INVESTIGADORES
CECCARELLI Eduardo Augusto
congresos y reuniones científicas
Título:
Swapping, adding and deleting proteins motifs to search for the structural backgrounds of the Ferredoxin NADP reductase catalytic efficiency
Autor/es:
MUSUMECI, M. A.; CATALANO DUPUY, D. L.; BOTTI, H.; BUSCHIAZZO, A.; CECCARELLI, E. A.
Lugar:
Salta
Reunión:
Congreso; XXIX Annual Meeting of the Argentinean Biophysical Society; 2010
Institución organizadora:
XXIX Annual Meeting of the Argentinean Biophysical Society
Resumen:
Ferredoxin-NADP(H)
reductase (FNR) is a FAD- containing protein that catalyzes the reversible
transfer of electrons between NADP(H) and ferredoxin or flavodoxin. FNR
participates in redox-based metabolisms of plastids, mitochondria and bacteria.
Some of the unresolved issues on these enzymes are the strong preference that
FNR displays for NADP(H) against NAD(H) and the structural backgrounds that
determine the high catalytic rates of plastidic FNRs. In order to investigate
these topics we have used several protein engineering approaches on FNRs from pea
and Escherichia coli. We have
constructed a chimeric pea FNR containing an artificial metal binding motif in its
carboxyl-terminus. This added structure binds Zn2+ and, as
consequence, the catalytic efficiency of the enzyme is modulated. In addition,
we have obtained several site directed mutants of FNR, some of which introduce changes
in the volume of the amino acids around the catalytic site. We have also mutually
exchanged structural traits involved in FAD binding between FNRs form pea and
bacteria. Several conclusions were obtained from our studies: 1) mobility of the
carboxyl terminal backbone, mainly the carboxyl-terminal tyrosine, is essential
for obtaining highly efficient FNRs; 2) interaction of the carboxyl-terminal
tyrosine and the prosthetic group FAD should be precisely regulated by adjusting
the volume of the amino acids surrounding the catalytic site; 3) in plastidic enzymes, the
b-hairpin 112-123 would
locate an aromatic residue in an optimal position for stacking with the FAD
adenine, stabilizing the extended prosthetic group conformation optimizing the
flavin conformation and the substrate accession to the catalytic site.