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PDBsum entry 2bjs
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Oxidoreductase
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PDB id
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2bjs
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References listed in PDB file
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Key reference
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Title
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Terminally truncated isopenicillin n synthase generates a dithioester product: evidence for a thioaldehyde intermediate during catalysis and a new mode of reaction for non-Heme iron oxidases.
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Authors
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L.A.Mcneill,
T.J.N.Brown,
M.Sami,
I.J.Clifton,
N.I.Burzlaff,
T.D.W.Claridge,
R.M.Adlington,
J.E.Baldwin,
P.J.Rutledge,
C.J.Schofield.
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Ref.
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Chemistry, 2017,
23,
12815-12824.
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PubMed id
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Abstract
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Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a
tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give
isopenicillin N (IPN), the first-formed β-lactam in penicillin and
cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II)
cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl
oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates
to the active-site metal iron. Substrate binding ablates the interaction between
Gln330 and the metal, triggering rearrangement of seven C-terminal residues,
which move to take up a conformation that extends the final α-helix and
encloses ACV in the active site. Mutagenesis studies are reported, which probe
the role of the C-terminal and other aspects of the substrate binding pocket in
IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding
pocket is important in catalysis. Deletion of seven C-terminal residues exposes
the active site and leads to formation of a new type of thiol oxidation product.
The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol
tautomer of a dithioester, made up from two molecules of ACV linked between the
thiol sulfur of one tripeptide and the oxidised cysteinyl β-carbon of the
other. A mechanism for its formation is proposed, supported by an X-ray crystal
structure, which shows the substrate ACV bound at the active site, its cysteinyl
β-carbon exposed to attack by a second molecule of substrate, adjacent.
Formation of this product constitutes a new mode of reaction for IPNS and
non-heme iron oxidases in general.
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