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PDBsum entry 1e5o
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Hydrolase/hydrolase inhibitor
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PDB id
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1e5o
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Contents |
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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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Structure of the catalytic core of the family f xylanase from pseudomonas fluorescens and identification of the xylopentaose-Binding sites.
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Authors
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G.W.Harris,
J.A.Jenkins,
I.Connerton,
N.Cummings,
L.Lo leggio,
M.Scott,
G.P.Hazlewood,
J.I.Laurie,
H.J.Gilbert,
R.W.Pickersgill.
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Ref.
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Structure, 1994,
2,
1107-1116.
[DOI no: ]
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PubMed id
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Abstract
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BACKGROUND: Sequence alignment suggests that xylanases evolved from two
ancestral proteins and therefore can be grouped into two families, designated F
and G. Family F enzymes show no sequence similarity with any known structure and
their architecture is unknown. Studies of an inactive enzyme-substrate complex
will help to elucidate the structural basis of binding and catalysis in the
family F xylanases. RESULTS: We have therefore determined the crystal structure
of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp.
cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of
0.20. The structure was solved using an engineered catalytic core in which the
nucleophilic glutamate was replaced by a cysteine. As expected, this yielded
both high-quality mercurial derivatives and an inactive enzyme which enabled the
preparation of the inactive enzyme-substrate complex in the crystal. We show
that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two
active-site glutamates, one of which is the nucleophile and the other the
acid-base. Xylopentaose binds to five subsites A-E with the cleaved bond between
subsites D and E. Ca2+ binding, remote from the active-site glutamates,
stabilizes the structure and may be involved in the binding of extended
substrates. CONCLUSIONS: The architecture of P. fluorescens subsp. cellulosa has
been determined crystallographically to be a commonly occurring enzyme fold, the
eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end
of the alpha/beta-barrel in an active-site cleft which contains the two
catalytic glutamates.
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Figure 3.
Figure 3. Reaction mechanism for a retaining
endo-β-1,4-xylanase. R is a number of xylose residues, HA is
the acid catalyst. The structures in brackets are possible
intermediates and R[1] is hydrogen or a number of xylose
residues. Intermediate (a) has the nucleophile stabilizing the
oxo-carbonium ion, whereas in (b) the covalent intermediate is
formed. Either of these intermediates could react with water and
be hydrolyzed or react with another xylo-oligosaccharide to
produce trans-glycosylation products. Figure 3. Reaction
mechanism for a retaining endo-β-1,4-xylanase. R is a number of
xylose residues, HA is the acid catalyst. The structures in
brackets are possible intermediates and R[1] is hydrogen or a
number of xylose residues. Intermediate (a) has the nucleophile
stabilizing the oxo-carbonium ion, whereas in (b) the covalent
intermediate is formed. Either of these intermediates could
react with water and be hydrolyzed or react with another
xylo-oligosaccharide to produce trans-glycosylation products.
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Figure 9.
Figure 9. The averaged 3.0 Å electron-density map, in the
region of β-strand 5 showing the quality of the map and the
density for aromatics which were used to initially align the
sequence with the electron-density map. The map is contoured at
1σ. Figure 9. The averaged 3.0 Å electron-density map,
in the region of β-strand 5 showing the quality of the map and
the density for aromatics which were used to initially align the
sequence with the electron-density map. The map is contoured at
1σ.
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The above figures are
reprinted
by permission from Cell Press:
Structure
(1994,
2,
1107-1116)
copyright 1994.
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