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PDBsum entry 2ign
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Oxidoreductase
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PDB id
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2ign
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References listed in PDB file
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Key reference
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Title
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Structural basis for substrate binding and regioselective oxidation of monosaccharides at c3 by pyranose 2-Oxidase.
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Authors
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M.Kujawa,
H.Ebner,
C.Leitner,
B.M.Hallberg,
M.Prongjit,
J.Sucharitakul,
R.Ludwig,
U.Rudsander,
C.Peterbauer,
P.Chaiyen,
D.Haltrich,
C.Divne.
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Ref.
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J Biol Chem, 2006,
281,
35104-35115.
[DOI no: ]
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PubMed id
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Abstract
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Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing
the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose-
and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to
the corresponding ketoaldoses. To investigate the structural determinants of
catalysis, covalent flavinylation, substrate binding, and regioselectivity,
wild-type and mutant P2Ox enzymes were produced and characterized biochemically
and structurally. Removal of the histidyl-FAD linkage resulted in a
catalytically competent enzyme containing tightly, but noncovalently bound FAD.
This mutant (H167A) is characterized by a 5-fold lower kcat, and a 35-mV lower
redox potential, although no significant structural changes were seen in its
crystal structure. In previous structures of P2Ox, the substrate loop (residues
452-457) covering the active site has been either disordered or in a
conformation incompatible with carbohydrate binding. We present here the crystal
structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose.
Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for
oxidation at C3, we also outline a probable binding mode for D-glucose
positioned for regioselective oxidation at C2. The tentative determinant for
discriminating between the two binding modes is the position of the O6 hydroxyl
group, which in the C2-oxidation mode can make favorable interactions with
Asp452 in the substrate loop and, possibly, a nearby arginine residue (Arg472).
We also substantiate our hypothesis with steady-state kinetics data for the
alanine replacements of Asp452 and Arg472 as well as the double alanine 452/472
mutant.
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Figure 3.
FIGURE 3. Details of ligand binding in nsP2Ox[ACT] and
His6-H167A[2FG]. Side-by-side view showing the active site in
the nsP2Ox[ACT] complex (A) and the His[6]-H167A[2FG] complex
(B). In B, the carbohydrate is positioned for oxidation at C3.
Firm interactions are noted for the C3 hydroxyl group and the
side chains of His^548 and Asn^593. The substrate loop is
highlighted in magenta. Atom-coloring scheme: carbon, beige
(protein), yellow (FAD), green (ligand); nitrogen, blue; oxygen,
red. C, superposition of the active sites in nsP2Ox[ACT] (beige)
and His[6]-H167A[2FG] (green).
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Figure 4.
FIGURE 4. Productive binding modes of D-glucose in the C2
and C3 orientations. A, observed binding of 2FG to the
His[6]-H167A[2FG] active site. Unbiased electron density for the
carbohydrate and part of the flavin ring. The electron density
was calculated using an early model where the ligand had not yet
been included. B, observed binding of 2FG in the C3-oxidation
orientation, and in the theoretical C2-oxidation orientation
(C). In C, the protein model from the His[6]-H167A[2FG] complex
was used for modeling the C2 orientation. The 2FG molecule was
rotated 180° about an axis defined roughly by a line running
through a point midway between the glucose atoms C5 and O5, and
a point midway between atoms C2 and C3. The same coloring scheme
is used as in Fig. 3.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
35104-35115)
copyright 2006.
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