 |
PDBsum entry 2wc7
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Structural features of the nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity.
|
|
|
Authors
|
|
A.B.Dumbrepatil,
J.H.Choi,
J.T.Park,
M.J.Kim,
T.J.Kim,
E.J.Woo,
K.H.Park.
|
|
|
Ref.
|
|
Proteins, 2010,
78,
348-356.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
The debranching enzyme Nostoc punctiforme debranching enzyme (NPDE) from the
cyanobacterium Nostoc punctiforme (PCC73102) hydrolyzes the alpha-1,6 glycosidic
linkages of malto-oligosaccharides. Despite its high homology to
cyclodextrin/pullulan (CD/PUL)-hydrolyzing enzymes from glycosyl hydrolase 13
family (GH-13), NPDE exhibits a unique catalytic preference for longer
malto-oligosaccharides (>G8), performing hydrolysis without the
transgylcosylation or CD-hydrolyzing activities of other GH-13 enzymes. To
investigate the molecular basis for the property of NPDE, we determined the
structure of NPDE at 2.37-A resolution. NPDE lacks the typical N-terminal domain
of other CD/PUL-hydrolyzing enzymes and forms an elongated dimer in a
head-to-head configuration. The unique orientation of residues 25-55 in NPDE
yields an extended substrate binding groove from the catalytic center to the
dimeric interface. The substrate binding groove with a lengthy cavity beyond the
-1 subsite exhibits a suitable architecture for binding longer
malto-oligosaccharides (>G8). These structural results may provide a
molecular basis for the substrate specificity and catalytic function of this
cyanobacterial enzyme, distinguishing it from the classical neopullulanases and
CD/PUL-hydrolyzing enzymes.
|
|
|
|
|
|
|
|
Figure 3.
Figure 3. Dimeric arrangement of NPDE monomers. a: The dimer
interface comprised of a monomer subunit is shown. Helix 6 is
shown in violet with their residues rendered as sticks. The
residues involved in the dimer interaction hydrogen bonding are
Gly100, Ala104, Arg86, Thr46, Ala82, Asp84, Phe132, His90,
Arg31, and Glu140. b: An NPDE dimer view from the side at the
active site is shown. The Helix 6 (131-150) and the loop region
of 23-56 are shown in green and magenta, respectively. The
violet residues are from Mol A, and those in orange color are
from Mol B of the NPDE dimer. c: Estimation of the molecular
weight of NPDE by gel permeation chromatography.
|
|
Figure 4.
Figure 4. Active site architecture of NPDE. a: The active site
of NPDE is shown as a cartoon. The catalytic residues and the
subsite site residues are shown as sticks. The residue R283 is
shown to interact by hydrogen bonding with D284 and D323. The
W242 residue creating the steep geometry is shown. The F213
residue is shown and the loop region 211-218 is highlighted in
red color. b: The active site steep geometry is highlighted,
which helps in the binding of kinked substrates, thus causing
the enzyme's preference for  ,
1-6 bond over ,
1-4 and the subsite -1 interacting residues in sticks along with
the active site residues. c: Molecular model of the NPDE in
complex with maltotriose (left). The catalytic residues (green),
Trp242 residue (red), and the Tyr 87 residue (dark pink) are
shown as sticks in the models. The reducing glucose unit of
maltotriose with -1,
4-glucosidic linkage is held in position by Trp242, Trp172, and
Arg283 at +1 subsite. The second sugar ring is slightly offset
at -1 subsite above the Tyr 87 residue in the model. d:
Molecular model of the NPDE in complex with isopanose. The
substrate with -1,
6-glucosidic linkage shows proper stacking interaction and
orientation at both +1 and -1 subsites with positioning of
glucosidic oxygen at the catalytic center in the model.
|
|
|
|
|
|
The above figures are
reprinted
by permission from John Wiley & Sons, Inc.:
Proteins
(2010,
78,
348-356)
copyright 2010.
|
|
|
|
|
|
|
