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

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

San Miguel de Tucumán, Tucumán

Congreso; XLV Reunión Anual de la Sociedad Argentina de investigación en Bioquímica y Biología Molecular (SAIB); 2009

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

The goal of this work was to search for the structural characteristicthat endows high catalytic efficiency to the plastidic ferredoxin-NADP+ reductases (FNRs). This features may be absents in the lowcatalytic efficiency bacterial FNRs (i.e. E. coli). The conformationadopted for the FAD prosthetic group (extended in plastidic andcontracted in bacterial FNRs) and the movement of C-terminaltyrosine both may be responsible for the high catalytic efficiency.Thus, we engineered FNR chimeras of pea FNR containingstructural features of the bacterial enzymes, and mutants E. coliFNRs that emulate the plastidic ones, in a effort to exchange thekinetic behavior between both FNRs. Surprisingly, only smallvariations of the kinetic parameters were observe for all chimericenzymes. Our results indicated that the analyzed structural featuresare involved in enzyme stability, FAD and NADP+ affinity and,related to the discrimination between NAD+ and natural substrateNADP+. Ab initio calculations and crystallographic studies suggestthat the strong interaction between FAD and a C-terminal tyrosine inE. coli FNR may limit its catalytic efficiency. Moreover, we foundthat E. coli FNR tightly bound NADP+ probably contributing to theenzyme stability and decreasing the catalytic efficiency of thebacterial enzyme.