PDBsum entry 1h42

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Oxidoreductase PDB id
Protein chain
295 a.a. *
Waters ×279
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: Ferredoxin:nadp+ reductase mutant with thr 155 replaced by gly, ala 160 replaced by thr and leu 263 replaced by pro (t155g-a160t-l263p)
Structure: Ferredoxin--NADP+ reductase. Chain: a. Synonym: fnr. Engineered: yes. Mutation: yes
Source: Anabaena sp.. Organism_taxid: 1168. Strain: pcc 7119. Expressed in: escherichia coli. Expression_system_taxid: 562
2.15Å     R-factor:   0.200     R-free:   0.231
Authors: J.A.Hermoso,T.Mayoral,M.Medina,J.Sanz-Aparicio, C.Gomez-Moreno
Key ref:
J.Tejero et al. (2003). Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity. J Biol Chem, 278, 49203-49214. PubMed id: 14500716 DOI: 10.1074/jbc.M307934200
26-Sep-02     Release date:   25-Sep-03    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P21890  (FENR_ANASO) -  Ferredoxin--NADP reductase
440 a.a.
295 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 4 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Ferredoxin--NADP(+) reductase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Methionine Synthase
      Reaction: 2 reduced ferredoxin + NADP+ + H+ = 2 oxidized ferredoxin + NADPH
2 × reduced ferredoxin
+ NADP(+)
+ H(+)
= 2 × oxidized ferredoxin
      Cofactor: FAD
Bound ligand (Het Group name = FAD) corresponds exactly
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     oxidation-reduction process   1 term 
  Biochemical function     oxidoreductase activity     1 term  


DOI no: 10.1074/jbc.M307934200 J Biol Chem 278:49203-49214 (2003)
PubMed id: 14500716  
Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity.
J.Tejero, M.Martínez-Julvez, T.Mayoral, A.Luquita, J.Sanz-Aparicio, J.A.Hermoso, J.K.Hurley, G.Tollin, C.Gómez-Moreno, M.Medina.
Previous studies indicated that the determinants of coenzyme specificity in ferredoxin-NADP+ reductase (FNR) from Anabaena are situated in the 2'-phosphate (2'-P) NADP+ binding region, and also suggested that other regions must undergo structural rearrangements of the protein backbone during coenzyme binding. Among the residues involved in such specificity could be those located in regions where interaction with the pyrophosphate group of the coenzyme takes place, namely loops 155-160 and 261-268 in Anabaena FNR. In order to learn more about the coenzyme specificity determinants, and to better define the structural basis of coenzyme binding, mutations in the pyrophosphate and 2'-P binding regions of FNR have been introduced. Modification of the pyrophosphate binding region, involving residues Thr-155, Ala-160, and Leu-263, indicates that this region is involved in determining coenzyme specificity and that selected alterations of these positions produce FNR enzymes that are able to bind NAD+. Thus, our results suggest that slightly different structural rearrangements of the backbone chain in the pyrophosphate binding region might determine FNR specificity for the coenzyme. Combined mutations at the 2'-P binding region, involving residues Ser-223, Arg-224, Arg-233, and Tyr-235, in combination with the residues mentioned above in the pyrophosphate binding region have also been carried out in an attempt to increase the FNR affinity for NAD+/H. However, in most cases the analyzed mutants lost the ability for NADP+/H binding and electron transfer, and no major improvements were observed with regard to the efficiency of the reactions with NAD+/H. Therefore, our results confirm that determinants for coenzyme specificity in FNR are also situated in the pyrophosphate binding region and not only in the 2'-P binding region. Such observations also suggest that other regions of the protein, yet to be identified, might also be involved in this process.
  Selected figure(s)  
Figure 2.
FIG. 2. Spectroscopic characterization of the complexes formed between the FNR[ox] forms and the coenzymes NADP+ or NAD^+. A, difference absorbance spectra elicited by binding of WT FNR (solid thick line) (31 µM) to NADP+ (89 µM), L263A FNR (closed circles) (56 µM) to NADP+ (353 µM), and L263P FNR (open triangles) (54 µM) to NADP+ (700 µM). B, difference absorbance spectra elicited by binding of T155G/A160T FNR (open squares) (36 µM) to NADP+ (418 µM) and T155G/A160T/L263P FNR (closed triangles) (60 µM) to NADP+ (700 µM). C, spectrophotometric titration of selected FNR forms with NADP+. L263A FNR (closed circles) (60 µM), L263P FNR (open triangles) (61 µM), T155G/A160T FNR (open squares) (36 µM), and T155G/A160T/L263P FNR (closed triangles) (69 µM). D, difference absorbance spectra elicited by the binding of L263A FNR (closed circles) (46 µM) to NAD^+ (12.7 mM), L263P FNR (open triangles) (47 µM) to NAD^+ (6.9 mM), T155G/A160T FNR (solid thick line) (30 µM) to NAD^+ (8.2 mM), and T155G/A160T/L263P FNR (closed triangles) (61 µM) to NAD^+ (2.5 mM).
Figure 6.
FIG. 6. Hydrogen bond pattern changes in the FAD environment after mutation at position 160. A, FNR wild type; B, T155G/A160T/L263P FNR mutant. Two new interactions are created between Thr-160 and FAD and Thr-157. This pattern is also maintained in the T155G/A160T FNR mutant. C, retraction of the 261-265 loop after site-directed mutagenesis. The C backbone of the 261-265 region is depicted with the Pro-263 residue drawn in a stick representation. Relative position of NADP+ according to the crystallographic data is shown in light blue stick representation. The L263P FNR mutant is colored in dark blue. This mutant shows a conformation for this region similar to that observed in the WT enzyme. However, a pronounced retraction is observed for this loop in the T155G/A160T/L263P FNR mutant (colored in orange), moving the structure in this region closer to that presented for the NAD^+/H-dependent nitrate reductase (colored in yellow). D, relative position of Leu-263 in the WT FNR (yellow), in the FNR-NADP+ co-crystallized complex (green) with regard to Pro-263 in the L263P FNR mutant (dark blue), and in the T155G/A160T/L263P FNR mutant (orange). C and D, the relative position of the C-terminal Tyr-303 (top of the figures) to the analyzed loop is shown.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 49203-49214) copyright 2003.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19583765 M.Medina (2009).
Structural and mechanistic aspects of flavoproteins: photosynthetic electron transfer from photosystem I to NADP+.
  FEBS J, 276, 3942-3958.  
18279389 M.A.Musumeci, A.K.Arakaki, D.V.Rial, D.L.Catalano-Dupuy, and E.A.Ceccarelli (2008).
Modulation of the enzymatic efficiency of ferredoxin-NADP(H) reductase by the amino acid volume around the catalytic site.
  FEBS J, 275, 1350-1366.  
18458342 Y.S.Choi, H.Zhang, J.S.Brunzelle, S.K.Nair, and H.Zhao (2008).
In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis.
  Proc Natl Acad Sci U S A, 105, 6858-6863.
PDB codes: 3chh 3chi 3cht 3chu
17958910 A.S.Nascimento, D.L.Catalano-Dupuy, A.Bernardes, M.d.e. .O.Neto, M.A.Santos, E.A.Ceccarelli, and I.Polikarpov (2007).
Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+.
  BMC Struct Biol, 7, 69.
PDB codes: 2rc5 2rc6
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