Identification of the N- and C-terminal substrate binding segments of ferredoxin-NADP+ reductase by NMR.
Ferredoxin-NADP(+) reductase (FNR) catalyzes the reduction of NADP(+) through the formation of an electron transfer complex with ferredoxin. To gain insight into the interaction of this enzyme with substrates at both ends of the polypeptide chain, we performed NMR analyses of a 314-residue maize leaf FNR with a nearly complete assignment of the backbone resonances. The chemical shift perturbation upon formation of the complex indicated that a flexible N-terminal region of FNR contributed to the interaction with maize ferredoxin, and an analysis of N-terminally truncated mutants of FNR confirmed the importance of this region for the binding of ferredoxin. Comparison between the spectra of FNR in the NADP(+)- and inhibitor-bound states also revealed that the nicotinamide moiety of NADP(+) was accessible to the C-terminal Tyr314. We propose that the formation of the catalytic competent complex of FNR and substrates is achieved through the interaction of the N- and C-terminal segments with ferredoxin and NADP(+), respectively. Since the ends of the polypeptide chain act as flexible regions of proteins, they may contribute to the search of a larger space for a binding partner and to the opening of active sites.
Maeda
Masahiro
M
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Lee
Young Ho
YH
Ikegami
Takahisa
T
Tamura
Kohsuke
K
Hoshino
Masaru
M
Yamazaki
Toshio
T
Nakayama
Masato
M
Hase
Toshiharu
T
Goto
Yuji
Y
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017384:Sequence Deletion; D013379:Substrate Specificity / Q000235:genetics; D017186:Titrimetry; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005288:Ferredoxins; D010446:Peptide Fragments; D010940:Plant Proteins; C089763:ferredoxin-NADP reductase-binding protein, Zea mays; D009249:NADP; D005287:Ferredoxin-NADP Reductase