INVESTIGADORES
CECCARELLI Eduardo Augusto
congresos y reuniones científicas
Título:
Structural Grounds For The Substrate Specificity Of The Ferredoxin NADP(H) Reductase
Autor/es:
PALADINI, S; CARRILLO, N; CECCARELLI, EDUARDO A.
Lugar:
Pinamar, Argentina
Reunión:
Congreso; 10th Congress Panamerican Association for Biochemistry and Molecular Biology; 2005
Institución organizadora:
SAIB - PABMB
Resumen:
Ferredoxin-NADP(H)
reductases (FNR) are ubiquitous flavoenzymes that catalyse the reversible
electron transfer between NADP(H) and obligatory one-electron carriers as
ferredoxin. A highly conserved C‑terminal tyrosine (Y308 in pea) is stacked
near to the flavin in plant type FNRs. This amino acid performs a fundamental
role in nucleotide discrimination and in increasing catalytic efficiency. Y308
is apparently displaced when the nicotinamide ring of NADP(H) is bound. We have
investigated the structural grounds of the FNRNADP(H) interaction and the
involvement of Y308 on it. Differential spectroscopy analysis of the mutant FNR
Y308S and analogues of NADP+ shows an autonomous binding of
2phospho‑AMP or nicotinamide moieties with similar spectroscopy changes. In
addition, nicotinamide mononucleotide reduced (NMNH) oxidase activity is
detected in Y308S but not in wild‑type FNR indicating that Y308 preventing of
NMNH to act as substrate. Moreover, the artificial substrate ferricyanide
inhibits FNR increasing NADPH and NADH Michaelis constants and, the specificity
for NADPH relative to NADH. Our results indicate that binding of any of the two
moieties of the substrate NADP(H) affects the prosthetic environment,
reflecting a conformational change on the enzyme which may tailor the enzyme
for catalysis