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PDBsum entry 2ogs
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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 the geobacillus stearothermophilus carboxylesterase est55 and its activation of prodrug cpt-11.
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Authors
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P.Liu,
H.E.Ewis,
P.C.Tai,
C.D.Lu,
I.T.Weber.
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Ref.
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J Mol Biol, 2007,
367,
212-223.
[DOI no: ]
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PubMed id
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Abstract
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Several mammalian carboxylesterases were shown to activate the prodrug
irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a
topoisomerase inhibitor used in cancer therapy. However, the potential use of
bacterial carboxylesterases, which have the advantage of high stability, has not
been explored. We present the crystal structure of the carboxyesterase Est55
from Geobacillus stearothermophilus and evaluation of its enzyme activity on
CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution
of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic
domain, an alpha/beta domain and a regulatory domain. The structure is in an
inactive form; the side-chain of His409, one of the catalytic triad residues, is
directed away from the other catalytic residues Ser194 and Glu310. Moreover, the
adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would
block the binding of substrate, suggesting a regulatory role. However, Cys408 is
not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic
side-chains were favorable, while polar serine was unfavorable for enzyme
activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The
mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore,
engineered thermostable Est55 is a candidate for use with irinotecan in
enzyme-prodrug cancer therapy.
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Figure 2.
Figure 2. Superposition of Est55 and human carboxylesterase
hCE1 structures. Est55 is in green and hCE1 is in red. The
active sites are within the ellipse. The black arrow indicates
the regulatory domain, which shows significant differences
between the two structures. Figure 2. Superposition of Est55
and human carboxylesterase hCE1 structures. Est55 is in green
and hCE1 is in red. The active sites are within the ellipse. The
black arrow indicates the regulatory domain, which shows
significant differences between the two structures.
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Figure 4.
Figure 4. (a) The active site region of Est55. Inter-atomic
interactions are shown as broken lines with distances in
Å. The catalytic residues Ser194, Glu310 and His409 are
labeled in red. (b) Comparison of active sites of Est55 (green)
and hCE1 (red). The catalytic triad residues of hCE1 are Ser221,
Glu354, and His468. Cys408 in Est55 is equivalent to Asp467 in
hCE1. Figure 4. (a) The active site region of Est55.
Inter-atomic interactions are shown as broken lines with
distances in Å. The catalytic residues Ser194, Glu310 and
His409 are labeled in red. (b) Comparison of active sites of
Est55 (green) and hCE1 (red). The catalytic triad residues of
hCE1 are Ser221, Glu354, and His468. Cys408 in Est55 is
equivalent to Asp467 in hCE1.
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The above figures are
reprinted
from an Open Access publication published by Elsevier:
J Mol Biol
(2007,
367,
212-223)
copyright 2007.
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