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PDBsum entry 1bqe
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Oxidoreductase
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PDB id
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1bqe
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Probing the determinants of coenzyme specificity in ferredoxin-Nadp+ reductase by site-Directed mutagenesis.
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Authors
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M.Medina,
A.Luquita,
J.Tejero,
J.Hermoso,
T.Mayoral,
J.Sanz-Aparicio,
K.Grever,
C.Gomez-Moreno.
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Ref.
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J Biol Chem, 2001,
276,
11902-11912.
[DOI no: ]
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PubMed id
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Abstract
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On the basis of sequence and three-dimensional structure comparison between
Anabaena PCC7119 ferredoxin-NADP(+) reductase (FNR) and other reductases from
its structurally related family that bind either NADP(+)/H or NAD(+)/H, a set of
amino acid residues that might determine the FNR coenzyme specificity can be
assigned. These residues include Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235.
Systematic replacement of these amino acids was done to identify which of them
are the main determinants of coenzyme specificity. Our data indicate that all of
the residues interacting with the 2'-phosphate of NADP(+)/H in Anabaena FNR are
not involved to the same extent in determining coenzyme specificity and
affinity. Thus, it is found that Ser-223 and Tyr-235 are important for
determining NADP(+)/H specificity and orientation with respect to the protein,
whereas Arg-224 and Arg-233 provide only secondary interactions in Anabaena FNR.
The analysis of the T155G FNR form also indicates that the determinants of
coenzyme specificity are not only situated in the 2'-phosphate NADP(+)/H
interacting region but that other regions of the protein must be involved. These
regions, although not interacting directly with the coenzyme, must produce
specific structural arrangements of the backbone chain that determine coenzyme
specificity. The loop formed by residues 261-268 in Anabaena FNR must be one of
these regions.
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Figure 9.
Fig. 9. Hydrogen bond network and structural differences
in the FNR WT (A) and in the T155G FNR mutant (B). In the native
state, OH (Thr-155) is making a bifurcated H-bond with the
Leu-263 residue. Two new interactions are created after
mutation: O (Leu-263) stabilizes a new interaction with N
(Met-266), and O  2
(Gly-267) stabilizes a new interaction with N (Gly-265). This
produces a less extended conformation for the 261-268 loop in
the mutated enzyme.
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Figure 10.
Fig. 10. MOLSCRIPT drawing of the superposition of
Anabaena FNR (light) and NADH-cytochrome b[5] reductase (dark)
(58) near the position of residue 155. In the NAD^+/H-dependent
enzymes, a Gly residue at this position is favored due to the
presence of a hairpin-like region (formed by a series of
prolines) that will not allow the space for a Thr to occupy
position 155 of FNR. On the contrary, the absence of this
hairpin in the NADP+/H-dependent enzymes permits the presence of
residues such as Thr or Pro at this position. Relevant residues
are labeled as chain A in FNR or chain B in cytochrome b[5]
reductase.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2001,
276,
11902-11912)
copyright 2001.
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Secondary reference #1
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Title
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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.
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Authors
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L.Serre,
F.M.Vellieux,
M.Medina,
C.Gomez-Moreno,
J.C.Fontecilla-Camps,
M.Frey.
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Ref.
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J Mol Biol, 1996,
263,
20-39.
[DOI no: ]
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PubMed id
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Figure 5.
Figure 5. Interaction between FNR and ferredoxin. The charged residues, which are probably involved in the binding
of FNR with ferredoxin and are currently mutated, are represented by thick lines.
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Figure 8.
Figure 8. A stereoscopic view of the difference electron density at the NADP
+
site (contoured at 2s) calculated with
phases from a model obtained by refining the native FNR X-ray model at 1.8 Å resolution (omitting residue 1 to 8,
the sulfate ion and the water molecules) by simulated annealing and energy minimization against the amplitudes from
the FNR-NADP
+
crystal. The final NADP
+
position is represented.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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