IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
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, MAT&2377AS AA; CATALANO DUPUY, DANIELA L; BOTTI, H; BUSCHIAZZO, A; CECCARELLI, EDUARDO A
Lugar:
Salta, Argentina
Reunión:
Conferencia; 3st Latin American Protein Society Meeting; 2010
Institución organizadora:
Latin American Protein 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 carboxylterminal 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 â-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.