Modulation of the enzymatic efficiency of ferredoxin-NADP(H) reductase by the amino acid volume around the catalytic site.
Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in plastids, mitochondria and bacteria. Plastidic FNRs are quite efficient reductases. In contrast, FNRs from organisms possessing a heterotrophic metabolism or anoxygenic photosynthesis display turnover numbers 20- to 100-fold lower than those of their plastidic and cyanobacterial counterparts. Several structural features of these enzymes have yet to be explained. The residue Y308 in pea FNR is stacked nearly parallel to the re-face of the flavin and is highly conserved amongst members of the family. By computing the relative free energy for the lumiflavin-phenol pair at different angles with the relative position found for Y308 in pea FNR, it can be concluded that this amino acid is constrained against the isoalloxazine. This effect is probably caused by amino acids C266 and L268, which face the other side of this tyrosine. Simple and double FNR mutants of these amino acids were obtained and characterized. It was observed that a decrease or increase in the amino acid volume resulted in a decrease in the catalytic efficiency of the enzyme without altering the protein structure. Our results provide experimental evidence that the volume of these amino acids participates in the fine-tuning of the catalytic efficiency of the enzyme.
Musumeci
Matías A
MA
Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Arakaki
Adrián K
AK
Rial
Daniela V
DV
Catalano-Dupuy
Daniela L
DL
Ceccarelli
Eduardo A
EA
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary
D000596:Amino Acids; D005182:Flavin-Adenine Dinucleotide; D005287:Ferredoxin-NADP Reductase