 |
PDBsum entry 1vyr
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Oxidoreductase
|
PDB id
|
|
|
|
1vyr
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Atomic resolution structures and solution behavior of enzyme-Substrate complexes of enterobacter cloacae pb2 pentaerythritol tetranitrate reductase. Multiple conformational states and implications for the mechanism of nitroaromatic explosive degradation.
|
|
|
Authors
|
|
H.Khan,
T.Barna,
R.J.Harris,
N.C.Bruce,
I.Barsukov,
A.W.Munro,
P.C.Moody,
N.S.Scrutton.
|
|
|
Ref.
|
|
J Biol Chem, 2004,
279,
30563-30572.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
The structure of pentaerythritol tetranitrate (PETN) reductase in complex with
the nitroaromatic substrate picric acid determined previously at 1.55 A
resolution indicated additional electron density between the indole ring of
residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the
presence of an unusual bond between substrate and the tryptophan side chain.
Herein, we have extended the resolution of the PETN reductase-picric acid
complex to 0.9 A. This high-resolution analysis indicates that the active site
is partially occupied with picric acid and that the anomalous density seen in
the original study is attributed to the population of multiple conformational
states of Trp-102 and not a formal covalent bond between the indole ring of
Trp-102 and picric acid. The significance of any interaction between Trp-102 and
nitroaromatic substrates was probed further in solution and crystal complexes
with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type
enzyme, in the crystalline form picric acid was bound at full occupancy in the
mutant enzymes, and there was no evidence for multiple conformations of active
site residues. Solution studies indicate tighter binding of picric acid in the
active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair
significantly enzyme reduction by NADPH, but the kinetics of decay of the
hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data
reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and
that the final distribution of reaction products for the mutant enzymes is
substantially different from wild-type enzyme. Implications for the mechanism of
high explosive degradation by PETN reductase are discussed.
|
|
|
|
|
|
|
|
Figure 1.
FIG. 1. Resonance forms of TNT. Panel A, structure of TNT
as the resonance hybrid of several canonical forms, illustrating
the enhancement in the electrophilicity of C3 and C5, the site
of hydride ion addition. Panel B, reduction of TNT by PETN
reductase to form the Meisenheimer-hydride complex.
|
|
Figure 2.
FIG. 2. Multiple conformational states of Trp-102 in PETN
reductase. Panel A, stereo pair of the electron density of
Trp-102, picrate, and FMN observed at 1.55 Å, showing the
apparent formation of a bond between the nitro group of picrate
and the indole of Trp-102. The 6-membered ring appears to have
puckered, consistent with the loss of aromatic character. Panel
B, same view of electron density observed at 0.9 Å,
showing that the side chain of Trp-102 adopts two conformations,
each with partial occupancy, thus avoiding a steric clash with
the partially occupied picrate.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
30563-30572)
copyright 2004.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.
|
|
|
Authors
|
|
T.M.Barna,
H.Khan,
N.C.Bruce,
I.Barsukov,
N.S.Scrutton,
P.C.Moody.
|
|
|
Ref.
|
|
J Mol Biol, 2001,
310,
433-447.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 2.
Figure 2. (a) Superposition of the active site illustrating
the conformation and environment of the flavin and the location
of small ligands bound close to the pyrimidine subnucleus of the
flavin isoalloxazine ring in the oxidised and reduced forms. The
oxidised forms have acetate, chloride or thiocyanate bound, the
reduced form (shown in blue) has water bound and shown
"butterfly" bending of the isoalloxazine ring along the N5-N10
axis, and into the active site. Hydrogen bonds to both the
active site histidine residues (181 and 184) are shown as broken
lines. The electron density for the acetate ion is shown in (b)
and the density for thoicyanate in (c). This Figure was prepared
with XTALVIEW[41] and Raster3D. [46]
|
|
Figure 4.
Figure 4. Chemical structures of the steroid substrates,
products and inhibitors of PETN reductase and nomenclature for
atom labelling in a general 3-oxo steroid nucleus.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
with permission from Elsevier
|
|
|
Secondary reference #2
|
|
|
Title
|
|
Crystallization and preliminary diffraction studies of pentaerythritol tetranitrate reductase from enterobacter cloacae pb2.
|
|
|
Authors
|
|
P.C.Moody,
N.Shikotra,
C.E.French,
N.C.Bruce,
N.S.Scrutton.
|
|
|
Ref.
|
|
Acta Crystallogr D Biol Crystallogr, 1998,
54,
675-677.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
Figure 2.
Fig. 2. 20 min exposure with 1
oscillation from a crystal of PETN
reductase,thecrystal-to-detectordistanceis150 mmandtheplateis
300 mm wide. The edge of the detector is at 2.0 A
Ê
.
|
|
|
|
|
|
The above figure is
reproduced from the cited reference
with permission from the IUCr
|
|
|
|
|
|
|
