bacterial enzymes, and mutants E. coli FNRs that emulate the plastidic ones, in a effort to exchange the kinetic behavior between both FNRs. Surprisingly, only small variations of the kinetic parameters were observe for all chimeric enzymes. Our results indicated that the analyzed structural features are involved in enzyme stability, FAD and NADP+ affinity and, related to the discrimination between NAD+ and natural substrate NADP+. Ab initio calculations and crystallographic studies suggest that the strong interaction between FAD and a C-terminal tyrosine in E. coli FNR may limit its catalytic efficiency. Moreover, we found that E. coli FNR tightly bound NADP+ probably contributing to the enzyme stability and decreasing the catalytic efficiency of the bacterial enzyme.