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PDBsum entry 2fhf
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References listed in PDB file
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Key reference
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Title
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Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site.
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Authors
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B.Mikami,
H.Iwamoto,
D.Malle,
H.J.Yoon,
E.Demirkan-Sarikaya,
Y.Mezaki,
Y.Katsuya.
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Ref.
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J Mol Biol, 2006,
359,
690-707.
[DOI no: ]
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PubMed id
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Abstract
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The crystal structures of Klebsiella pneumoniae pullulanase and its complex with
glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or
maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a
synchrotron radiation source at SPring-8. The refined models contained 920-1052
amino acid residues, 942-1212 water molecules, four or five calcium ions, and
the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A,
and C). The N1 domain was clearly visible only in the structure of the complex
with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while
the N3, A, and C domains have weak similarity with those of Pseudomonas
isoamylase. The N1 domain was found to be a new type of carbohydrate-binding
domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2
bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately
-3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately
-1'. The two bound G3 and G4 molecules in the active cleft are almost parallel
and interact with each other. The subsites -1 approximately -4 and +1
approximately +2, including catalytic residues Glu706 and Asp677, are conserved
between pullulanase and alpha-amylase, indicating that pullulanase strongly
recognizes branched point and branched sugar residues, while subsites 0' and
-1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are
specific to pullulanase and isoamylase. The comparison suggested that the
conformational difference around the active cleft, together with the domain
organization, determines the different substrate specificities between
pullulanase and isoamylase.
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Figure 7.
Figure 7. The crystallographic dimer of the pullulanase/G4
model (stereo view). The monomers of the dimer are coloured cyan
and yellow. The bound sugar moieties are shown as red stick
models The side-chains of Ser66, Ser67, and Thr68 interacting
with the sugar moieties in the active site of the other monomer
are shown as blue and orange CPK models. The calcium ions are
shown as purple spheres.
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Figure 8.
Figure 8. (a) Superimposition of pullulanase/G4 on
pancreatic a-amylase/acarbose complex. (stereo view). The
protein model and bound sugar moieties are coloured light grey
and blue in pullulanase/G4 and dark grey and orange in
a-amylase/acarbose, respectively. (b) A cartoon showing the
glucose units of oligosaccharides and pullulan and their
position at numbered subsites. G, glucose unit of G4 (blue),
estimated position of pullulan (black) and estimated position of
substrate in a-amylase (red). The arrow in pullan indicates the
a-1,6 linkage. The triangle indicates the catalytic site.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
359,
690-707)
copyright 2006.
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