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 (Tyr308 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, thek 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 thek 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.

[1]  P. Karplus,et al.  A productive NADP+ binding mode of ferredoxin–NADP + reductase revealed by protein engineering and crystallographic studies , 1999, Nature Structural Biology.

[2]  P. Karplus,et al.  Probing the Function of the Invariant Glutamyl Residue 312 in Spinach Ferredoxin-NADP+ Reductase* , 1998, The Journal of Biological Chemistry.

[3]  C. Croy,et al.  Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase. , 1998, Archives of biochemistry and biophysics.

[4]  A. Aubry,et al.  A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+. , 1997, Journal of molecular biology.

[5]  H. Eklund,et al.  The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 A resolution. , 1997, Journal of molecular biology.

[6]  A. Arakaki,et al.  Plant‐type ferredoxin‐NADP+ reductases: a basal structural framework and a multiplicity of functions , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[7]  J. Fontecilla-Camps,et al.  X-ray structure of the ferredoxin:NADP+ reductase from the cyanobacterium Anabaena PCC 7119 at 1.8 A resolution, and crystallographic studies of NADP+ binding at 2.25 A resolution. , 1996, Journal of molecular biology.

[8]  A. Dean,et al.  A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A M Lesk,et al.  NAD-binding domains of dehydrogenases. , 1995, Current opinion in structural biology.

[10]  E. Ceccarelli,et al.  Contribution of the FAD binding site residue tyrosine 308 to the stability of pea ferredoxin-NADP+ oxidoreductase. , 1995, Biochemistry.

[11]  P. Karplus,et al.  Involvement of serine 96 in the catalytic mechanism of ferredoxin-NADP+ reductase: structure--function relationship as studied by site-directed mutagenesis and X-ray crystallography. , 1995, Biochemistry.

[12]  P. Karplus,et al.  Refined crystal structure of spinach ferredoxin reductase at 1.7 A resolution: oxidized, reduced and 2'-phospho-5'-AMP bound states. , 1995, Journal of molecular biology.

[13]  Yang Wang,et al.  Structural organization of the human neuronal nitric oxide synthase gene (NOS1). , 1994, The Journal of biological chemistry.

[14]  G. Schneider,et al.  Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5 A resolution: relationship to other flavoprotein reductases. , 1994, Structure.

[15]  G. Schulz,et al.  Anatomy of an engineered NAD‐binding site , 1994, Protein science : a publication of the Protein Society.

[16]  P. Karplus,et al.  Structure-function relations for ferredoxin reductase , 1994, Journal of bioenergetics and biomembranes.

[17]  P. Karplus,et al.  Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin , 1993, Protein science : a publication of the Protein Society.

[18]  D. Sem,et al.  Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH. , 1993, Biochemistry.

[19]  E. Ceccarelli,et al.  Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis. , 1993, The Journal of biological chemistry.

[20]  R. Herrmann,et al.  The role of cysteine residues of spinach ferredoxin-NADP+ reductase As assessed by site-directed mutagenesis. , 1993, Biochemistry.

[21]  N S Scrutton,et al.  Creation of an NADP-dependent pyruvate dehydrogenase multienzyme complex by protein engineering. , 1993, Biochemistry.

[22]  M. Nishiyama,et al.  Alteration of coenzyme specificity of malate dehydrogenase from Thermus flavus by site-directed mutagenesis. , 1993, The Journal of biological chemistry.

[23]  B. Plapp,et al.  An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme. , 1991, Biochemistry.

[24]  P. Karplus,et al.  Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. , 1991, Science.

[25]  R. Feeney,et al.  A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme. , 1990, Biochemical and biophysical research communications.

[26]  Nigel S. Scrutton,et al.  Redesign of the coenzyme specificity of a dehydrogenase by protein engineering , 1990, Nature.

[27]  T. Lee,et al.  Structural and functional analysis of NADPH-cytochrome P-450 reductase from human liver: complete sequence of human enzyme and NADPH-binding sites. , 1989, Biochemistry.

[28]  D. Rueger,et al.  Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. , 1989, The Journal of biological chemistry.

[29]  Y. Sakaki,et al.  The organization and the complete nucleotide sequence of the human NADH-cytochrome b5 reductase gene. , 1989, Gene.

[30]  H. Kamin,et al.  Association of ferredoxin-NADP+ reductase with NADP(H) specificity and oxidation-reduction properties. , 1986, The Journal of biological chemistry.

[31]  D. Arnon,et al.  ENZYMIC MECHANISMS OF PYRIDINE NUCLEOTIDE REDUCTION IN CHLOROPLASTS. , 1965, The Journal of biological chemistry.

[32]  A. R. Fresht Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding , 1999 .

[33]  G. Salmond,et al.  The methane monooxygenase gene cluster of Methylococcus capsulatus (Bath). , 1990, Gene.