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PDBsum entry 1e5h
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Oxidoreductase
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PDB id
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1e5h
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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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Kinetic and crystallographic studies on deacetoxycephalosporin c synthase (daocs).
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Authors
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H.J.Lee,
M.D.Lloyd,
K.Harlos,
I.J.Clifton,
J.E.Baldwin,
C.J.Schofield.
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Ref.
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J Mol Biol, 2001,
308,
937-948.
[DOI no: ]
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PubMed id
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Abstract
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Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and
2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N
to deacetoxycephalosporin C, the committed step in the biosynthesis of
cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C
terminus of one molecule is inserted into the active site of its neighbor in a
cyclical fashion within a trimeric unit. This arrangement has hindered the
generation of crystalline enzyme-substrate complexes. Therefore, we constructed
a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate
was significantly uncoupled from oxidation of the penicillin substrate in
certain truncated mutants. The extent of uncoupling varied with the number of
residues deleted and the penicillin substrate used. Crystal structures were
determined for the DeltaR306 mutant complexed with iron(II) and 2-oxoglutarate
(to 2.10 A) and the DeltaR306A mutant complexed with iron(II), succinate and
unhydrated carbon dioxide (to 1.96 A). The latter may mimic a product complex,
and supports proposals for a metal-bound CO(2) intermediate during catalysis.
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Figure 1.
Figure 1. Structure of (a) iron(II) and 2-oxoglutarate
complex of wild-type enzyme [Valegard et al 1998]; (b) iron(II)
and 2-oxoglutarate complex of the DR306 mutant; and (c)
iron(II), succinate and CO[2] complex of the DR307A mutant. The
jelly-roll motif is shown in red, C termini are shown in blue,
and the C terminus from symmetry related molecules in green.
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Figure 3.
Figure 3. A view of the structure of the DR307A mutant
iron(II), succinate and CO[2] complex. (a) A similar view to
that in Figure 2, with the above residues omitted for clarity.
The electron density map is contoured to 1.29s; (b) close up
view of the CO[2]-binding site with succinate and the
side-chains of Val262 and Phe264 enclosing one face. The
remaining face of the molecule is bordered by residues in the C
terminus, the conformation of which may be significantly
different in solution (see the text for a discussion). The
electron density map is contoured to 1.69s.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2001,
308,
937-948)
copyright 2001.
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Secondary reference #1
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Title
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Studies on the active site of deacetoxycephalosporin c synthase.
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Authors
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M.D.Lloyd,
H.J.Lee,
K.Harlos,
Z.H.Zhang,
J.E.Baldwin,
C.J.Schofield,
J.M.Charnock,
C.D.Garner,
T.Hara,
A.C.Terwisscha van scheltinga,
K.Valegård,
J.A.Viklund,
J.Hajdu,
I.Andersson,
A.Danielsson,
R.Bhikhabhai.
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Ref.
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J Mol Biol, 1999,
287,
943-960.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4. Conversion of adipoyl-6-APA (8) to adipoyl-7-ADCA
(9), and penicillin G (10) to phenylacetyl-7-ADCA (11) by DAOCS.
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Figure 10.
Figure 10. Mechanism for the expansion of penicillins by
DAOCS to cephalosporins. The binding of substrate and release of
products is probably accompanied by significant conformational
changes between “open” and “closed” forms of the active
site (see the text). R = Image -δ-(α-aminoadipoyl)-.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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