Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase.
Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2'-phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.
Piubelli
L
L
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
Aliverti
A
A
Arakaki
A K
AK
Carrillo
N
N
Ceccarelli
E A
EA
Karplus
P A
PA
Zanetti
G
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D001667:Binding, Competitive; D002736:Chloroplasts / Q000201:enzymology*; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D009536:Niacinamide; D018532:Pisum sativum / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014443:Tyrosine
D011994:Recombinant Proteins; D009243:NAD; D009536:Niacinamide; D014443:Tyrosine; D009249:NADP; D005287:Ferredoxin-NADP Reductase