Searching of the source for the high catalytic efficiency of plastidic ferredoxin-NADP+ reductases by swapping ofstructural traits between bacterial and plant Ferredoxin NADP(H) Reductases

MATÍAS A. MUSUMECI; HORACIO BOTTI; ALEJANDRO BUSCHIAZZO; EDUARDO A. CECCARELLI

Salta

Congreso; 3rd Latin American Protein Society Meeting; 2010

Latin American Protein Society

Plant type ferredoxin-NADP(H) reductases are classified into a plastidic class, having an extended FAD conformation and high catalytic rates and a bacterial class whose members display a folded FAD conformation and low turnover rates. The β-hairpin between amino acids 112 to 124 from pea FNR and the carboxyl-terminal tryptophan of the bacterial enzyme (FPR) responsible for the FAD conformation have been exchanged. Pea FNR lacking the β-hairpin was unable to fold properly. Inclusion of a tryptophan residue at the carboxyl-terminal position emulating the bacterial FPR recovered the protein folding, but displayed less stability and less affinity for FAD, showing a decrease in the catalytic activities with respect to the wild-type enzyme. The reverse exchange, constructed by inserting the β-hairpin into the appropriate location in Escherichia coli FPR, displayed an increase FAD affinity and protein stability and, a minor reduction in catalytic activities. A FPR chimera in which the β-hairpin was inserted and the carboxyl terminal tryptophan was deleted showed a 2.6 times increase of the NADPH diaphorase activity, only 10% of cytochrome c reductase activity with ferredoxin and undetectable activity with flavodoxin. A similar kinetic behavior was observed for the mutant FPR in which only the tryptophan was deleted. This variant showed lower stability than the other enzyme variants. Crystallographic structures of the chimeras showed no significant changes to their overall structure. The obtained data highlight the important roles of the FAD conformation and the structures involved in obtaining it on the function of ferredoxin-NADP+ reductases.