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
Isopenicillin N synthase (IPNS) catalyses the oxidation of a tripeptide, L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), to isopenicillin N (IPN), the first-formed β-lactam in penicillin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as co-substrate. In the absence o...
| Main Authors: | , , , , , , , , , |
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| Format: | Journal article |
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Wiley
2017
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| _version_ | 1797086184910880768 |
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| author | McNeill, LA Brown, TJN Sami, M Clifton, IJ Burzlaff, NI Claridge, TDW Adlington, RM Baldwin, JE Rutledge, PJ Schofield, CJ |
| author_facet | McNeill, LA Brown, TJN Sami, M Clifton, IJ Burzlaff, NI Claridge, TDW Adlington, RM Baldwin, JE Rutledge, PJ Schofield, CJ |
| author_sort | McNeill, LA |
| collection | OXFORD |
| description | Isopenicillin N synthase (IPNS) catalyses the oxidation of a tripeptide, L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), to isopenicillin N (IPN), the first-formed β-lactam in penicillin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as 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. Substrate binding ablates this interaction, triggering rearrangement of seven C-terminal residues which move to take up a conformation that extends the final α-helix and encloses the active site. We report mutagenesis studies probing the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. Unexpectedly, 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 X-ray crystal data 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. |
| first_indexed | 2024-03-07T02:18:28Z |
| format | Journal article |
| id | oxford-uuid:a315639f-c94d-44e6-808c-992fb5960d31 |
| institution | University of Oxford |
| last_indexed | 2024-03-07T02:18:28Z |
| publishDate | 2017 |
| publisher | Wiley |
| record_format | dspace |
| spelling | oxford-uuid:a315639f-c94d-44e6-808c-992fb5960d312022-03-27T02:24:27ZTerminally 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 oxidasesJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:a315639f-c94d-44e6-808c-992fb5960d31Symplectic Elements at OxfordWiley2017McNeill, LABrown, TJNSami, MClifton, IJBurzlaff, NIClaridge, TDWAdlington, RMBaldwin, JERutledge, PJSchofield, CJIsopenicillin N synthase (IPNS) catalyses the oxidation of a tripeptide, L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), to isopenicillin N (IPN), the first-formed β-lactam in penicillin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as 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. Substrate binding ablates this interaction, triggering rearrangement of seven C-terminal residues which move to take up a conformation that extends the final α-helix and encloses the active site. We report mutagenesis studies probing the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. Unexpectedly, 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 X-ray crystal data 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. |
| spellingShingle | McNeill, LA Brown, TJN Sami, M Clifton, IJ Burzlaff, NI Claridge, TDW Adlington, RM Baldwin, JE Rutledge, PJ Schofield, CJ 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 |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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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