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PDBsum entry 2x8h
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Oxidoreductase
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PDB id
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2x8h
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References listed in PDB file
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Key reference
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Title
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Mapping the catalytic cycle of schistosoma mansoni thioredoxin glutathione reductase by x-Ray crystallography.
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Authors
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F.Angelucci,
D.Dimastrogiovanni,
G.Boumis,
M.Brunori,
A.E.Miele,
F.Saccoccia,
A.Bellelli.
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Ref.
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J Biol Chem, 2010,
285,
32557-32567.
[DOI no: ]
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PubMed id
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Abstract
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Schistosomiasis is the second most widespread human parasitic disease. It is
principally treated with one drug, praziquantel, that is administered to 100
million people each year; less sensitive strains of schistosomes are emerging.
One of the most appealing drug targets against schistosomiasis is thioredoxin
glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion
of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing
reductase domain. Selenocysteine is located on a flexible C-terminal arm that is
usually disordered in the available structures of the protein and is essential
for the full catalytic activity of TGR. In this study, we dissect the catalytic
cycle of Schistosoma mansoni TGR by structural and functional analysis of the
U597C mutant. The crystallographic data presented herein include the following:
the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3
and 1.9 Å, respectively); and a different crystal form of the (partially)
reduced enzyme (3.1 Å), showing the physiological dimer and the entire C
terminus of one subunit. Whenever possible, we determined the rate constants for
the interconversion between the different oxidation states of TGR by kinetic
methods. By combining the crystallographic analysis with computer modeling, we
were able to throw further light on the mechanism of action of S. mansoni TGR.
In particular, we hereby propose the putative functionally relevant
conformational change of the C terminus after the transfer of reducing
equivalents from NADPH to the redox sites of the enzyme.
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Secondary reference #1
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Title
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Glutathione reductase and thioredoxin reductase at the crossroad: the structure of schistosoma mansoni thioredoxin glutathione reductase.
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Authors
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F.Angelucci,
A.E.Miele,
G.Boumis,
D.Dimastrogiovanni,
M.Brunori,
A.Bellelli.
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Ref.
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Proteins, 2008,
72,
936-945.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. The structure of truncated Schistosoma mansoni TGR in
ribbon representation, produced with CCP4MG.[23] Panel A:
Overall structural organization of the biological unit of the
enzyme: the W-shaped SmTGR dimer. One monomer is in blue and the
other monomer is colored according to the division in domains
from N- to C- terminus: Grx domain in light blue (1-107); FAD
binding domain in gold (108-257 and 391-461); NADPH binding
domain in green (258-358 and 364-390); the interface domain in
magenta (462-493). The linker regions between Grx and TR
(103-107) domains in magenta and the insertion (359-363)
peculiar to SmTGRs in red lie at the top and at the bottom,
respectively. Panel B: as in A, the view is from the top,
looking down the twofold dimerization axis (in black for
clarity). Panel C: The FAD binding site. View of the 2Fo-Fc
electron density map around the FAD cofactor contoured at 1.0
 .
The backbone of SmTGR around the active site is in ribbon, the
residues (K162 and Y296) that stabilize the position of the
flavine ring, together with the catalytic disulphide
(C154-C159), packing the isoalloxazine ring of the FAD, are
shown in sticks. Residues from the symmetrical monomer B, which
are implicated in the catalytic active site (H571B and E576B)
are also shown. Distances of the H-bond network range from 2.7
to 3.4 Å.
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Figure 2.
Figure 2. The putative orientation of NADPH and GSSG in SmTGR.
The SmTGR dimer is shown in a same orientation as in Figure
1(B), and is superimposed to hGR (olive-green) and hTR1 (grey).
For clarity the superimposition is displayed only for one
monomer. NADPH and GSSG from the superposed structures are
showed in CPK. Color coding as in Figure 1.
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The above figures are
reproduced from the cited reference
with permission from John Wiley & Sons, Inc.
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Secondary reference #2
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Title
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Inhibition of schistosoma mansoni thioredoxin-Glutathione reductase by auranofin: structural and kinetic aspects.
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Authors
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F.Angelucci,
A.A.Sayed,
D.L.Williams,
G.Boumis,
M.Brunori,
D.Dimastrogiovanni,
A.E.Miele,
F.Pauly,
A.Bellelli.
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Ref.
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J Biol Chem, 2009,
284,
28977-28985.
[DOI no: ]
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PubMed id
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Figure 1.
The three gold-binding sites of wild type SmTGR.A, the
three-dimensional model of one subunit is shown with the three
gold-binding sites. Site 1 shows the gold in between Cys^154 and
Cys^159; site 2 shows the gold in between Cys^520 and Cys^574;
site 3 shows the gold in the putative NADPH-binding pocket. The
glutaredoxin domain of TGR is shown in green, whereas the
thioredoxin domain is shown in red. The bound flavin is also
highlighted. B, site 1. The linear geometry of the
Cys^154-gold-Cys^159 adduct is shown. The occupancy of the gold
atom is about 50%. Both distances of the sulfur-gold bond are
2.3 Å, as expected for this type of coordination moiety.
C, site 2. The electron density map (2F[o] − F[c]) contoured
at 1 σ shows the possible charge transfer complex between the
gold and Phe^505. D, site 2. The gold atom between Cys^574 and
Cys^520 is shown together with the other residues that surround
the metal, i.e. Phe^505, Pro^507, and Pro^542. E, site 3. Gold
in the putative NADPH-binding site of SmTGR. Tyr^296 is known to
swing upon NADPH binding in thiol reductase enzymes (30).
Ser^295 is the residue closest to the gold (Ser^295(OG)-gold:
3.2 Å). Other van der Waals' contacts are with the main
chain atoms of the polypeptide (Ala^390, Val^391, Gly^392, and
Arg^393).
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Figure 2.
The SmTGR crystal structure in complex with gold ions (in
green) is superimposed to the mouse TR with NADPH bound (in
magenta; Protein Data Bank code 1zdl (30)). The root mean square
deviation is 0.82 Å over the 462 aligned residues. The
residues surrounding NADPH in mouse TR are conserved in SmTGR
(sequence alignment not shown). The structural comparison shows
the change in conformation of the loop 293–296 and in
particular of Tyr^296 and Ser^295, highlighted for the two
enzymes as balls and sticks (the other amino acid side chains
are omitted for clarity). The OG atom of Ser^295 is the closest
contact with the gold ion in the SmTGR crystal structure (see
“Results” and Fig. 1). In the mouse TR structure, Ser^295
shifts in position to make room for the bound NADPH; the clash
between the metal in site 3 and the phosphate of the cofactor in
SmTGR is self-evident.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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