Modulation of the enzyme catalytic efficiency by amino acid volume.

MUSUMECI, MATÍAS; RIAL, DANIELA V.; CATALANO DUPUY, DANIELA L.; ARAKAKI, ADRIÁN K.; CECCARELLI, EDUARDO A.

Pinamar, Buenos Aires, ARGENTINA

Congreso; XLI Reunión Anual de SAIB; 2005

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that participate in a broad range of redox metabolic pathways in a wide variety of organisms. Several features in the enzymology of these enzymes remain yet to be explained in relation to their structural features. In that context, chloroplast ferredoxin-NADP(H) reductase has a 32,000- fold preference for NADPH over NADH, displaying turnover numbers that are 20- to 100-fold higher than those reductases 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. The residue Y308 in pea FNR is stacked near parallel to the re-face of the flavin and highly conserved among members of the family. Computing the relative free energy for the lumiflavin-phenol pair with the relative positions found for Tyr308 in pea FNR we have concluded that this amino acid is constrained against the isoalloxazine. This effect is probably performed 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. Simple and double FNR mutants of amino acids C266 and L268 were obtained and characterized. Our results allows to suggest that these amino acids have been evolutively selected by volume and that they participate in the fine tuning of the enzyme efficiency, modulating the interaction of the Tyr308 with the isoalloxazine.