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PDBsum entry 4cel
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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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Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase i from trichoderma reesei.
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Authors
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J.Ståhlberg,
C.Divne,
A.Koivula,
K.Piens,
M.Claeyssens,
T.T.Teeri,
T.A.Jones.
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Ref.
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J Mol Biol, 1996,
264,
337-349.
[DOI no: ]
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PubMed id
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Abstract
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The roles of the residues in the catalytic trio Glu212-Asp214-Glu217 in
cellobiohydrolase I (CBHI) from Trichoderma reesei have been investigated by
changing these residues to their isosteric amide counterparts. Three mutants,
E212Q, D214N and E217Q, were constructed and expressed in T. reesei. All three
point mutations significantly impair the catalytic activity of the enzyme,
although all retain some residual activity. On the small chromophoric substrate
CNP-Lac, the kcat values were reduced to 1/2000, 1/85 and 1/370 of the wild-type
activity, respectively, whereas the KM values remained essentially unchanged. On
insoluble crystalline cellulose, BMCC, no significant activity was detected for
the E212Q and E217Q mutants, whereas the D214N mutant retained residual
activity. The consequences of the individual mutations on the active-site
structure were assessed for two of the mutants, E212Q and D214N, by X-ray
crystallography at 2.0 A and 2.2 A resolution, respectively. In addition, the
structure of E212Q CBHI in complex with the natural product, cellobiose, was
determined at 2.0 A resolution. The active-site structure of each mutant is very
similar to that of the wild-type enzyme. In the absence of ligand, the active
site of the D214N mutant contains a calcium ion firmly bound to Glu212, whereas
that of E212Q does not. This supports our hypothesis that Glu212 is the charged
species during catalysis. As in the complex of wild-type CBHI with bound
o-iodobenzyl-1-thio-beta-D-glucoside, cellobiose is bound to the two product
sites in the complex with E212Q. However, the binding of cellobiose differs from
that of the glucoside in that the cellobiose is shifted away from the trio of
catalytic residues to interact more intimately with a loop that is part of the
outer wall of the active site.
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Figure 2.
Figure 2. Close-up view of a superposition of the CBHI
wild-type and mutant active sites: wild-type/IBTG
(beige), E212Q (blue), D214N (magenta) and E212Q/cel-
lobiose (green). Only the residues close to the cleavage
site are shown. For clarity, the ligands and water
molecules have been omitted. The residue types given
refer to those of wild-type CBHI. In the D214N model, a
calcium ion is bound to Glu212. The side-chain of Gln175
flips to participate in metal co-ordination. The illustration
was created using the program O (Jones et al., 1991).
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Figure 5.
Figure 5. Superposition of residues in the active site of
CBHI (beige) and the Bacillus macerans 1,3-1,4-b-glu-
canase (blue; PDB accession code 1MAC). The side-chains
are presented as ball-and-stick models.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1996,
264,
337-349)
copyright 1996.
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Secondary reference #1
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Title
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The three-Dimensional crystal structure of the catalytic core of cellobiohydrolase i from trichoderma reesei.
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Authors
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C.Divne,
J.Ståhlberg,
T.Reinikainen,
L.Ruohonen,
G.Pettersson,
J.K.Knowles,
T.T.Teeri,
T.A.Jones.
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Ref.
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Science, 1994,
265,
524-528.
[DOI no: ]
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PubMed id
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Secondary reference #2
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Title
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Crystallization and preliminary X-Ray studies on the core proteins of cellobiohydrolase i and endoglucanase i from trichoderma reesei.
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Authors
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C.Divne,
I.Sinning,
J.Ståhlberg,
G.Pettersson,
M.Bailey,
M.Siika-Aho,
E.Margolles-Clark,
T.Teeri,
T.A.Jones.
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Ref.
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J Mol Biol, 1993,
234,
905-907.
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PubMed id
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