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PDBsum entry 2c1f
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References listed in PDB file
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Key reference
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Title
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Understanding the structural basis for substrate and inhibitor recognition in eukaryotic gh11 xylanases.
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Authors
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M.Vardakou,
C.Dumon,
J.W.Murray,
P.Christakopoulos,
D.P.Weiner,
N.Juge,
R.J.Lewis,
H.J.Gilbert,
J.E.Flint.
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Ref.
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J Mol Biol, 2008,
375,
1293-1305.
[DOI no: ]
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PubMed id
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Abstract
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Endo-beta1,4-xylanases (xylanases) hydrolyse the beta1,4 glycosidic bonds in the
backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11)
have been extensively studied, several issues remain unresolved. Thus, the
mechanism by which these enzymes hydrolyse decorated xylans is unclear and the
structural basis for the variation in catalytic activity within this family is
unknown. Furthermore, the mechanism for the differences in the inhibition of
fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these
issues we report the crystal structure and biochemical properties of the
Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high
catalytic activity and is one of the few fungal GH11 proteins not inhibited by
XIP-I. Although the structure of NpXyn11A could not be determined in complex
with substrates, we have been able to investigate how GH11 enzymes hydrolyse
decorated substrates by solving the crystal structure of a second GH11 xylanase,
EnXyn11A (encoded by an environmental DNA sample), bound to ferulic
acid-1,5-arabinofuranose-alpha1,3-xylotriose (FAX(3)). The crystal structure of
the EnXyn11A-FAX(3) complex shows that solvent exposure of the backbone xylose
O2 and O3 groups at subsites -3 and +2 allow accommodation of alpha1,2-linked
4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the
ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic
interactions at the +2 subsite, indicating that the decoration may represent a
specificity determinant at this aglycone subsite. The structure of NpXyn11A
reveals potential -3 and +3 subsites that are kinetically significant. The
extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases,
may explain why the Neocallimastix enzyme displays unusually high catalytic
activity. Finally, the crystal structure of NpXyn11A shows that the resistance
of the enzyme to XIP-I is not due solely to insertions in the loop connecting
beta strands 11 and 12, as suggested previously, but is highly complex.
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Figure 4.
Figure 4. The interactions between EnXyn11A and FAX[3.] The
subsites are labelled, the broken lines represent hydrogen
bonding interactions, and the interatomic distances are noted.
(a) The glycone and (b) the aglycone region of the active site.
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Figure 6.
Figure 6. Overlay of the XIP-I/P. funiculosum GH11 xylanase
complex with wild-type and mutant NpXyn11A. (a) The structure of
the XIP-I (green)/P. funiculosum GH11 xylanase (red) complex was
overlaid with wild-type NpXyn11A (cyan). The loops connecting β
strand 3 with 4 and β strand 11 with 12 appear to clash with
the xylanase inhibitor. (b) Wild-type NpXyn11 (cyan) and the
Δ3-4NpXyn11A mutant (magenta) were overlaid with the structure
of XIP-I when in complex with the P. funiculosum GH11 xylanase.
The mutation has reduced the size of the loop connecting β
strands 3 and 4, which now no longer make steric clashes with
XIP-I.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2008,
375,
1293-1305)
copyright 2008.
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