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PDBsum entry 1ljl
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Oxidoreductase
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PDB id
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1ljl
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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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All intermediates of the arsenate reductase mechanism, Including an intramolecular dynamic disulfide cascade.
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Authors
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J.Messens,
J.C.Martins,
K.Van belle,
E.Brosens,
A.Desmyter,
M.De gieter,
J.M.Wieruszeski,
R.Willem,
L.Wyns,
I.Zegers.
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Ref.
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Proc Natl Acad Sci U S A, 2002,
99,
8506-8511.
[DOI no: ]
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PubMed id
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Abstract
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The mechanism of pI258 arsenate reductase (ArsC) catalyzed arsenate reduction,
involving its P-loop structural motif and three redox active cysteines, has been
unraveled. All essential intermediates are visualized with x-ray
crystallography, and NMR is used to map dynamic regions in a key disulfide
intermediate. Steady-state kinetics of ArsC mutants gives a view of the crucial
residues for catalysis. ArsC combines a phosphatase-like nucleophilic
displacement reaction with a unique intramolecular disulfide bond cascade.
Within this cascade, the formation of a disulfide bond triggers a reversible
"conformational switch" that transfers the oxidative equivalents to
the surface of the protein, while releasing the reduced substrate.
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Figure 3.
Fig. 3. (A) Scheme of the reaction mechanism of pI258
ArsC. (1) The nucleophilic attack of the thiol of Cys-10; (2)
the formation of a covalent Cys-10-HAsO[  -
]intermediate; (3) the nucleophilic attack of the thiol of
Cys-82 with arsenite release; (4) the formation of a
Cys-10-Cys-82 intermediate and the nucleophilic attack of the
thiol of Cys-89; (5) the formation of a Cys-82-Cys-89 disulfide.
(B-F) A stereo view of the 2F[o]  F[c]
electron density maps contoured at 1.0  placed
next to its corresponding reaction step in A. (B) The P-loop
(residues 10-17) in the structure of reduced wild-type ArsC with
Cys-10 in the center of the image. The P-loop is fully
structured, despite the absence of bound oxyanion (2.0 Å).
(C) In the structure of C15A ArsC-HAsO[ -
], an arsenic is covalently bound on Cys-10, surrounded by three
oxygens in a plane and a water molecule opposite the sulfur of
Cys-10 (1.4 Å). (D) Oxidized ArsC C89L with the
intermediate Cys-10-Cys-82 disulfide bond (1.6 Å). (E) A
view on the flexible looped-out region of oxidized ArsC C89L,
where Cys-89 has left the hydrophobic core and is replaced by
Leu-92 upon Cys-10-Cys-82 formation. The electron density in
this highly flexible region is not so well defined. (F) A view
on the surface of oxidized ArsC C10SC15A (6) with the
Cys-82-Cys-89 disulfide bond.
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Figure 4.
Fig. 4. The movement of the "conformational switch" in
the flexible segment (residues 80-98) trapped in four different
ArsC crystals. Starting with a helix in the reduced ArsC wild
type (blue), via oxidized ArsC C89L (Cys-10-Cys-82) in the first
(yellow) and in the second (red) molecule in the asymmetric unit
to finally looping out to form the C82-C89 disulfide (green).
The two arrows indicate the movement of L92 and C89.
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