IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
artículos
Título:
Swapping FAD Binding Motifs Between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases.
Autor/es:
MATIAS A. MUSUMECI; HORACIO BOTTI; ALEJANDRO BUSCHIAZZO; EDUARDO A. CECCARELLI
Revista:
BIOCHEMISTRY
Editorial:
AMER CHEMICAL SOC
Referencias:
Lugar: Washington D.C.; Año: 2010
ISSN:
0006-2960
Resumen:
Plant-type ferredoxin-NADP(H) reductases (FNRs) group in two classes, plastidic with
an extended FAD conformation and high catalytic rates, and bacterial with a folded flavin
nucleotide and low turnover rates. The beta-hairpin between amino acids 112 and 124 from
pea FNR and the carboxy-terminal tryptophan of the Escherichia coli ferredoxin-
NADP(H) reductase (FPR), suggested as responsible for the FAD differential
conformation, were mutually exchanged. Pea FNR lacking the beta-hairpin was unable to
fold properly. The inclusion of an extra tryptophan residue at the carboxy terminus,
emulating the bacterial FPR, recovered the protein folding, decreased the affinity for
FAD and reduced the diaphorase and ferredoxin-dependent cytochrome c reductase
activities to 67% and 44% respectively. The sole inclusion of a carboxy-terminal
tryptophan in pea FNR reduced the cytochrome c reductase activity without altering the
FAD binding affinity. The insertion of the beta-hairpin into the corresponding position in
FPR resulted in increased FAD affinity. Finally, a chimeric FPR in which the beta-hairpin
was inserted and the carboxy-terminal tryptophan was simultaneously deleted showed a
2.6 and 8.6-fold increase in kcat and catalytic efficiency for the NADPH diaphorase
activity respectively. This variant retained 10% of the cytochrome c reductase activity
with ferredoxina and was unable to use flavodoxin as substrate. Crystallographic
structures of the chimeras showed no significant changes in their overall structure,
although alterations in the FAD conformations were observed. This report highlights the
role of the FAD moiety conformation and the structural determinants involved in
stabilizing it, ultimately modulating the functional output of FNRs.