INVESTIGADORES
CECCARELLI Eduardo Augusto
congresos y reuniones científicas
Título:
Searching for the structural determinants of the substrate specificity and catalytic efficiency of the ferredoxin-NADP(H) reductase
Autor/es:
CECCARELLI, EDUARDO A.; ARAKAKI, A; CATALANO DUPUY, D; RIAL, D; MUSUMECI, MATIAS M.; PALADINI, S
Lugar:
Río de Janeiro
Reunión:
Congreso; First Latin-American Protein Society 8 - 12 de noviembre de 2004 Meeting, Angra dos Reis; 2004
Institución organizadora:
Latinamerican Protein Society
Resumen:
Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are
ubiquitous flavoenzymes that participate in a wide range of redox metabolic
pathways in an ample variety of organisms. Several structural and functional
aspects of these enzymes remain yet to be explained. In that context,
chloroplast FNR has a 32,000- fold preference for NADP(H) over NAD(H),
displaying turnover numbers that are 20- to 100-fold higher than FNRs from
bacteria, albeit they share extensive structural and conformational identity.
FNRs consist of two domains; one involved in the binding of the prosthetic
group FAD and the other responsible for binding of NADP+. Noteworthy, two tyrosine
residues lie close to each side of the isoalloxazine. The highly conserved Y308
in pea FNR is stacked near parallel to the re-face of the avin and should be
displaced by the nicotinamide ring of NADP(H) for productive binding to the
enzyme.
The other side of the flavin is facing Y89, which
participates in a network of interactions with other amino acids and with the
FAD itself. We have investigated the function of these two tyrosines performing
ab initio molecular orbital calculations and site directed
mutagenesis. Our results indicate that the position of Y89 is mainly governed by
the energetic minimum of the pairwise interaction between the phenol ring and
the flavin, displaying geometries that correspond to the more negative free
energy theoretical value. By the contrary, the Y308 is constrained against the
isoalloxazine by amino acids C266 and L268, which are facing the other side of
this tyrosine, forcing the Y308 to adopt a more planar orientation with respect
to the flavin. A set of aromatic and non aromatic mutants of both amino acids
were obtained. Analyses of these FNR mutants indicate that aromaticity on residue
89 is essential for FAD binding and proper folding of the protein and, that
hydrogen bonding through the Y89 hydroxyl group may be responsible for the
correct positioning of FAD and the substrate NADP+. On the other hand, our
results indicate that the Y308 plays a key role in modulating pyridine
nucleotide discrimination and enhancing the enzyme efficiency. Simple and
double mutants of amino acids C266 and L268 allow us to suggest that these
amino acids participate in the fine-tuning of the enzyme efficiency, modulating
the interaction of the Y308 with the isoaloxazine.