The reaction cycle of isopenicillin N synthase observed by X-ray diffraction

Nicolai I. Burzlaff, Peter J. Rutledge, Ian J. Clifton, Charles M. H. Hensgens, Michael Pickford, Robert M. Adlington, Peter L. Roach and Jack E. Baldwin ()
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Nicolai I. Burzlaff: The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford
Peter J. Rutledge: The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford
Ian J. Clifton: The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford
Charles M. H. Hensgens: The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford
Michael Pickford: The Laboratory of Molecular Biophysics, University of Oxford
Robert M. Adlington: The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford
Peter L. Roach: The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford
Jack E. Baldwin: The Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford

Nature, 1999, vol. 401, issue 6754, 721-724

Abstract: Abstract Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the biosynthesis of isopenicillin N (IPN), the precursor of all penicillins and cephalosporins1. The key steps in this reaction are the two iron-dioxygen-mediated ring closures of the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV). It has been proposed that the four-membered β-lactam ring forms initially, associated with a highly oxidized iron(IV)-oxo (ferryl) moiety, which subsequently mediates closure of the five-membered thiazolidine ring2. Here we describe observation of the IPNS reaction in crystals by X-ray crystallography. IPNS·Fe2+·substrate crystals were grown anaerobically3,4, exposed to high pressures of oxygen to promote reaction and frozen, and their structures were elucidated by X-ray diffraction. Using the natural substrate ACV, this resulted in the IPNS·Fe2+·IPN product complex. With the substrate analogue, δ-(L-α-aminoadipoyl)-L-cysteinyl-L-S-methylcysteine (ACmC) in the crystal, the reaction cycle was interrupted at the monocyclic stage. These mono- and bicyclic structures support our hypothesis of a two-stage reaction sequence leading to penicillin. Furthermore, the formation of a monocyclic sulphoxide product from ACmC is most simply explained by the interception of a high-valency iron-oxo species.

Date: 1999
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DOI: 10.1038/44400

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