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PDBsum entry 2w3x
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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 and catalytic mechanism of the thioesterase cale7 in enediyne biosynthesis.
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Authors
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M.Kotaka,
R.Kong,
I.Qureshi,
Q.S.Ho,
H.Sun,
C.W.Liew,
L.P.Goh,
P.Cheung,
Y.Mu,
J.Lescar,
Z.X.Liang.
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Ref.
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J Biol Chem, 2009,
284,
15739-15749.
[DOI no: ]
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PubMed id
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Abstract
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The biosynthesis of the enediyne moiety of the antitumor natural product
calicheamicin involves an iterative polyketide synthase (CalE8) and other
ancillary enzymes. In the proposed mechanism for the early stage of 10-membered
enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the
assistance of a putative thioesterase (CalE7). We have determined the x-ray
crystal structure of CalE7 and found that the subunit adopts a hotdog fold with
an elongated and kinked substrate-binding channel embedded between two subunits.
The 1.75-A crystal structure revealed that CalE7 does not contain a critical
catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases.
Based on biochemical and site-directed mutagenesis studies, we proposed a
catalytic mechanism in which the conserved Arg(37) plays a crucial role in the
hydrolysis of the thioester bond, and that Tyr(29) and a hydrogen-bonded water
network assist the decarboxylation of the beta-ketocarboxylic acid intermediate.
Moreover, computational docking suggested that the substrate-binding channel
binds a polyene substrate that contains a single cis double bond at the C4/C5
position, raising the possibility that the C4=C5 double bond in the enediyne
moiety could be generated by the iterative polyketide synthase. Together, the
results revealed a hotdog fold thioesterase distinct from the common type I and
type II thioesterases associated with polyketide biosynthesis and provided
interesting insight into the enediyne biosynthetic mechanism.
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Figure 1.
Calicheamicin and its biosynthesis. A, structure of
calicheamicin γ′[1] with the incorporated acetate units in
the 10-membered enediyne moiety highlighted in bold sticks. B,
early steps of the biosynthetic pathway of the 10-membered
enediyne as proposed by Kong et al. (13). The incorporated
acetate units are highlighted in bold sticks with the
configuration of the double bonds in the intermediates
arbitrarily assigned. (AT, acyl transferase; KS, ketoacyl
synthase; ACP, acyl carrier protein; KR, ketoreductase; DH,
dehydratase; and PPTase, phosphopantetheinyl transferase.).
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Figure 3.
Sequence and structure of CalE7. A, sequence alignment of
CalE7 with SgcE10 and the hypothetical protein (AQ1494) from A.
aeolicus (PDB code 2EGI). Strictly conserved residues are
highlighted in red, and partially conserved residues are yellow.
The residues mutated in the present study are indicated by blue
asterisks. B, representation of the CalE7 monomer. The protein
secondary structures elements are labeled and colored from blue
at the N terminus to red at the C terminus. C, schematic
representation of the CalE7 tetramer showing the Jeffamine
fragment (sticks) binding site between two subunits. Subunits
are colored gray and cyan for one dimer and dark blue and dark
gray for the other. The β5–α3 loops at the active sites is
colored red. The inset shows a close-up view of the Jeffamine
binding site superposed with 4-hydroxybenzoyl-CoA-bound 4-BHT
(PDB code: 1LO9). D, simulated annealing difference Fourier map
with coefficients |F[obs]| − |F[calc]| and phases calculated
from the protein model with atoms from the Jeffamine fragment
omitted and contoured at a level of 3σ.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
15739-15749)
copyright 2009.
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