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Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction

Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction
Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction
Background: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-((L)-alpha-aminoadipoyl)-(L)-cysteinyl-(D)-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring.
Results: Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed.
Conclusions: In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding P-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.
enzymic oxidation, high-pressure oxygen, non-haem iron enzyme, penicillin biosynthesis, time-resolved crystallographyphotorealistic molecular graphics, time-resolved crystallography, electron-density maps, crystal-structure, enzymes, resolution, raster3d, pathway
1074-5521
1231-1237
Ogle, James M.
3156362c-ba56-4dc2-849d-1ee716febc61
Clifton, Ian J.
d5677cec-7406-4f6a-92a6-5e2b2670ba54
Rutledge, Peter J.
1bb10fcb-ce8b-4f02-8fe0-756ded021a40
Elkins, Jonathan M.
103a8855-1ca0-4abb-9ce2-a7c4f350fe39
Burzlaff, Nicolai I.
6a717e4c-c9aa-4b95-8170-73c99b01e2fd
Adlington, Robert M.
b383bda5-2be6-4b0c-8402-90ce98374cd4
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9
Baldwin, Jack E.
8ee0f163-e32e-4e77-bf41-a2de55778ef6
Ogle, James M.
3156362c-ba56-4dc2-849d-1ee716febc61
Clifton, Ian J.
d5677cec-7406-4f6a-92a6-5e2b2670ba54
Rutledge, Peter J.
1bb10fcb-ce8b-4f02-8fe0-756ded021a40
Elkins, Jonathan M.
103a8855-1ca0-4abb-9ce2-a7c4f350fe39
Burzlaff, Nicolai I.
6a717e4c-c9aa-4b95-8170-73c99b01e2fd
Adlington, Robert M.
b383bda5-2be6-4b0c-8402-90ce98374cd4
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9
Baldwin, Jack E.
8ee0f163-e32e-4e77-bf41-a2de55778ef6

Ogle, James M., Clifton, Ian J., Rutledge, Peter J., Elkins, Jonathan M., Burzlaff, Nicolai I., Adlington, Robert M., Roach, Peter L. and Baldwin, Jack E. (2001) Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction. Chemistry & Biology, 8 (12), 1231-1237. (doi:10.1016/S1074-5521(01)00090-4).

Record type: Article

Abstract

Background: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-((L)-alpha-aminoadipoyl)-(L)-cysteinyl-(D)-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring.
Results: Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed.
Conclusions: In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding P-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.

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More information

Published date: 1 December 2001
Keywords: enzymic oxidation, high-pressure oxygen, non-haem iron enzyme, penicillin biosynthesis, time-resolved crystallographyphotorealistic molecular graphics, time-resolved crystallography, electron-density maps, crystal-structure, enzymes, resolution, raster3d, pathway

Identifiers

Local EPrints ID: 19583
URI: http://eprints.soton.ac.uk/id/eprint/19583
ISSN: 1074-5521
PURE UUID: 2b3c69a1-4409-4a4e-8ec4-f17684de965a
ORCID for Peter L. Roach: ORCID iD orcid.org/0000-0001-9880-2877

Catalogue record

Date deposited: 14 Feb 2006
Last modified: 03 Dec 2019 01:53

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Contributors

Author: James M. Ogle
Author: Ian J. Clifton
Author: Peter J. Rutledge
Author: Jonathan M. Elkins
Author: Nicolai I. Burzlaff
Author: Robert M. Adlington
Author: Peter L. Roach ORCID iD
Author: Jack E. Baldwin

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