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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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Interactions of a family 18 chitinase with the designed inhibitor hm508 and its degradation product, Chitobiono-Delta-Lactone.
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Authors
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G.Vaaje-Kolstad,
A.Vasella,
M.G.Peter,
C.Netter,
D.R.Houston,
B.Westereng,
B.Synstad,
V.G.Eijsink,
D.M.Van aalten.
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Ref.
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J Biol Chem, 2004,
279,
3612-3619.
[DOI no: ]
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PubMed id
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Abstract
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We describe enzymological and structural analyses of the interaction between the
family 18 chitinase ChiB from Serratia marcescens and the designed inhibitor
N,N'-diacetylchitobionoxime-N-phenylcarbamate (HM508). HM508 acts as a
competitive inhibitor of this enzyme with a K(i) in the 50 microM range. Active
site mutants of ChiB show K(i) values ranging from 1 to 200 microM, providing
insight into some of the interactions that determine inhibitor affinity.
Interestingly, the wild type enzyme slowly degrades HM508, but the inhibitor is
essentially stable in the presence of the moderately active D142N mutant of
ChiB. The crystal structure of the D142N-HM508 complex revealed that the two
sugar moieties bind to the -2 and -1 subsites, whereas the phenyl group
interacts with aromatic side chains that line the +1 and +2 subsites. Enzymatic
degradation of HM508, as well as a Trp --> Ala mutation in the +2 subsite of
ChiB, led to reduced affinity for the inhibitor, showing that interactions
between the phenyl group and the enzyme contribute to binding. Interestingly, a
complex of enzymatically degraded HM508 with the wild type enzyme showed a
chitobiono-delta-lactone bound in the -2 and -1 subsites, despite the fact that
the equilibrium between the lactone and the hydroxy acid forms in solution lies
far toward the latter. This shows that the active site preferentially binds the
(4)E conformation of the -1 sugar, which resembles the proposed transition state
of the reaction.
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Figure 1.
FIG. 1. Chemical structure of relevant compounds. A,
oxazolinium ion reaction intermediate. B, HM508
(N,N'-diacetyl-chitobionoxime-N-phenylcarbamate) (M[r] = 556).
C, chitobionolactone (M[r] = 421). D, the putative structure of
the transition state (11-13, 40).
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Figure 5.
FIG. 5. Proposed reaction scheme for HM508 degradation by
ChiB.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
3612-3619)
copyright 2004.
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Secondary reference #1
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Title
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Structural insights into the catalytic mechanism of a family 18 exo-Chitinase.
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Authors
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D.M.Van aalten,
D.Komander,
B.Synstad,
S.Gåseidnes,
M.G.Peter,
V.G.Eijsink.
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Ref.
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Proc Natl Acad Sci U S A, 2001,
98,
8979-8984.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Proposed catalytic mechanism. Asp-140, Asp-142,
and Glu-144, conserved in most family 18 chitinases, are shown
during separate stages of catalysis. The mechanism is based on
proposals by Tews et al. (9) and Brameld and Goddard (15) and
refined/expanded on the basis of the results presented in this
paper. A three-dimensional view of the changing interactions in
the crystal structures described here is shown in Fig. 2. (A)
Resting enzyme. Asp-142 is too far away to interact with
Glu-144. (B) Binding of substrate (only  1 binding
NAG residue is shown) causes distortion of the pyranose ring to
a boat or skewed boat conformation (see also Fig. 2) and
rotation of Asp-142 toward Glu-144, enabling hydrogen bond
interactions between the hydrogen of the acetamido group,
Asp-142, and Glu-144. (C) Hydrolysis of the oxazolinium ion
intermediate leads to protonation of Glu-144 and rotation of
Asp-142 to its original position where it shares a proton with
Asp-140.
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Figure 2.
Fig. 2. Structures of the ChiB complexes. The EQ,
EQ_NAG5, WT_ALLO, and WT_RX structures are shown as stereo
images in the sequence as they would occur along the reaction
coordinate (see also Fig. 1). In the stereo images, side chains
(carbons in black) interacting with the sugars are shown as
sticks, together with relevant stretches of backbone (gray). The
sugars are drawn in a stick model with green carbons. Water
molecules discussed in the text are shown as blue spheres.
Unbiased F[o] F[c] maps
(i.e., before inclusion of any ligand atom) are contoured at
2.25  [magenta,
except for the uninterpreted density at 1 in WT_RX
(yellow)]. Hydrogen bonds discussed in the text are drawn as
dotted lines. Labels identify amino acid side chains in EQ, and
the sugars bound to subsites 2 to +3 in
EQ_NAG5.
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Secondary reference #2
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Title
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Structure of a two-Domain chitotriosidase from serratia marcescens at 1.9-A resolution.
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Authors
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D.M.Van aalten,
B.Synstad,
M.B.Brurberg,
E.Hough,
B.W.Riise,
V.G.Eijsink,
R.K.Wierenga.
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Ref.
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Proc Natl Acad Sci U S A, 2000,
97,
5842-5847.
[DOI no: ]
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PubMed id
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Figure 2.
Fig. 2. (A) Comparison of experimental and final maps. An
area around the active site residue Glu144 is drawn in a stick
representation. A 1 contoured
F[o],  MLPHARE map
is shown in black, calculated by using the phases at the end of
heavy atom refinement with MLPHARE. A 2F[o]-F[c], [calc] map is
shown at the end of refinement with CNS, contoured at 1.4 (in red).
(B) The two molecules in the asymmetric unit, color-coded to
identify various regions. The TIM barrel (gray), the  /  -domain
(yellow), the support loop (red), the linker (blue), and the
ChBD (green). (C) ChiB, as in Fig. 2B, with the flexible loop
covering the active site (green), the active site residue (red
sticks), the porch loop (orange), and the exposed aromatic
residues (black sticks). (D) Superposition of the ChBD of ChiB
(blue ribbon) and the CeBD of endoglucanase Cel5 (gray ribbon).
Most of the support loop of the catalytic domain of ChiB is
shown as a dark-blue ribbon. Trp252 also is shown in magenta.
The substrate-binding residues for the CeBD are shown in yellow,
and the equivalent residues in the ChBD are shown in magenta.
The disulfide bond between the termini of the CeBD is shown in
green. Polar residues lining the path of aromatic residues in
ChiB are shown in magenta. Labels correspond to the ChiB
sequence. Note the almost exact overlap of the conserved -strands.
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Figure 3.
Fig. 3. (A) Stereo view of the active site with the
modeled chitotetraose (same view as in Fig. 1C). The ChiB
backbone is shown as a yellow ribbon. The modeled chitotetraose
is shown in a stick representation, with the carbons colored
green. Side chains within 5 Å of the chitotetraose are
depicted by gray sticks, and also are indicated in Fig. 1.
Possible hydrogen bonds are drawn as black dashed lines, and the
residues involved are indicated in Fig. 1. The four water
molecules that are predicted to be replaced by the substrate are
shown as blue transparent spheres. The GlcNAc residues are
labeled from 3 to +1,
corresponding to their location with respect to the active site
residue (15). The loop around residue 316, partially covering
the active site, is shown in magenta. (B) Stereo view of the
interior of the ChiB TIM barrel. The strands forming the TIM
barrel are shown as a yellow ribbon. Side chains of residues
lining the inside of the barrel are shown as sticks. Side chains
conserved in ChiA, ChiB, and hevamine are colored magenta. Water
molecules in the structure are shown as red spheres. Hydrogen
bonds are shown as black dashed lines. Conserved residues are
labeled according to the ChiB sequence. Part of the
chitotetraose model is shown as sticks, with carbon atoms
colored orange. (C) Stereo view of a superposition of ChiA and
ChiB. Both structures are shown in a ribbon representation. ChiB
is colored yellow, except for residues that correspond to
insertions in ChiB with respect to ChiA, which are colored red.
ChiA is colored gray except for residues that correspond to
insertions in ChiA with respect to ChiB, which are colored
green. Some insertions are indicated with two-letter labels. AA,
active site covering loop in ChiA; AB, active site covering loop
in ChiB; CD, ChBD in ChiB; DL, ChBD support loop in ChiB; FD,
fibronectin domain in ChiA; LI, linker in ChiB; PO, porch loop
in ChiB.
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